CA2170638A1 - Improvements in or relating to haemoglobin - Google Patents
Improvements in or relating to haemoglobinInfo
- Publication number
- CA2170638A1 CA2170638A1 CA002170638A CA2170638A CA2170638A1 CA 2170638 A1 CA2170638 A1 CA 2170638A1 CA 002170638 A CA002170638 A CA 002170638A CA 2170638 A CA2170638 A CA 2170638A CA 2170638 A1 CA2170638 A1 CA 2170638A1
- Authority
- CA
- Canada
- Prior art keywords
- globin
- residues
- beta
- leu
- haemoglobin
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
- 108060003196 globin Proteins 0.000 claims abstract description 68
- 102000018146 globin Human genes 0.000 claims abstract description 67
- BVKZGUZCCUSVTD-UHFFFAOYSA-M Bicarbonate Chemical compound OC([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-M 0.000 claims abstract description 38
- 230000000694 effects Effects 0.000 claims abstract description 38
- 230000004075 alteration Effects 0.000 claims abstract description 10
- 125000000539 amino acid group Chemical group 0.000 claims abstract description 7
- 230000004048 modification Effects 0.000 claims abstract description 6
- 238000012986 modification Methods 0.000 claims abstract description 6
- LNNWVNGFPYWNQE-GMIGKAJZSA-N desomorphine Chemical compound C1C2=CC=C(O)C3=C2[C@]24CCN(C)[C@H]1[C@@H]2CCC[C@@H]4O3 LNNWVNGFPYWNQE-GMIGKAJZSA-N 0.000 claims description 47
- 241000270722 Crocodylidae Species 0.000 claims description 46
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 44
- 229910052760 oxygen Inorganic materials 0.000 claims description 44
- 239000001301 oxygen Substances 0.000 claims description 44
- 238000006467 substitution reaction Methods 0.000 claims description 11
- 239000002473 artificial blood Substances 0.000 claims description 6
- 125000003275 alpha amino acid group Chemical group 0.000 claims 1
- 125000003588 lysine group Chemical group [H]N([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])(N([H])[H])C(*)=O 0.000 claims 1
- 230000035772 mutation Effects 0.000 description 15
- 241000588724 Escherichia coli Species 0.000 description 7
- 150000001413 amino acids Chemical group 0.000 description 7
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 6
- 241000270311 Crocodylus niloticus Species 0.000 description 6
- 241000251539 Vertebrata <Metazoa> Species 0.000 description 6
- 150000003278 haem Chemical class 0.000 description 6
- 108090000623 proteins and genes Proteins 0.000 description 6
- 230000014509 gene expression Effects 0.000 description 5
- 210000004369 blood Anatomy 0.000 description 4
- 239000008280 blood Substances 0.000 description 4
- 238000000034 method Methods 0.000 description 4
- TZCGZYWNIDZZMR-UHFFFAOYSA-N Ile-Arg-Ala Natural products CCC(C)C(N)C(=O)NC(C(=O)NC(C)C(O)=O)CCCN=C(N)N TZCGZYWNIDZZMR-UHFFFAOYSA-N 0.000 description 3
- DDNIHOWRDOXXPF-NGZCFLSTSA-N Val-Asp-Pro Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)N1CCC[C@@H]1C(=O)O)N DDNIHOWRDOXXPF-NGZCFLSTSA-N 0.000 description 3
- 108010044940 alanylglutamine Proteins 0.000 description 3
- 239000003633 blood substitute Substances 0.000 description 3
- 229910002092 carbon dioxide Inorganic materials 0.000 description 3
- 210000003743 erythrocyte Anatomy 0.000 description 3
- 108010049041 glutamylalanine Proteins 0.000 description 3
- 108010050848 glycylleucine Proteins 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 230000009467 reduction Effects 0.000 description 3
- 241000894007 species Species 0.000 description 3
- COHPSYLINFUPSS-HHHIRMLJSA-N (2s)-2-[[(2s)-2-[[(2s)-6-amino-2-[[(2s)-2-[[(2s,3r)-2-amino-3-hydroxybutanoyl]amino]-3-hydroxypropanoyl]amino]hexanoyl]amino]-3-(4-hydroxyphenyl)propanoyl]amino]-5-(diaminomethylideneamino)pentanoic acid Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCCN)C(=O)N[C@H](C(=O)N[C@@H](CCCN=C(N)N)C(O)=O)CC1=CC=C(O)C=C1 COHPSYLINFUPSS-HHHIRMLJSA-N 0.000 description 2
- XOHUEYCVLUUEJJ-UHFFFAOYSA-I 2,3-Diphosphoglycerate Chemical compound [O-]P(=O)([O-])OC(C(=O)[O-])COP([O-])([O-])=O XOHUEYCVLUUEJJ-UHFFFAOYSA-I 0.000 description 2
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 2
- BYXHQQCXAJARLQ-ZLUOBGJFSA-N Ala-Ala-Ala Chemical compound C[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](C)C(O)=O BYXHQQCXAJARLQ-ZLUOBGJFSA-N 0.000 description 2
- YLTKNGYYPIWKHZ-ACZMJKKPSA-N Ala-Ala-Glu Chemical compound C[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@H](C(O)=O)CCC(O)=O YLTKNGYYPIWKHZ-ACZMJKKPSA-N 0.000 description 2
- WQVFQXXBNHHPLX-ZKWXMUAHSA-N Ala-Ala-His Chemical compound C[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](Cc1cnc[nH]1)C(O)=O WQVFQXXBNHHPLX-ZKWXMUAHSA-N 0.000 description 2
- OKEWAFFWMHBGPT-XPUUQOCRSA-N Ala-His-Gly Chemical compound OC(=O)CNC(=O)[C@@H](NC(=O)[C@@H](N)C)CC1=CN=CN1 OKEWAFFWMHBGPT-XPUUQOCRSA-N 0.000 description 2
- NMXKFWOEASXOGB-QSFUFRPTSA-N Ala-Ile-His Chemical compound C[C@H](N)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@H](C(O)=O)CC1=CN=CN1 NMXKFWOEASXOGB-QSFUFRPTSA-N 0.000 description 2
- XSLGWYYNOSUMRM-ZKWXMUAHSA-N Ala-Val-Asn Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(N)=O)C(O)=O XSLGWYYNOSUMRM-ZKWXMUAHSA-N 0.000 description 2
- RVHGJNGNKGDCPX-KKUMJFAQSA-N Asn-Phe-Lys Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CC(=O)N)N RVHGJNGNKGDCPX-KKUMJFAQSA-N 0.000 description 2
- WNGZKSVJFDZICU-XIRDDKMYSA-N Asp-Leu-Trp Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CC1=CNC2=CC=CC=C21)C(=O)O)NC(=O)[C@H](CC(=O)O)N WNGZKSVJFDZICU-XIRDDKMYSA-N 0.000 description 2
- QJHOOKBAHRJPPX-QWRGUYRKSA-N Asp-Phe-Gly Chemical compound OC(=O)C[C@H](N)C(=O)N[C@H](C(=O)NCC(O)=O)CC1=CC=CC=C1 QJHOOKBAHRJPPX-QWRGUYRKSA-N 0.000 description 2
- QTIZKMMLNUMHHU-DCAQKATOSA-N Asp-Pro-His Chemical compound C1C[C@H](N(C1)C(=O)[C@H](CC(=O)O)N)C(=O)N[C@@H](CC2=CN=CN2)C(=O)O QTIZKMMLNUMHHU-DCAQKATOSA-N 0.000 description 2
- MBPKYKSYUAPLMY-DCAQKATOSA-N Cys-Arg-Leu Chemical compound [H]N[C@@H](CS)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(O)=O MBPKYKSYUAPLMY-DCAQKATOSA-N 0.000 description 2
- 108020004414 DNA Proteins 0.000 description 2
- ZDJZEGYVKANKED-NRPADANISA-N Gln-Cys-Val Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CS)C(=O)N[C@@H](C(C)C)C(O)=O ZDJZEGYVKANKED-NRPADANISA-N 0.000 description 2
- TWTWUBHEWQPMQW-ZPFDUUQYSA-N Gln-Ile-Arg Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O TWTWUBHEWQPMQW-ZPFDUUQYSA-N 0.000 description 2
- SBYVDRJAXWSXQL-AVGNSLFASA-N Glu-Asn-Phe Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O SBYVDRJAXWSXQL-AVGNSLFASA-N 0.000 description 2
- UJMNFCAHLYKWOZ-DCAQKATOSA-N Glu-Lys-Gln Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCC(N)=O)C(O)=O UJMNFCAHLYKWOZ-DCAQKATOSA-N 0.000 description 2
- XBWMTPAIUQIWKA-BYULHYEWSA-N Gly-Asp-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)CN XBWMTPAIUQIWKA-BYULHYEWSA-N 0.000 description 2
- MOJKRXIRAZPZLW-WDSKDSINSA-N Gly-Glu-Ala Chemical compound [H]NCC(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(O)=O MOJKRXIRAZPZLW-WDSKDSINSA-N 0.000 description 2
- IRJWAYCXIYUHQE-WHFBIAKZSA-N Gly-Ser-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@H](CO)NC(=O)CN IRJWAYCXIYUHQE-WHFBIAKZSA-N 0.000 description 2
- DZMVESFTHXSSPZ-XVYDVKMFSA-N His-Ala-Ser Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](C)C(=O)N[C@@H](CO)C(O)=O DZMVESFTHXSSPZ-XVYDVKMFSA-N 0.000 description 2
- VTZYMXGGXOFBMX-DJFWLOJKSA-N His-Ile-Asp Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC(O)=O)C(O)=O VTZYMXGGXOFBMX-DJFWLOJKSA-N 0.000 description 2
- RLAOTFTXBFQJDV-KKUMJFAQSA-N His-Phe-Asp Chemical compound C([C@H](N)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CC(O)=O)C(O)=O)C1=CN=CN1 RLAOTFTXBFQJDV-KKUMJFAQSA-N 0.000 description 2
- QCBYAHHNOHBXIH-UWVGGRQHSA-N His-Pro-Gly Chemical compound C([C@H](N)C(=O)N1[C@@H](CCC1)C(=O)NCC(O)=O)C1=CN=CN1 QCBYAHHNOHBXIH-UWVGGRQHSA-N 0.000 description 2
- BBQABUDWDUKJMB-LZXPERKUSA-N Ile-Ile-Ile Chemical compound CC[C@H](C)[C@H]([NH3+])C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)CC)C([O-])=O BBQABUDWDUKJMB-LZXPERKUSA-N 0.000 description 2
- MYGQXVYRZMKRDB-SRVKXCTJSA-N Leu-Asp-Lys Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@H](C(O)=O)CCCCN MYGQXVYRZMKRDB-SRVKXCTJSA-N 0.000 description 2
- WMTOVWLLDGQGCV-GUBZILKMSA-N Leu-Glu-Cys Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](CS)C(=O)O)N WMTOVWLLDGQGCV-GUBZILKMSA-N 0.000 description 2
- KXODZBLFVFSLAI-AVGNSLFASA-N Leu-His-Glu Chemical compound OC(=O)CC[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)[C@@H](N)CC(C)C)CC1=CN=CN1 KXODZBLFVFSLAI-AVGNSLFASA-N 0.000 description 2
- QJXHMYMRGDOHRU-NHCYSSNCSA-N Leu-Ile-Gly Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(O)=O QJXHMYMRGDOHRU-NHCYSSNCSA-N 0.000 description 2
- ADJWHHZETYAAAX-SRVKXCTJSA-N Leu-Ser-His Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)N ADJWHHZETYAAAX-SRVKXCTJSA-N 0.000 description 2
- FBNPMTNBFFAMMH-UHFFFAOYSA-N Leu-Val-Arg Natural products CC(C)CC(N)C(=O)NC(C(C)C)C(=O)NC(C(O)=O)CCCN=C(N)N FBNPMTNBFFAMMH-UHFFFAOYSA-N 0.000 description 2
- XTONYTDATVADQH-CIUDSAMLSA-N Lys-Cys-Asn Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CS)C(=O)N[C@@H](CC(N)=O)C(O)=O XTONYTDATVADQH-CIUDSAMLSA-N 0.000 description 2
- QQPSCXKFDSORFT-IHRRRGAJSA-N Lys-Lys-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@@H](N)CCCCN QQPSCXKFDSORFT-IHRRRGAJSA-N 0.000 description 2
- DBXMFHGGHMXYHY-DCAQKATOSA-N Met-Leu-Ser Chemical compound CSCC[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(O)=O DBXMFHGGHMXYHY-DCAQKATOSA-N 0.000 description 2
- FIZZULTXMVEIAA-IHRRRGAJSA-N Met-Ser-Phe Chemical compound CSCC[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 FIZZULTXMVEIAA-IHRRRGAJSA-N 0.000 description 2
- BJEYSVHMGIJORT-NHCYSSNCSA-N Phe-Ala-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@@H](N)CC1=CC=CC=C1 BJEYSVHMGIJORT-NHCYSSNCSA-N 0.000 description 2
- UEHNWRNADDPYNK-DLOVCJGASA-N Phe-Cys-Ala Chemical compound C[C@@H](C(=O)O)NC(=O)[C@H](CS)NC(=O)[C@H](CC1=CC=CC=C1)N UEHNWRNADDPYNK-DLOVCJGASA-N 0.000 description 2
- KXUZHWXENMYOHC-QEJZJMRPSA-N Phe-Leu-Ala Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C)C(O)=O KXUZHWXENMYOHC-QEJZJMRPSA-N 0.000 description 2
- VPEVBAUSTBWQHN-NHCYSSNCSA-N Pro-Glu-Val Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C(C)C)C(O)=O VPEVBAUSTBWQHN-NHCYSSNCSA-N 0.000 description 2
- MESDJCNHLZBMEP-ZLUOBGJFSA-N Ser-Asp-Asp Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(O)=O)C(O)=O MESDJCNHLZBMEP-ZLUOBGJFSA-N 0.000 description 2
- MUJQWSAWLLRJCE-KATARQTJSA-N Ser-Leu-Thr Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O MUJQWSAWLLRJCE-KATARQTJSA-N 0.000 description 2
- UUSQVWOVUYMLJA-PPCPHDFISA-N Thr-Lys-Ile Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O UUSQVWOVUYMLJA-PPCPHDFISA-N 0.000 description 2
- WURLIFOWSMBUAR-SLFFLAALSA-N Tyr-Phe-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CC2=CC=CC=C2)NC(=O)[C@H](CC3=CC=C(C=C3)O)N)C(=O)O WURLIFOWSMBUAR-SLFFLAALSA-N 0.000 description 2
- ARMNWLJYHCOSHE-KKUMJFAQSA-N Tyr-Pro-Gln Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCC(N)=O)C(O)=O ARMNWLJYHCOSHE-KKUMJFAQSA-N 0.000 description 2
- MNWINJDPGBNOED-ULQDDVLXSA-N Tyr-Pro-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1C(=O)[C@@H](N)CC1=CC=C(O)C=C1 MNWINJDPGBNOED-ULQDDVLXSA-N 0.000 description 2
- RWOGENDAOGMHLX-DCAQKATOSA-N Val-Lys-Ala Chemical compound C[C@@H](C(=O)O)NC(=O)[C@H](CCCCN)NC(=O)[C@H](C(C)C)N RWOGENDAOGMHLX-DCAQKATOSA-N 0.000 description 2
- SVLAAUGFIHSJPK-JYJNAYRXSA-N Val-Trp-Ser Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC1=CNC2=CC=CC=C21)C(=O)N[C@@H](CO)C(=O)O)N SVLAAUGFIHSJPK-JYJNAYRXSA-N 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 108010070944 alanylhistidine Proteins 0.000 description 2
- 230000003281 allosteric effect Effects 0.000 description 2
- 125000003277 amino group Chemical group 0.000 description 2
- 230000000692 anti-sense effect Effects 0.000 description 2
- 239000001569 carbon dioxide Substances 0.000 description 2
- 239000013604 expression vector Substances 0.000 description 2
- 108010000434 glycyl-alanyl-leucine Proteins 0.000 description 2
- 108010082286 glycyl-seryl-alanine Proteins 0.000 description 2
- 108010015792 glycyllysine Proteins 0.000 description 2
- 108010028295 histidylhistidine Proteins 0.000 description 2
- 108010025306 histidylleucine Proteins 0.000 description 2
- 230000003993 interaction Effects 0.000 description 2
- 108010044374 isoleucyl-tyrosine Proteins 0.000 description 2
- 108010060857 isoleucyl-valyl-tyrosine Proteins 0.000 description 2
- 108010003700 lysyl aspartic acid Proteins 0.000 description 2
- 108010038320 lysylphenylalanine Proteins 0.000 description 2
- 108010017391 lysylvaline Proteins 0.000 description 2
- 229910052753 mercury Inorganic materials 0.000 description 2
- 239000002207 metabolite Substances 0.000 description 2
- 108010072397 neo-kyotorphin Proteins 0.000 description 2
- 108020004707 nucleic acids Proteins 0.000 description 2
- 102000039446 nucleic acids Human genes 0.000 description 2
- 150000007523 nucleic acids Chemical class 0.000 description 2
- 229920001184 polypeptide Polymers 0.000 description 2
- 108090000765 processed proteins & peptides Proteins 0.000 description 2
- 102000004196 processed proteins & peptides Human genes 0.000 description 2
- 108010048397 seryl-lysyl-leucine Proteins 0.000 description 2
- 108010029384 tryptophyl-histidine Proteins 0.000 description 2
- 241000251468 Actinopterygii Species 0.000 description 1
- NXSFUECZFORGOG-CIUDSAMLSA-N Ala-Asn-Leu Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(C)C)C(O)=O NXSFUECZFORGOG-CIUDSAMLSA-N 0.000 description 1
- LGFCAXJBAZESCF-ACZMJKKPSA-N Ala-Gln-Ala Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](C)C(O)=O LGFCAXJBAZESCF-ACZMJKKPSA-N 0.000 description 1
- BTBUEVAGZCKULD-XPUUQOCRSA-N Ala-Gly-His Chemical compound C[C@H](N)C(=O)NCC(=O)N[C@H](C(O)=O)CC1=CN=CN1 BTBUEVAGZCKULD-XPUUQOCRSA-N 0.000 description 1
- ANGAOPNEPIDLPO-XVYDVKMFSA-N Ala-His-Cys Chemical compound C[C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)N[C@@H](CS)C(=O)O)N ANGAOPNEPIDLPO-XVYDVKMFSA-N 0.000 description 1
- FAJIYNONGXEXAI-CQDKDKBSSA-N Ala-His-Phe Chemical compound C([C@H](NC(=O)[C@@H](N)C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(O)=O)C1=CNC=N1 FAJIYNONGXEXAI-CQDKDKBSSA-N 0.000 description 1
- CCDFBRZVTDDJNM-GUBZILKMSA-N Ala-Leu-Glu Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(O)=O CCDFBRZVTDDJNM-GUBZILKMSA-N 0.000 description 1
- SOBIAADAMRHGKH-CIUDSAMLSA-N Ala-Leu-Ser Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(O)=O SOBIAADAMRHGKH-CIUDSAMLSA-N 0.000 description 1
- PEEYDECOOVQKRZ-DLOVCJGASA-N Ala-Ser-Phe Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O PEEYDECOOVQKRZ-DLOVCJGASA-N 0.000 description 1
- BOKLLPVAQDSLHC-FXQIFTODSA-N Ala-Val-Cys Chemical compound C[C@@H](C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CS)C(=O)O)N BOKLLPVAQDSLHC-FXQIFTODSA-N 0.000 description 1
- REWSWYIDQIELBE-FXQIFTODSA-N Ala-Val-Ser Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CO)C(O)=O REWSWYIDQIELBE-FXQIFTODSA-N 0.000 description 1
- 241000270728 Alligator Species 0.000 description 1
- YFWTXMRJJDNTLM-LSJOCFKGSA-N Arg-Ala-His Chemical compound C[C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)NC(=O)[C@H](CCCN=C(N)N)N YFWTXMRJJDNTLM-LSJOCFKGSA-N 0.000 description 1
- YUIGJDNAGKJLDO-JYJNAYRXSA-N Arg-Arg-Tyr Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O YUIGJDNAGKJLDO-JYJNAYRXSA-N 0.000 description 1
- OISWSORSLQOGFV-AVGNSLFASA-N Arg-Met-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](CCSC)NC(=O)[C@@H](N)CCCN=C(N)N OISWSORSLQOGFV-AVGNSLFASA-N 0.000 description 1
- LFWOQHSQNCKXRU-UFYCRDLUSA-N Arg-Tyr-Phe Chemical compound C([C@H](NC(=O)[C@H](CCCN=C(N)N)N)C(=O)N[C@@H](CC=1C=CC=CC=1)C(O)=O)C1=CC=C(O)C=C1 LFWOQHSQNCKXRU-UFYCRDLUSA-N 0.000 description 1
- OLGCWMNDJTWQAG-GUBZILKMSA-N Asn-Glu-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](N)CC(N)=O OLGCWMNDJTWQAG-GUBZILKMSA-N 0.000 description 1
- IVPNEDNYYYFAGI-GARJFASQSA-N Asp-Leu-Pro Chemical compound CC(C)C[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CC(=O)O)N IVPNEDNYYYFAGI-GARJFASQSA-N 0.000 description 1
- XWKBWZXGNXTDKY-ZKWXMUAHSA-N Asp-Val-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@H](C(C)C)NC(=O)[C@@H](N)CC(O)=O XWKBWZXGNXTDKY-ZKWXMUAHSA-N 0.000 description 1
- 108020004705 Codon Proteins 0.000 description 1
- UWZLBXOBVKRUFE-HGNGGELXSA-N Gln-Ala-His Chemical compound C[C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)NC(=O)[C@H](CCC(=O)N)N UWZLBXOBVKRUFE-HGNGGELXSA-N 0.000 description 1
- LKUWAWGNJYJODH-KBIXCLLPSA-N Gln-Ala-Ile Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](C)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O LKUWAWGNJYJODH-KBIXCLLPSA-N 0.000 description 1
- FKXCBKCOSVIGCT-AVGNSLFASA-N Gln-Lys-Leu Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(O)=O FKXCBKCOSVIGCT-AVGNSLFASA-N 0.000 description 1
- ZZLDMBMFKZFQMU-NRPADANISA-N Gln-Val-Ala Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](C)C(O)=O ZZLDMBMFKZFQMU-NRPADANISA-N 0.000 description 1
- ITYRYNUZHPNCIK-GUBZILKMSA-N Glu-Ala-Leu Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(O)=O ITYRYNUZHPNCIK-GUBZILKMSA-N 0.000 description 1
- KEBACWCLVOXFNC-DCAQKATOSA-N Glu-Arg-Met Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCSC)C(O)=O KEBACWCLVOXFNC-DCAQKATOSA-N 0.000 description 1
- BUAKRRKDHSSIKK-IHRRRGAJSA-N Glu-Glu-Tyr Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 BUAKRRKDHSSIKK-IHRRRGAJSA-N 0.000 description 1
- ILWHFUZZCFYSKT-AVGNSLFASA-N Glu-Lys-Leu Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(O)=O ILWHFUZZCFYSKT-AVGNSLFASA-N 0.000 description 1
- SUIAHERNFYRBDZ-GVXVVHGQSA-N Glu-Lys-Val Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C(C)C)C(O)=O SUIAHERNFYRBDZ-GVXVVHGQSA-N 0.000 description 1
- MFVQGXGQRIXBPK-WDSKDSINSA-N Gly-Ala-Glu Chemical compound NCC(=O)N[C@@H](C)C(=O)N[C@@H](CCC(O)=O)C(O)=O MFVQGXGQRIXBPK-WDSKDSINSA-N 0.000 description 1
- VSVZIEVNUYDAFR-YUMQZZPRSA-N Gly-Ala-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)CN VSVZIEVNUYDAFR-YUMQZZPRSA-N 0.000 description 1
- GDOZQTNZPCUARW-YFKPBYRVSA-N Gly-Gly-Glu Chemical compound NCC(=O)NCC(=O)N[C@H](C(O)=O)CCC(O)=O GDOZQTNZPCUARW-YFKPBYRVSA-N 0.000 description 1
- FSPVILZGHUJOHS-QWRGUYRKSA-N Gly-His-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)CN)CC1=CNC=N1 FSPVILZGHUJOHS-QWRGUYRKSA-N 0.000 description 1
- SXJHOPPTOJACOA-QXEWZRGKSA-N Gly-Ile-Arg Chemical compound NCC(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@H](C(O)=O)CCCN=C(N)N SXJHOPPTOJACOA-QXEWZRGKSA-N 0.000 description 1
- VEPBEGNDJYANCF-QWRGUYRKSA-N Gly-Lys-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)CN)CCCCN VEPBEGNDJYANCF-QWRGUYRKSA-N 0.000 description 1
- BIAKMWKJMQLZOJ-ZKWXMUAHSA-N His-Ala-Ala Chemical compound C[C@H](NC(=O)[C@H](C)NC(=O)[C@@H](N)Cc1cnc[nH]1)C(O)=O BIAKMWKJMQLZOJ-ZKWXMUAHSA-N 0.000 description 1
- IPIVXQQRZXEUGW-UWJYBYFXSA-N His-Ala-His Chemical compound C([C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1NC=NC=1)C(O)=O)C1=CN=CN1 IPIVXQQRZXEUGW-UWJYBYFXSA-N 0.000 description 1
- CYHWWHKRCKHYGQ-GUBZILKMSA-N His-Cys-Glu Chemical compound C1=C(NC=N1)C[C@@H](C(=O)N[C@@H](CS)C(=O)N[C@@H](CCC(=O)O)C(=O)O)N CYHWWHKRCKHYGQ-GUBZILKMSA-N 0.000 description 1
- OHOXVDFVRDGFND-YUMQZZPRSA-N His-Cys-Gly Chemical compound N[C@@H](Cc1cnc[nH]1)C(=O)N[C@@H](CS)C(=O)NCC(O)=O OHOXVDFVRDGFND-YUMQZZPRSA-N 0.000 description 1
- NQKRILCJYCASDV-QWRGUYRKSA-N His-Gly-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)CNC(=O)[C@@H](N)CC1=CN=CN1 NQKRILCJYCASDV-QWRGUYRKSA-N 0.000 description 1
- VFBZWZXKCVBTJR-SRVKXCTJSA-N His-Leu-Asp Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)O)NC(=O)[C@H](CC1=CN=CN1)N VFBZWZXKCVBTJR-SRVKXCTJSA-N 0.000 description 1
- TVMNTHXFRSXZGR-IHRRRGAJSA-N His-Lys-Val Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C(C)C)C(O)=O TVMNTHXFRSXZGR-IHRRRGAJSA-N 0.000 description 1
- PZAJPILZRFPYJJ-SRVKXCTJSA-N His-Ser-Leu Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(O)=O PZAJPILZRFPYJJ-SRVKXCTJSA-N 0.000 description 1
- KDDKJKKQODQQBR-NHCYSSNCSA-N His-Val-Asp Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)O)NC(=O)[C@H](CC1=CN=CN1)N KDDKJKKQODQQBR-NHCYSSNCSA-N 0.000 description 1
- 101000899111 Homo sapiens Hemoglobin subunit beta Proteins 0.000 description 1
- NNVXABCGXOLIEB-PYJNHQTQSA-N Ile-Met-His Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@@H](CCSC)C(=O)N[C@H](C(O)=O)CC1=CN=CN1 NNVXABCGXOLIEB-PYJNHQTQSA-N 0.000 description 1
- JODPUDMBQBIWCK-GHCJXIJMSA-N Ile-Ser-Asn Chemical compound [H]N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(N)=O)C(O)=O JODPUDMBQBIWCK-GHCJXIJMSA-N 0.000 description 1
- UYODHPPSCXBNCS-XUXIUFHCSA-N Ile-Val-Leu Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@H](C(O)=O)CC(C)C UYODHPPSCXBNCS-XUXIUFHCSA-N 0.000 description 1
- QSXSHZIRKTUXNG-STECZYCISA-N Ile-Val-Tyr Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 QSXSHZIRKTUXNG-STECZYCISA-N 0.000 description 1
- HGCNKOLVKRAVHD-UHFFFAOYSA-N L-Met-L-Phe Natural products CSCCC(N)C(=O)NC(C(O)=O)CC1=CC=CC=C1 HGCNKOLVKRAVHD-UHFFFAOYSA-N 0.000 description 1
- FADYJNXDPBKVCA-UHFFFAOYSA-N L-Phenylalanyl-L-lysin Natural products NCCCCC(C(O)=O)NC(=O)C(N)CC1=CC=CC=C1 FADYJNXDPBKVCA-UHFFFAOYSA-N 0.000 description 1
- 241000880493 Leptailurus serval Species 0.000 description 1
- BQSLGJHIAGOZCD-CIUDSAMLSA-N Leu-Ala-Ser Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](CO)C(O)=O BQSLGJHIAGOZCD-CIUDSAMLSA-N 0.000 description 1
- ULXYQAJWJGLCNR-YUMQZZPRSA-N Leu-Asp-Gly Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)NCC(O)=O ULXYQAJWJGLCNR-YUMQZZPRSA-N 0.000 description 1
- IIKJNQWOQIWWMR-CIUDSAMLSA-N Leu-Cys-Ala Chemical compound C[C@@H](C(=O)O)NC(=O)[C@H](CS)NC(=O)[C@H](CC(C)C)N IIKJNQWOQIWWMR-CIUDSAMLSA-N 0.000 description 1
- WIDZHJTYKYBLSR-DCAQKATOSA-N Leu-Glu-Glu Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(O)=O WIDZHJTYKYBLSR-DCAQKATOSA-N 0.000 description 1
- LAPSXOAUPNOINL-YUMQZZPRSA-N Leu-Gly-Asp Chemical compound CC(C)C[C@H](N)C(=O)NCC(=O)N[C@H](C(O)=O)CC(O)=O LAPSXOAUPNOINL-YUMQZZPRSA-N 0.000 description 1
- PBGDOSARRIJMEV-DLOVCJGASA-N Leu-His-Ala Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](C)C(O)=O PBGDOSARRIJMEV-DLOVCJGASA-N 0.000 description 1
- DDEMUMVXNFPDKC-SRVKXCTJSA-N Leu-His-Cys Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)N[C@@H](CS)C(=O)O)N DDEMUMVXNFPDKC-SRVKXCTJSA-N 0.000 description 1
- UCBPDSYUVAAHCD-UWVGGRQHSA-N Leu-Pro-Gly Chemical compound CC(C)C[C@H](N)C(=O)N1CCC[C@H]1C(=O)NCC(O)=O UCBPDSYUVAAHCD-UWVGGRQHSA-N 0.000 description 1
- IDGZVZJLYFTXSL-DCAQKATOSA-N Leu-Ser-Arg Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@H](C(O)=O)CCCN=C(N)N IDGZVZJLYFTXSL-DCAQKATOSA-N 0.000 description 1
- AMSSKPUHBUQBOQ-SRVKXCTJSA-N Leu-Ser-Lys Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCCN)C(=O)O)N AMSSKPUHBUQBOQ-SRVKXCTJSA-N 0.000 description 1
- FBNPMTNBFFAMMH-AVGNSLFASA-N Leu-Val-Arg Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@H](C(O)=O)CCCN=C(N)N FBNPMTNBFFAMMH-AVGNSLFASA-N 0.000 description 1
- AIRZWUMAHCDDHR-KKUMJFAQSA-N Lys-Leu-Leu Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(O)=O AIRZWUMAHCDDHR-KKUMJFAQSA-N 0.000 description 1
- VWPJQIHBBOJWDN-DCAQKATOSA-N Lys-Val-Ala Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](C)C(O)=O VWPJQIHBBOJWDN-DCAQKATOSA-N 0.000 description 1
- DRRXXZBXDMLGFC-IHRRRGAJSA-N Lys-Val-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@@H](N)CCCCN DRRXXZBXDMLGFC-IHRRRGAJSA-N 0.000 description 1
- FGAMAYQCWQCUNF-DCAQKATOSA-N Met-His-Asn Chemical compound CSCC[C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)N[C@@H](CC(=O)N)C(=O)O)N FGAMAYQCWQCUNF-DCAQKATOSA-N 0.000 description 1
- OSZTUONKUMCWEP-XUXIUFHCSA-N Met-Leu-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CCSC OSZTUONKUMCWEP-XUXIUFHCSA-N 0.000 description 1
- 125000000729 N-terminal amino-acid group Chemical group 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 108091034117 Oligonucleotide Proteins 0.000 description 1
- WSXKXSBOJXEZDV-DLOVCJGASA-N Phe-Ala-Asn Chemical compound NC(=O)C[C@@H](C([O-])=O)NC(=O)[C@H](C)NC(=O)[C@@H]([NH3+])CC1=CC=CC=C1 WSXKXSBOJXEZDV-DLOVCJGASA-N 0.000 description 1
- FMMIYCMOVGXZIP-AVGNSLFASA-N Phe-Glu-Asn Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(N)=O)C(O)=O FMMIYCMOVGXZIP-AVGNSLFASA-N 0.000 description 1
- JEBWZLWTRPZQRX-QWRGUYRKSA-N Phe-Gly-Asp Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)NCC(=O)N[C@@H](CC(O)=O)C(O)=O JEBWZLWTRPZQRX-QWRGUYRKSA-N 0.000 description 1
- ZLGQEBCCANLYRA-RYUDHWBXSA-N Phe-Gly-Glu Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)NCC(=O)N[C@@H](CCC(O)=O)C(O)=O ZLGQEBCCANLYRA-RYUDHWBXSA-N 0.000 description 1
- TXKWKTWYTIAZSV-KKUMJFAQSA-N Phe-Leu-Cys Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CS)C(=O)O)NC(=O)[C@H](CC1=CC=CC=C1)N TXKWKTWYTIAZSV-KKUMJFAQSA-N 0.000 description 1
- DOXQMJCSSYZSNM-BZSNNMDCSA-N Phe-Lys-Leu Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(O)=O DOXQMJCSSYZSNM-BZSNNMDCSA-N 0.000 description 1
- KIPIKSXPPLABPN-CIUDSAMLSA-N Pro-Glu-Asn Chemical compound NC(=O)C[C@@H](C(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H]1CCCN1 KIPIKSXPPLABPN-CIUDSAMLSA-N 0.000 description 1
- FYXCBXDAMPEHIQ-FHWLQOOXSA-N Pro-Trp-Lys Chemical compound C1C[C@H](NC1)C(=O)N[C@@H](CC2=CNC3=CC=CC=C32)C(=O)N[C@@H](CCCCN)C(=O)O FYXCBXDAMPEHIQ-FHWLQOOXSA-N 0.000 description 1
- XVAUJOAYHWWNQF-ZLUOBGJFSA-N Ser-Asn-Ala Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](C)C(O)=O XVAUJOAYHWWNQF-ZLUOBGJFSA-N 0.000 description 1
- QYSFWUIXDFJUDW-DCAQKATOSA-N Ser-Leu-Arg Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O QYSFWUIXDFJUDW-DCAQKATOSA-N 0.000 description 1
- XUDRHBPSPAPDJP-SRVKXCTJSA-N Ser-Lys-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@@H](N)CO XUDRHBPSPAPDJP-SRVKXCTJSA-N 0.000 description 1
- YEDSOSIKVUMIJE-DCAQKATOSA-N Ser-Val-Leu Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(C)C)C(O)=O YEDSOSIKVUMIJE-DCAQKATOSA-N 0.000 description 1
- ZHZLQVLQBDBQCQ-WDSOQIARSA-N Trp-Lys-Arg Chemical compound C1=CC=C2C(=C1)C(=CN2)C[C@@H](C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCN=C(N)N)C(=O)O)N ZHZLQVLQBDBQCQ-WDSOQIARSA-N 0.000 description 1
- WZQZUVWEPMGIMM-JYJNAYRXSA-N Tyr-Gln-Lys Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)N[C@@H](CCCCN)C(=O)O)N)O WZQZUVWEPMGIMM-JYJNAYRXSA-N 0.000 description 1
- CNLKDWSAORJEMW-KWQFWETISA-N Tyr-Gly-Ala Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)NCC(=O)N[C@@H](C)C(O)=O CNLKDWSAORJEMW-KWQFWETISA-N 0.000 description 1
- JIODCDXKCJRMEH-NHCYSSNCSA-N Val-Arg-Gln Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CCC(=O)N)C(=O)O)N JIODCDXKCJRMEH-NHCYSSNCSA-N 0.000 description 1
- SCBITHMBEJNRHC-LSJOCFKGSA-N Val-Asp-Val Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](C(C)C)C(=O)O)N SCBITHMBEJNRHC-LSJOCFKGSA-N 0.000 description 1
- YLHLNFUXDBOAGX-DCAQKATOSA-N Val-Cys-His Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CS)C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)N YLHLNFUXDBOAGX-DCAQKATOSA-N 0.000 description 1
- CFSSLXZJEMERJY-NRPADANISA-N Val-Gln-Ala Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](C)C(O)=O CFSSLXZJEMERJY-NRPADANISA-N 0.000 description 1
- SYSWVVCYSXBVJG-RHYQMDGZSA-N Val-Leu-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](C(C)C)N)O SYSWVVCYSXBVJG-RHYQMDGZSA-N 0.000 description 1
- PZTZYZUTCPZWJH-FXQIFTODSA-N Val-Ser-Ser Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)O)N PZTZYZUTCPZWJH-FXQIFTODSA-N 0.000 description 1
- PMKQKNBISAOSRI-XHSDSOJGSA-N Val-Tyr-Pro Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)N2CCC[C@@H]2C(=O)O)N PMKQKNBISAOSRI-XHSDSOJGSA-N 0.000 description 1
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 1
- 108010047495 alanylglycine Proteins 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 108010008355 arginyl-glutamine Proteins 0.000 description 1
- 108010068380 arginylarginine Proteins 0.000 description 1
- 108010040443 aspartyl-aspartic acid Proteins 0.000 description 1
- 108010093581 aspartyl-proline Proteins 0.000 description 1
- 108010047857 aspartylglycine Proteins 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- OWMVSZAMULFTJU-UHFFFAOYSA-N bis-tris Chemical compound OCCN(CCO)C(CO)(CO)CO OWMVSZAMULFTJU-UHFFFAOYSA-N 0.000 description 1
- 229940125691 blood product Drugs 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 230000021235 carbamoylation Effects 0.000 description 1
- 238000012219 cassette mutagenesis Methods 0.000 description 1
- 230000004186 co-expression Effects 0.000 description 1
- 238000004891 communication Methods 0.000 description 1
- 238000012926 crystallographic analysis Methods 0.000 description 1
- 108010016616 cysteinylglycine Proteins 0.000 description 1
- 230000002939 deleterious effect Effects 0.000 description 1
- 239000000539 dimer Substances 0.000 description 1
- 230000009982 effect on human Effects 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- ZZUFCTLCJUWOSV-UHFFFAOYSA-N furosemide Chemical compound C1=C(Cl)C(S(=O)(=O)N)=CC(C(O)=O)=C1NCC1=CC=CO1 ZZUFCTLCJUWOSV-UHFFFAOYSA-N 0.000 description 1
- 108010040030 histidinoalanine Proteins 0.000 description 1
- 108010018006 histidylserine Proteins 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 230000002163 immunogen Effects 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 108010087810 leucyl-seryl-glutamyl-leucine Proteins 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- QSHDDOUJBYECFT-UHFFFAOYSA-N mercury Chemical compound [Hg] QSHDDOUJBYECFT-UHFFFAOYSA-N 0.000 description 1
- MYWUZJCMWCOHBA-VIFPVBQESA-N methamphetamine Chemical compound CN[C@@H](C)CC1=CC=CC=C1 MYWUZJCMWCOHBA-VIFPVBQESA-N 0.000 description 1
- 108010068488 methionylphenylalanine Proteins 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- GWUSZQUVEVMBPI-UHFFFAOYSA-N nimetazepam Chemical compound N=1CC(=O)N(C)C2=CC=C([N+]([O-])=O)C=C2C=1C1=CC=CC=C1 GWUSZQUVEVMBPI-UHFFFAOYSA-N 0.000 description 1
- 108010012581 phenylalanylglutamate Proteins 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000000241 respiratory effect Effects 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- 230000003019 stabilising effect Effects 0.000 description 1
- 239000013598 vector Substances 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/795—Porphyrin- or corrin-ring-containing peptides
- C07K14/805—Haemoglobins; Myoglobins
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P7/00—Drugs for disorders of the blood or the extracellular fluid
- A61P7/08—Plasma substitutes; Perfusion solutions; Dialytics or haemodialytics; Drugs for electrolytic or acid-base disorders, e.g. hypovolemic shock
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Abstract
Disclosed is a .beta. globin modified so as to exhibit, when present as part of a haemoglobin molecule, an altered bicarbonate effect compared to the unmodified .beta. globin, the modification comprising alteration of at least two of the amino acid residues in the group consisting of residues (29-41), and a haemoglobin molecule comprising said .beta. globin, with or without further modifications to the .alpha. globin component thereof.
Description
PcT/Gss4lo 1 996 WO 9510793~ 7 ~ 6 3 ~
TITLE: IMPROVEMENTS ~N OR RELATING TO HAEMOGLOBIN
Field of the Invention This invention relates to improved forms of haemoglobin and to improved blood substitutes comprising haemoglobin.
Back~round to the Invention All vertebrate haemoglobins (Hb) are tetramers consisting of two alpha (~) globin chains and two beta (~B) globin sub-units each carrying an iron-cont~ining prosthetic group, haem.
The oxygen binding to the four sub-units within the tetramer is co-operative (ie binding of oxygen at one haem group facilitates the binding of oxygen at the other haem groups on the same globin tetramer and conversely, the unloading of oxygen at one haem unit facilitates the unloading of oxygen at the others).
During vertebrate evolution the amino acid sequence of globins has diverged considerably, and the sequence identity between Hbs from the most distantly-related vertebrate species, (eg human and fish), is only about 50%. That between human and crocodile haemaglobins, for example, is about 60%. Despite this sequence variation, vertebrate Hbs have ~enerally retained their tertiary and quaternary structures (Camardella et al., 1992 J. Mol. Biol. _'~4, 449-460) and the key residues necessary for haem-haem interaction .
The oxygen affinity of human Hb is modulated by various metabolites, such as 2,3-diphosphoglycerate (DPG) and H+. They facilitate unloading of oxygen to activelyrespiring tissues. This general phenomenon is called the heterotropic allosteric effect.
-
TITLE: IMPROVEMENTS ~N OR RELATING TO HAEMOGLOBIN
Field of the Invention This invention relates to improved forms of haemoglobin and to improved blood substitutes comprising haemoglobin.
Back~round to the Invention All vertebrate haemoglobins (Hb) are tetramers consisting of two alpha (~) globin chains and two beta (~B) globin sub-units each carrying an iron-cont~ining prosthetic group, haem.
The oxygen binding to the four sub-units within the tetramer is co-operative (ie binding of oxygen at one haem group facilitates the binding of oxygen at the other haem groups on the same globin tetramer and conversely, the unloading of oxygen at one haem unit facilitates the unloading of oxygen at the others).
During vertebrate evolution the amino acid sequence of globins has diverged considerably, and the sequence identity between Hbs from the most distantly-related vertebrate species, (eg human and fish), is only about 50%. That between human and crocodile haemaglobins, for example, is about 60%. Despite this sequence variation, vertebrate Hbs have ~enerally retained their tertiary and quaternary structures (Camardella et al., 1992 J. Mol. Biol. _'~4, 449-460) and the key residues necessary for haem-haem interaction .
The oxygen affinity of human Hb is modulated by various metabolites, such as 2,3-diphosphoglycerate (DPG) and H+. They facilitate unloading of oxygen to activelyrespiring tissues. This general phenomenon is called the heterotropic allosteric effect.
-
2 ~ 7 ~ PCT/GB94101~96 ~
Wo 95/07932 ~. 7 In particular, the oxygen affinity of Hb is affected by the concentration of C0~, such thathigh concentrations of C0~, (with a partial pressure, "PC0," of about 40mm Hg, as will be found in actively respiring tissues) cause a reduction in the oxygen affinity, thereby facilitating the unloading of oxygen to the tissues. This phenomenon is known as the C0~
effect. In human Hb, C0. binds directly to the ~x-amino groups of the ~ and ,B globin submits to form carbamino groups and reduces the oxygen affinity by stabilising the low affinity (T) quaternary structure (Kilmartin & Rossi-Bernardi, 1969 Nature ~ ~, 1243-1746). The C01 effect is one of the principal heterotropic allosteric effects.
Vertebrate species have adapted to strikingly diverse environmental conditions, and the haemoglobin molecule has evolved to meet a wide range of respiratory needs in such environments. For example, the oxygen affinity of crocodile Hb is marketly reduced by physiological concentrations of C07 (40 torr). Bauer et al. (1981, J. Biol. Chem. 756, 8479-8435) showed that the large reduction in the oxygen affinity of crocodile Hb is not caused by the binding of C0~ to the a amino groups as in human Hb. Carbon dioxide (C0~) dissolves in aqueous solutions (such as blood) to give HC03-. The marked reduction in the oxygen affinity in crocodile Hb is caused by the binding of bicarbonate ions and this phenomenon is called the "bicarbonate effect". A much larger proportion of Hb-bound oxygen can be released and utilised in the tissues and this enables crocodiles to stay under water for a long time. It has been postulated (Perutz et al., 1981 ~atue 291, 682-684) that this effect is due to bicarbonate ions binding to residues Lys 82 and Glu 144 of the ,B globin molecule.
There is interest in developing "artificial blood" products. Such products would elimin~te the chance of infection due to a patient receiving infected blood from a donor, and generally simplify the blood transfusion process. Clearly, in order to perform all the functions of natural blood, the artificial product must be capable of transporting oxygen.
Ijnfortunately, haemoglobin outside the environment of a red blood cell (RBC) has too high an affinity to release much oxygen to the tissues. This is because RBCs contain large amounts of DPG which, as mentioned above, is one of the metabolites which lowers the oxygen affinitv of Hb.
217 ~ pcTlGBs~lol996 95l07932 Consequently, there is a need for a stable, artificial blood product with improved oxygen affinity characteristics which will deliver oxygen more effectively to respiring tissues.
Summarv of the Invention In a first aspect the invention provides a ~ globin modified so as to exhibit, when present as part of a haemoglobin molecule, an altered bicarbonate effect compared to theunmodified ,B globin, the modification comprising alteration of at least two of the amino acid residues in the group con~i~ting of residues ~9-41.
It was found by the present inventors that human haemoglobin comprising ~i' globin subunits altered in accordance with the invention, by substitution with equivalent crocodile ,B globin residues, exhibited a greatly enh~rlce~l bicarbonate effect analogous to that displayed by native crocodile haemoglobin. This is surprising because haemoglobin is a complex tetrameric molecule with prosthetic groups, yet very small changes in just the ,~
globin primary structure in a (crocodile) ~5 (human) haemoglobin can cause large changes in the bicarbonate effect, whilst the a globin and prosthetic groups are unaltered.
Further, most of residues ~9-41 of ,~ globin are located at the interface of the a~ 2 globin subunits and changes therein might thus be expected to cause the molecule to lose stability or permanently reduce its oxygen affinity. It was surprisingly found, however, that an alteration in accordance with the invention did not cause such deleterious effects.
In a second aspect the invention provides a method of modifying a ~ globin so as to exhibit, when present as part of an haemoglobin molecule, an altered bicarbonate effect compared to the unmodified ,B globin, the modification comprising substitution of at least two of the amino acid residues in the group consisting of residues '~9-41.
Preferably the ~5 globin is human ,6' globin. Preferably the ,~ globin is modified so as to exhibit an enhanced bicarbonate efect (ie a greater decrease in oxygen affinity in the presence of aqueous carbon dioxide) compared to the unmodified ,~ globin.
23L~3~
PCTIGBs~/0l99 WO 95/0?932 It is preferred that the at least two altered arnino acid residues comprise at least two residues from the group consisting o~ residues '~9, 31. 33, 38, 39 and 41. Most preferably the modification comprises substitution of residues 38 and 41.
In a preferred embodiment, residues ~9-41 of human ~B globin are replaced with the equivalent residues from crocodile ,B globin. It will be apparent to those skilled in the art that some of the amino acid residues are common to the ~Ib of both species. Accordingly, it is not necessary to alter all the residues 29-41 but simply those residues which are not common to both (ie residues '79, 31, 33, 38, 39 and 41). Moreover residues Ser '~9,B and Met 31,~ are not in the al~ contact and substitutions of these residues not be required in order to introduce the full bicarbonate effect.
In another aspect the invention provides a haemoglobin molecule for use as an artificial blood substitute, comprising a,~? globin modified so as to exhibit an altered bicarbonate effect compared to a haemoglobin molecule comprising an unmodified ~ globin, themodification comprising substitution of at least two of the amino acid residues in the group consisting of residues 29-41.
Conveniently the haemoglobin molecule also comprises a modified c~ globin chain.
Typically the ~ globin chain will comprise a substantially human c~ globin se~uence modified at one or more residues so as to optimise the oxygen-transport characteristics of the haemoglobin molecule for use as an artifical blood substitute. Conveniently the a globin chain will be modified in a manner as disclosed in UK Patent Application No 941477'7.5. or similar, so as to exhibit an altered bicarbonate effect compared to the unmodified c globin.
Conveniently the modified c~ globin will comprise an alteration at one or more of the following residues: 34, 35, 36. 37, 41, 100 and 103.
Desirably the modified c~ globin will comprise one or more (preferably all) of the following substitutions: Leu 34~Cvs. Ser 35 ~Ala, Phe 36 ~Tyr, Thr 37 ~Gln, Thr 41 ~Ile, 217 0 ~ 3 ~ pcTlGBs4lol996 ~ Wo 95/07932 s Leu 100~Phe and His 103 ~Gln.
In a further aspect, the invention provides an artificial blood substitute comprising the haemoglobin molecule defined above.
The invention will now be further described by way of illustrative example and by reference to the drawings, in which:
Figure 1 shows the nucleotide sequences encoding the a and ,B globins of the Nile crocodile (as optimised for expression in E. coli) and the amino acid sequences of the encoded polypeptides;
Figure 7 is a schematic representation of the crocodile a and ~5 globin expression constructs pOmega-5 and pManuo-4, and of the hybrid expression construct pSC4-11;
Figure 3a is a graph of the oxygen binding curve (% 07 saturation against log pO~ in mm Hg) for recombinant crocodile (circles) and human (squares) haemoglobins in the absence (open symbols) or presence (filled-in symbols) of 5% CO~;
Figure 3b is a similar graph comparing the oxygen binding curves of hybrid a(crocodile)~/,B(human)7 (circles) and a(crocodile)~/~B(SC4)~ (squares) haemoglobins;
Figure 3c is a similar graph comparing the oxygen binding curve of recombinant "Scuba"
haemoglobin with (circles) or without (squares) alterations at residues 100a and 103a; and Figure 4 is a schematic representation of human deoxyhaemoglobin.
Example 1 As described previously, Perutz et al. (1981, cited above) proposed bicarbonate ion binding sites to be located in the central cavity between two ~B subunits and to involve hydro en bonds to Lys (87 ,B) and Glu (14 f~) of one ~ subunit and the N-terminal residues 217~3~
PCT/GB94/nl996 _ of its partner. Four differences (relative to human haemoglobin) in crocodile Hb, His-143 ~' ~AIa, Lys-144 k'~Gly. Val-1 ,6' Se~ and His-~ Pro, are thought to be important to promote formation of these hydrogen bonds. The present inventors first introduced these mutations into human ,6' globin but the CO, effect was the same as in native human Hb.
It was considered that some additional mutations may be needed to create the appropriate electrostatic environment for the interaction of these residues with bicarbonate ions.
Accordingly, three additional mutations, Asp-94 ~6' to Glu, Glu-90 ,~ ~Lys and His-135 k' ~Arg, were introduced into this mutant but they still failed to show a large bicarbonate effect in human Hb. At this stage this strategy was abandoned and it was decided to first make crocodile Hb in E. coli and to find mutations which abolish the bicarbonate effect.
As a first step, protein coding sequences of Nile crocodile a and ,~ globins were chemically synthesised by assembly of 14 oligonucleotides with codons optimal for expression in E. coli (Ikemura et al., 1982 J. Mol. Biol. 158, 573-597). All mutations were introduced by cassette mutagenesis (Wells et al., 1985 Gene 34, 315). The synthetic a and ,6' globin coding sequences, and the polypeptides encoded thereby, are shown in Figure 1 and as Seq. ID Nos. 1-4 in the attached sequence listing. These genes were cloned separately into the Hb expression vector developed by Somatogen, (Hoffman et al., 1990 Proc Nat. Acad. Sci. USA 87 8521-8525). This vector was derived from pKK223-3 (available from Pharmacia). The expression constructs pOmega-5 and pManuo-4 (expressing the Nile crocodile a and ,~ globin genes respectively, under the control of the tac promoter) are illustrated schematically in Figure '~, together with the co-expression construct pSC4-11, described below. These expression vectors were also derived from pKK~3-3. The method of their preparation will be apparent to those skilled in the art from the teaching disclosed herein in combination with the disclosures of Hoffman et al., (cited previously).
When a (crocodile) and ,6' (crocodile) chains were co-expressed, the resulting E. coli product was soluble, but brown. The haem group was either oxidised in vivo or anunnatural haem was incorporated into Hb. It has not been posible to produce either the a or ,6' chain or human Hb in E. coli unless they are co-expressed (Hoffman et al., 1990 cited above). However. when the crocodile a or ~' genes were expressed on their own in 217 () G ~ 8 PCT/GBg~/0l996 ~ WO 95/07932 E. coli, soluble single chain Hbs were produced. P ification of recombinant haemoglobins was performed according to the method of Komiyama et al., (1991 Nature 35~, 349-351) By co-expressing mixtures of hur~ and crocodile a and ~ globins in E. coli, it was possible to obtain hybrid haemoglobin molecules: a (crocodile),/~ (human), and a(human),/,~ (crocodile),. These hybrids were compared with the "single species"
molecules, a (human),/,6' (human), and a (crocodile)~/~ (crocodile)7 which were made by mixing the respective chains.
Oxygen equilibrium curves were obtained using the automatic recording apparatus described by Imai (1981 Meth. Enzymol. 76, 438-449). All measurements were performed at pH7.4 in 50mM bis-Tris with 0.1M Cl at 25C, either in the presence or absence of 5% CO" which was in equilibrium with ~lmM bicarbonate ion in the buffer.
The results are shown in Table I, which illustrates the oxygen affinity (as measured by P50 mmHg, i.e. the partial pressure of oxygen, in millimetres of mercury, at which the haemoglobin became 50% saturated with oxygen) for these molecules either in the absence of added CO, or in the presence of 5% added CO2. The table also shows these values for two other haemoglobin molecules, a (crocodile)J,B (SC4), and a (crocodile),/~ (crocodile:
8~K-Q)" the significance of which is discussed below. The table also shows the value of Hill's coefficient (n) for each hybrid, which is a measure of the co-operativity of oxygen binding.
Table I shows that wild type human Hb 5hows only a small effect of C07. Kilmartin &
Rossi-Bernardi (1969, cited previously) showed that this is due to carbamylation of a amino groups. As reported bv Bauer & Jelkman (1977 Nature '769, 825-877), the oxygen affinity of a (crocodile),/~ (crocodile)~ is reduce~ ;l-fold by 5% CO.. Bauer et al.
(1981, cited previously) have shown that nliS large . ~ction in oxygen affinity is caused by binding two bicarbonate ions per molecule of crocodile Hb. In Perutz's model, residue Lys-8'7 ~ forms a salt bridge with a bicarbonate ion. The present inventors introduced the Lys-8'~ ~6' to Gln mutation into Nile crocodile Hb [a (crocodile),/~ (crocodile:8~k-Q),] but the magnitude of the bicarbonate effect was ony slightly reduced as shown by Table I.
~ ~ 8 PCTIGBs~/0l996 wo 95/07932 2 1 ~
This shows the bicarbonate ions do not bind to the site proposed by Perutz et al.
Table I shows that neither the ~ (crocodile),/~5~(human), nor the a (human)7/~B (crocodile), hybrid molecule exhibited a large CO. effect. It therefore seemed that neither the ~
subunit nor the ,5 subunit were largely responsible but that the bicarbonate ion binding site may be located at the subunit interface between the a and ,B subunits.
TABLE I Oxygen binding parameters of engineered Hbs with no CO~ with 5% CO~
Pso (mmHg) n P~o (mmHg) n a (human), ,B (human), 6.7 2.6 10.0 2.6 a (croco), ,B (human), 1.4 1.8 1.4 1.7 a (human), ,B (croco), 8.6 1.6 16.6 2.0 ~x (croco), ,B (croco)~ 6.3 1.9 42.2 1.9 a (croco)~
(SC4), 1.1 1.4 11.0 1.7 a (croco), (croco:8~K-Q), 12.0 1.9 43.0 1.9 2 ~ 7 ~ ~ 3 ~ PCT/Gl~g~/01996 ~ WO 95/07932 T.9BLE II Comparison of amino acid cequences of ,~ subunits of human and crocodile Hb in the region between residue3 9 and 41.
crocodile S R M L I V Y P W K R R Y
* * * * * *
human ,~ G R L L V V Y P W T Q R F
Arnino acid residue No 30 40 The globin sequences of crocodilian haemoglobins (caim~n Nile crocodile and ~iC~jccippi alligator) were compared with those of other vertebrate Hbs. Residues between 29 and -11 of,~ subunits (located at the c~ , contacts), are all conserved in these three species and in particular, Arg-39 ,B and Tyr-41~ are found only in crocodilian Hbs. This region (29-41) of the human sequence was replaced with that of the crocodile sequence (Table II) in ~ (crocodile)"B (human)" and the resulting hybrid Hb, named ct (croco)"B (SC4)~, exhibited a large CO, effect, as shown in Table I. This hybrid haemoglobin was expressed by the construct pSC4-11 as shown in Figure 2.
To further investigate these finc~ing~ the inventors also introduced the Lys-38 ~Thr, Arg-39 ,~ ~Gln and Tyr-41 ,~ ~Phe mutations into ,~(crocodile), and these three mutations completely abolished the bicarbonate ion effect but the Arg-39 ,B~Gln mutation alone did not effect the bicarbonate effect substantially. Thus the two alterations at positions 38 and 41 are sufficient to confer most of the bicarbonate ion effect on human beta globin. These results unequivocally show that the binding site for bicarbonate ion is located at the a~
interface and the Lys-38 ~B and Tyr-41 ,B residues are important for bicarbonate ion binding .
The introduction of these t~hO mutations into human ~ globin will result in a modified human haemoglobin which displays a large bicarbonate effect and will therefore be more efficient in delivering oxygen when used as an artifical blood substitute. In this way it should be possible to deliver the same amount of oxygen with a lesser dose of haemoglobin and thus reduce the effective cost and potential side effects of such an _ WO 9s/07g3~ 3 g PCT/GB94/0l996 artificial oxygen carrier.
Ideally the ,B globin should also contain a mutatio~i-a2 position 108 (Asn ~Lys). This is Known as the Presbyterian mutation (a naturally occurring mutant form of human haemoglobin) and results in increasing the Bohr effect (the pH-dependency of haemoglobin's 0, affinity). The mutant ~ chains are preferably co-expressed with human di-a globin dimers (as described, for example, by Looker et al., 1997 Nature 356, '~58-'~60) to make a stable (substantially human) haemoglobin molecule with desirableproperties. As the molecule would contain only '7 ~' globin residues from a non-human haemoglobin it would be predicted to have virtually no immunogenic capacity.
Having demonstrated the potential to modify human ~6' globin in this way using crocodile haemoglohin it should be possible to use residues in the region ,~ globin ~9-41 from other species' haemoglohin to introduce the CO. effect from other ~nim~l~ into human ,~' globin.
Example ~
The results presented in Example 1 showed that it was possible to introduce a substantial (but not a full) bicarbonate effect into human haemoglobin by making alterations at certain residues in the ,t3 globin chain, indicating that additional changes in the a globin subunit may also be desirable.
In order to find the minim~l number of crocodile residues required to create thebicarbonate effect in human haemoglobin, the present inventors h~-m~ni7~d the a globin sequence in chimeric haemoglobin, (a(crocodile),a(SC4)~ stepwise. The number of crocodile residues rem~ining in the a subunit could be reduced to just 7 without losing the full bicarbonate effect. This new engineered haemoglobin, named haemoglobin Scuba, consists of human a subunits with the substitutions Leu-34a ~Cys. Ser-35a Ala, Phe-36a ~Tyr~ Thr-37a ~Gln, Thr-41a ~Ile, Leu-lOOa Phe, His-103a Gln mutations and the ~(SC4) subunits. Without the Leu-lOOa ~Phe and His 103a IGln mutations this engineered haemoglobin loses not only some of the bicarbonate effect, but also some of the cooperative oxygen binding effect.
217 0 6~ 8 PCT/GB94lûl996 Typical results are shown in Figures 3a-3c.
Figure 3a compares the oxygen binding curve for recombinant crocodile haemoglobin (circles) with that for human haemoglobin (squares), in the presence (filled symbols) or absence (open symbols) of 5 % CO, . The crocodile haemoglobin exhibits a large bicarbonate effect with a considerable "shift" of the curve to the left in the presence of S~o CO" indicating a greatly reduced oxygen affinity, whilst the shift for the humanhaemoglobin plot is very much smaller.
Figure 3b compares the curves for the hybrid haemoglobins: a(crocodile)J~B(human)7 (circles) and a(crocodile),/,B(SC4).. The former shows no bicarbonate effect, whilst the bicarbonate effect of the latter hybrid is almost as great as that exhibited by recombinant crocodile haemoglobin.
Figure 3c shows the oxygen binding curves for haemoglobin scuba with (circles) or without (squares) the Leu 100a ~Phe and His 103a~G1n substitutions, in the absence (open symbols) or presence (filled symbols) of 5% CO7 Although haemoglobin Scuba retains the full bicarbonate effect its oxygen affinity is one order of magnitude higher than that of Nile crocodile haemoglobin, both in the presence and absence of CO, (Fig.3c). Additional mutations are therefore desirable to lower the oxygen affinity. Figure 4 shows a schematic drawing of human deoxyhaemoglobin inwhich the position of the 1~ mutations in haemoglobin scuba are shown. Most of these residues are clustered at the a,~'7 subunit interface where the two subunits slide with respect to each other upon oxygen binding. Perutz and Fermi (Personal communication) have modelled a stereochemically plausible binding site con~ ting of the two mutant residues Lys-38 and Tyr-41 ,6', together with the conserved Tyr-42 a, but crystallographic analysis of Hb Scuba in the presence of bi . - .bonate ions is necessarv to determine the precise binding site.
Wo 95/07932 ~. 7 In particular, the oxygen affinity of Hb is affected by the concentration of C0~, such thathigh concentrations of C0~, (with a partial pressure, "PC0," of about 40mm Hg, as will be found in actively respiring tissues) cause a reduction in the oxygen affinity, thereby facilitating the unloading of oxygen to the tissues. This phenomenon is known as the C0~
effect. In human Hb, C0. binds directly to the ~x-amino groups of the ~ and ,B globin submits to form carbamino groups and reduces the oxygen affinity by stabilising the low affinity (T) quaternary structure (Kilmartin & Rossi-Bernardi, 1969 Nature ~ ~, 1243-1746). The C01 effect is one of the principal heterotropic allosteric effects.
Vertebrate species have adapted to strikingly diverse environmental conditions, and the haemoglobin molecule has evolved to meet a wide range of respiratory needs in such environments. For example, the oxygen affinity of crocodile Hb is marketly reduced by physiological concentrations of C07 (40 torr). Bauer et al. (1981, J. Biol. Chem. 756, 8479-8435) showed that the large reduction in the oxygen affinity of crocodile Hb is not caused by the binding of C0~ to the a amino groups as in human Hb. Carbon dioxide (C0~) dissolves in aqueous solutions (such as blood) to give HC03-. The marked reduction in the oxygen affinity in crocodile Hb is caused by the binding of bicarbonate ions and this phenomenon is called the "bicarbonate effect". A much larger proportion of Hb-bound oxygen can be released and utilised in the tissues and this enables crocodiles to stay under water for a long time. It has been postulated (Perutz et al., 1981 ~atue 291, 682-684) that this effect is due to bicarbonate ions binding to residues Lys 82 and Glu 144 of the ,B globin molecule.
There is interest in developing "artificial blood" products. Such products would elimin~te the chance of infection due to a patient receiving infected blood from a donor, and generally simplify the blood transfusion process. Clearly, in order to perform all the functions of natural blood, the artificial product must be capable of transporting oxygen.
Ijnfortunately, haemoglobin outside the environment of a red blood cell (RBC) has too high an affinity to release much oxygen to the tissues. This is because RBCs contain large amounts of DPG which, as mentioned above, is one of the metabolites which lowers the oxygen affinitv of Hb.
217 ~ pcTlGBs~lol996 95l07932 Consequently, there is a need for a stable, artificial blood product with improved oxygen affinity characteristics which will deliver oxygen more effectively to respiring tissues.
Summarv of the Invention In a first aspect the invention provides a ~ globin modified so as to exhibit, when present as part of a haemoglobin molecule, an altered bicarbonate effect compared to theunmodified ,B globin, the modification comprising alteration of at least two of the amino acid residues in the group con~i~ting of residues ~9-41.
It was found by the present inventors that human haemoglobin comprising ~i' globin subunits altered in accordance with the invention, by substitution with equivalent crocodile ,B globin residues, exhibited a greatly enh~rlce~l bicarbonate effect analogous to that displayed by native crocodile haemoglobin. This is surprising because haemoglobin is a complex tetrameric molecule with prosthetic groups, yet very small changes in just the ,~
globin primary structure in a (crocodile) ~5 (human) haemoglobin can cause large changes in the bicarbonate effect, whilst the a globin and prosthetic groups are unaltered.
Further, most of residues ~9-41 of ,~ globin are located at the interface of the a~ 2 globin subunits and changes therein might thus be expected to cause the molecule to lose stability or permanently reduce its oxygen affinity. It was surprisingly found, however, that an alteration in accordance with the invention did not cause such deleterious effects.
In a second aspect the invention provides a method of modifying a ~ globin so as to exhibit, when present as part of an haemoglobin molecule, an altered bicarbonate effect compared to the unmodified ,B globin, the modification comprising substitution of at least two of the amino acid residues in the group consisting of residues '~9-41.
Preferably the ~5 globin is human ,6' globin. Preferably the ,~ globin is modified so as to exhibit an enhanced bicarbonate efect (ie a greater decrease in oxygen affinity in the presence of aqueous carbon dioxide) compared to the unmodified ,~ globin.
23L~3~
PCTIGBs~/0l99 WO 95/0?932 It is preferred that the at least two altered arnino acid residues comprise at least two residues from the group consisting o~ residues '~9, 31. 33, 38, 39 and 41. Most preferably the modification comprises substitution of residues 38 and 41.
In a preferred embodiment, residues ~9-41 of human ~B globin are replaced with the equivalent residues from crocodile ,B globin. It will be apparent to those skilled in the art that some of the amino acid residues are common to the ~Ib of both species. Accordingly, it is not necessary to alter all the residues 29-41 but simply those residues which are not common to both (ie residues '79, 31, 33, 38, 39 and 41). Moreover residues Ser '~9,B and Met 31,~ are not in the al~ contact and substitutions of these residues not be required in order to introduce the full bicarbonate effect.
In another aspect the invention provides a haemoglobin molecule for use as an artificial blood substitute, comprising a,~? globin modified so as to exhibit an altered bicarbonate effect compared to a haemoglobin molecule comprising an unmodified ~ globin, themodification comprising substitution of at least two of the amino acid residues in the group consisting of residues 29-41.
Conveniently the haemoglobin molecule also comprises a modified c~ globin chain.
Typically the ~ globin chain will comprise a substantially human c~ globin se~uence modified at one or more residues so as to optimise the oxygen-transport characteristics of the haemoglobin molecule for use as an artifical blood substitute. Conveniently the a globin chain will be modified in a manner as disclosed in UK Patent Application No 941477'7.5. or similar, so as to exhibit an altered bicarbonate effect compared to the unmodified c globin.
Conveniently the modified c~ globin will comprise an alteration at one or more of the following residues: 34, 35, 36. 37, 41, 100 and 103.
Desirably the modified c~ globin will comprise one or more (preferably all) of the following substitutions: Leu 34~Cvs. Ser 35 ~Ala, Phe 36 ~Tyr, Thr 37 ~Gln, Thr 41 ~Ile, 217 0 ~ 3 ~ pcTlGBs4lol996 ~ Wo 95/07932 s Leu 100~Phe and His 103 ~Gln.
In a further aspect, the invention provides an artificial blood substitute comprising the haemoglobin molecule defined above.
The invention will now be further described by way of illustrative example and by reference to the drawings, in which:
Figure 1 shows the nucleotide sequences encoding the a and ,B globins of the Nile crocodile (as optimised for expression in E. coli) and the amino acid sequences of the encoded polypeptides;
Figure 7 is a schematic representation of the crocodile a and ~5 globin expression constructs pOmega-5 and pManuo-4, and of the hybrid expression construct pSC4-11;
Figure 3a is a graph of the oxygen binding curve (% 07 saturation against log pO~ in mm Hg) for recombinant crocodile (circles) and human (squares) haemoglobins in the absence (open symbols) or presence (filled-in symbols) of 5% CO~;
Figure 3b is a similar graph comparing the oxygen binding curves of hybrid a(crocodile)~/,B(human)7 (circles) and a(crocodile)~/~B(SC4)~ (squares) haemoglobins;
Figure 3c is a similar graph comparing the oxygen binding curve of recombinant "Scuba"
haemoglobin with (circles) or without (squares) alterations at residues 100a and 103a; and Figure 4 is a schematic representation of human deoxyhaemoglobin.
Example 1 As described previously, Perutz et al. (1981, cited above) proposed bicarbonate ion binding sites to be located in the central cavity between two ~B subunits and to involve hydro en bonds to Lys (87 ,B) and Glu (14 f~) of one ~ subunit and the N-terminal residues 217~3~
PCT/GB94/nl996 _ of its partner. Four differences (relative to human haemoglobin) in crocodile Hb, His-143 ~' ~AIa, Lys-144 k'~Gly. Val-1 ,6' Se~ and His-~ Pro, are thought to be important to promote formation of these hydrogen bonds. The present inventors first introduced these mutations into human ,6' globin but the CO, effect was the same as in native human Hb.
It was considered that some additional mutations may be needed to create the appropriate electrostatic environment for the interaction of these residues with bicarbonate ions.
Accordingly, three additional mutations, Asp-94 ~6' to Glu, Glu-90 ,~ ~Lys and His-135 k' ~Arg, were introduced into this mutant but they still failed to show a large bicarbonate effect in human Hb. At this stage this strategy was abandoned and it was decided to first make crocodile Hb in E. coli and to find mutations which abolish the bicarbonate effect.
As a first step, protein coding sequences of Nile crocodile a and ,~ globins were chemically synthesised by assembly of 14 oligonucleotides with codons optimal for expression in E. coli (Ikemura et al., 1982 J. Mol. Biol. 158, 573-597). All mutations were introduced by cassette mutagenesis (Wells et al., 1985 Gene 34, 315). The synthetic a and ,6' globin coding sequences, and the polypeptides encoded thereby, are shown in Figure 1 and as Seq. ID Nos. 1-4 in the attached sequence listing. These genes were cloned separately into the Hb expression vector developed by Somatogen, (Hoffman et al., 1990 Proc Nat. Acad. Sci. USA 87 8521-8525). This vector was derived from pKK223-3 (available from Pharmacia). The expression constructs pOmega-5 and pManuo-4 (expressing the Nile crocodile a and ,~ globin genes respectively, under the control of the tac promoter) are illustrated schematically in Figure '~, together with the co-expression construct pSC4-11, described below. These expression vectors were also derived from pKK~3-3. The method of their preparation will be apparent to those skilled in the art from the teaching disclosed herein in combination with the disclosures of Hoffman et al., (cited previously).
When a (crocodile) and ,6' (crocodile) chains were co-expressed, the resulting E. coli product was soluble, but brown. The haem group was either oxidised in vivo or anunnatural haem was incorporated into Hb. It has not been posible to produce either the a or ,6' chain or human Hb in E. coli unless they are co-expressed (Hoffman et al., 1990 cited above). However. when the crocodile a or ~' genes were expressed on their own in 217 () G ~ 8 PCT/GBg~/0l996 ~ WO 95/07932 E. coli, soluble single chain Hbs were produced. P ification of recombinant haemoglobins was performed according to the method of Komiyama et al., (1991 Nature 35~, 349-351) By co-expressing mixtures of hur~ and crocodile a and ~ globins in E. coli, it was possible to obtain hybrid haemoglobin molecules: a (crocodile),/~ (human), and a(human),/,~ (crocodile),. These hybrids were compared with the "single species"
molecules, a (human),/,6' (human), and a (crocodile)~/~ (crocodile)7 which were made by mixing the respective chains.
Oxygen equilibrium curves were obtained using the automatic recording apparatus described by Imai (1981 Meth. Enzymol. 76, 438-449). All measurements were performed at pH7.4 in 50mM bis-Tris with 0.1M Cl at 25C, either in the presence or absence of 5% CO" which was in equilibrium with ~lmM bicarbonate ion in the buffer.
The results are shown in Table I, which illustrates the oxygen affinity (as measured by P50 mmHg, i.e. the partial pressure of oxygen, in millimetres of mercury, at which the haemoglobin became 50% saturated with oxygen) for these molecules either in the absence of added CO, or in the presence of 5% added CO2. The table also shows these values for two other haemoglobin molecules, a (crocodile)J,B (SC4), and a (crocodile),/~ (crocodile:
8~K-Q)" the significance of which is discussed below. The table also shows the value of Hill's coefficient (n) for each hybrid, which is a measure of the co-operativity of oxygen binding.
Table I shows that wild type human Hb 5hows only a small effect of C07. Kilmartin &
Rossi-Bernardi (1969, cited previously) showed that this is due to carbamylation of a amino groups. As reported bv Bauer & Jelkman (1977 Nature '769, 825-877), the oxygen affinity of a (crocodile),/~ (crocodile)~ is reduce~ ;l-fold by 5% CO.. Bauer et al.
(1981, cited previously) have shown that nliS large . ~ction in oxygen affinity is caused by binding two bicarbonate ions per molecule of crocodile Hb. In Perutz's model, residue Lys-8'7 ~ forms a salt bridge with a bicarbonate ion. The present inventors introduced the Lys-8'~ ~6' to Gln mutation into Nile crocodile Hb [a (crocodile),/~ (crocodile:8~k-Q),] but the magnitude of the bicarbonate effect was ony slightly reduced as shown by Table I.
~ ~ 8 PCTIGBs~/0l996 wo 95/07932 2 1 ~
This shows the bicarbonate ions do not bind to the site proposed by Perutz et al.
Table I shows that neither the ~ (crocodile),/~5~(human), nor the a (human)7/~B (crocodile), hybrid molecule exhibited a large CO. effect. It therefore seemed that neither the ~
subunit nor the ,5 subunit were largely responsible but that the bicarbonate ion binding site may be located at the subunit interface between the a and ,B subunits.
TABLE I Oxygen binding parameters of engineered Hbs with no CO~ with 5% CO~
Pso (mmHg) n P~o (mmHg) n a (human), ,B (human), 6.7 2.6 10.0 2.6 a (croco), ,B (human), 1.4 1.8 1.4 1.7 a (human), ,B (croco), 8.6 1.6 16.6 2.0 ~x (croco), ,B (croco)~ 6.3 1.9 42.2 1.9 a (croco)~
(SC4), 1.1 1.4 11.0 1.7 a (croco), (croco:8~K-Q), 12.0 1.9 43.0 1.9 2 ~ 7 ~ ~ 3 ~ PCT/Gl~g~/01996 ~ WO 95/07932 T.9BLE II Comparison of amino acid cequences of ,~ subunits of human and crocodile Hb in the region between residue3 9 and 41.
crocodile S R M L I V Y P W K R R Y
* * * * * *
human ,~ G R L L V V Y P W T Q R F
Arnino acid residue No 30 40 The globin sequences of crocodilian haemoglobins (caim~n Nile crocodile and ~iC~jccippi alligator) were compared with those of other vertebrate Hbs. Residues between 29 and -11 of,~ subunits (located at the c~ , contacts), are all conserved in these three species and in particular, Arg-39 ,B and Tyr-41~ are found only in crocodilian Hbs. This region (29-41) of the human sequence was replaced with that of the crocodile sequence (Table II) in ~ (crocodile)"B (human)" and the resulting hybrid Hb, named ct (croco)"B (SC4)~, exhibited a large CO, effect, as shown in Table I. This hybrid haemoglobin was expressed by the construct pSC4-11 as shown in Figure 2.
To further investigate these finc~ing~ the inventors also introduced the Lys-38 ~Thr, Arg-39 ,~ ~Gln and Tyr-41 ,~ ~Phe mutations into ,~(crocodile), and these three mutations completely abolished the bicarbonate ion effect but the Arg-39 ,B~Gln mutation alone did not effect the bicarbonate effect substantially. Thus the two alterations at positions 38 and 41 are sufficient to confer most of the bicarbonate ion effect on human beta globin. These results unequivocally show that the binding site for bicarbonate ion is located at the a~
interface and the Lys-38 ~B and Tyr-41 ,B residues are important for bicarbonate ion binding .
The introduction of these t~hO mutations into human ~ globin will result in a modified human haemoglobin which displays a large bicarbonate effect and will therefore be more efficient in delivering oxygen when used as an artifical blood substitute. In this way it should be possible to deliver the same amount of oxygen with a lesser dose of haemoglobin and thus reduce the effective cost and potential side effects of such an _ WO 9s/07g3~ 3 g PCT/GB94/0l996 artificial oxygen carrier.
Ideally the ,B globin should also contain a mutatio~i-a2 position 108 (Asn ~Lys). This is Known as the Presbyterian mutation (a naturally occurring mutant form of human haemoglobin) and results in increasing the Bohr effect (the pH-dependency of haemoglobin's 0, affinity). The mutant ~ chains are preferably co-expressed with human di-a globin dimers (as described, for example, by Looker et al., 1997 Nature 356, '~58-'~60) to make a stable (substantially human) haemoglobin molecule with desirableproperties. As the molecule would contain only '7 ~' globin residues from a non-human haemoglobin it would be predicted to have virtually no immunogenic capacity.
Having demonstrated the potential to modify human ~6' globin in this way using crocodile haemoglohin it should be possible to use residues in the region ,~ globin ~9-41 from other species' haemoglohin to introduce the CO. effect from other ~nim~l~ into human ,~' globin.
Example ~
The results presented in Example 1 showed that it was possible to introduce a substantial (but not a full) bicarbonate effect into human haemoglobin by making alterations at certain residues in the ,t3 globin chain, indicating that additional changes in the a globin subunit may also be desirable.
In order to find the minim~l number of crocodile residues required to create thebicarbonate effect in human haemoglobin, the present inventors h~-m~ni7~d the a globin sequence in chimeric haemoglobin, (a(crocodile),a(SC4)~ stepwise. The number of crocodile residues rem~ining in the a subunit could be reduced to just 7 without losing the full bicarbonate effect. This new engineered haemoglobin, named haemoglobin Scuba, consists of human a subunits with the substitutions Leu-34a ~Cys. Ser-35a Ala, Phe-36a ~Tyr~ Thr-37a ~Gln, Thr-41a ~Ile, Leu-lOOa Phe, His-103a Gln mutations and the ~(SC4) subunits. Without the Leu-lOOa ~Phe and His 103a IGln mutations this engineered haemoglobin loses not only some of the bicarbonate effect, but also some of the cooperative oxygen binding effect.
217 0 6~ 8 PCT/GB94lûl996 Typical results are shown in Figures 3a-3c.
Figure 3a compares the oxygen binding curve for recombinant crocodile haemoglobin (circles) with that for human haemoglobin (squares), in the presence (filled symbols) or absence (open symbols) of 5 % CO, . The crocodile haemoglobin exhibits a large bicarbonate effect with a considerable "shift" of the curve to the left in the presence of S~o CO" indicating a greatly reduced oxygen affinity, whilst the shift for the humanhaemoglobin plot is very much smaller.
Figure 3b compares the curves for the hybrid haemoglobins: a(crocodile)J~B(human)7 (circles) and a(crocodile),/,B(SC4).. The former shows no bicarbonate effect, whilst the bicarbonate effect of the latter hybrid is almost as great as that exhibited by recombinant crocodile haemoglobin.
Figure 3c shows the oxygen binding curves for haemoglobin scuba with (circles) or without (squares) the Leu 100a ~Phe and His 103a~G1n substitutions, in the absence (open symbols) or presence (filled symbols) of 5% CO7 Although haemoglobin Scuba retains the full bicarbonate effect its oxygen affinity is one order of magnitude higher than that of Nile crocodile haemoglobin, both in the presence and absence of CO, (Fig.3c). Additional mutations are therefore desirable to lower the oxygen affinity. Figure 4 shows a schematic drawing of human deoxyhaemoglobin inwhich the position of the 1~ mutations in haemoglobin scuba are shown. Most of these residues are clustered at the a,~'7 subunit interface where the two subunits slide with respect to each other upon oxygen binding. Perutz and Fermi (Personal communication) have modelled a stereochemically plausible binding site con~ ting of the two mutant residues Lys-38 and Tyr-41 ,6', together with the conserved Tyr-42 a, but crystallographic analysis of Hb Scuba in the presence of bi . - .bonate ions is necessarv to determine the precise binding site.
3 ~
WO 95/07932 PCT/GB9~/01996 SEQUENCE LISTING
(1) GENERAL INFORMATION:
~i) APPLICANT~
(A) NAME: Medical Research Counci~ .
(B) STREET: 20 Park Crescent `-(C) CITY: London (E) COUNTRY: United Kingdom (F) POSTAL CODE (ZIP): WlN 4AL
(G) TELEPHONE: (071) 636 5422 (H) TELEFAX: (071) 323 1331 (ii) TITLE OF INVENTION: Improvements in or Relating to Haemoglobin (I) (iii) NUMBER OF SEQUENCES: 4 (iv) COMPUTER READABLE FORM:
(A) MEDIUM TYP~: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1Ø Version #1.25 (EPO) (2) INFORMATION FOR SEQ ID NO: 1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 423 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(iii) ANTI-SENSE: NO
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1. 423 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:
ATG CTG TCT TCT GAC GAC AAA TGC AAC GTT AAA GCT GTr TGG TCT M A 48 Met Leu Ser Ser Asp Asp Lys Cys Asn Val Lys Ala Val Trp Ser Lys Val Ala Gly His Leu Glu Glu Tyr Gly Ala Glu Ala Leu Glu Arg Met Wo 9~/07932 2 17 a ~ ~ ~ PCT/GB94/01996 Phe Cys Ala Tyr Pro Gln Thr Lys Ile Tyr Phe Pro His Phe Asp Leu Ser His Gly Ser Ala Gln Ile Arg Ala His Gly Lys Lys Val Phe Ala Ala Leu His Glu Ala Val Asn His Ile Asp Asp Leu Pro Gly Ala Leu Cys Arg Leu Ser Glu Leu His Ala His Ser Leu Arg Val Asp Pro Val Asn Phe Lys Phe Leu Ala Gln Cys Val Leu Val Val Val Ala Ile His His Pro Gly Ser Leu Thr Pro Glu Val His Ala Ser Leu Asp Lys Phe Leu Cys Ala Val Ser Ser Val Leu Thr Ser Lys Tyr Arg (2) INFORMATION FOR SEQ ID NO: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 141 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii~ MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
Met Leu Ser Ser Asp Asp Lys Cys Asn Val Lys Ala Val Trp Ser Lys al Ala Gly His Leu Glu Glu Tyr Gly Ala Glu Ala Leu Glu Arg Met Phe Cys Ala Tyr Pro Gln Thr Lys Ile Tyr Phe Pro His Phe Asp Leu Ser His Gly Ser Ala Gln Ile Arg Ala His Gly Lys Lys Val Phe Ala Ala Leu His Glu Ala Val Asn His Ile Asp ASD Leu Pro Gly Ala Leu W0 25/07932 ..~ ;, PCT/GB9 1/01996 0 Cys Arg Leu Ser G15u Leu His Ala His Ser Leu Arg Val Asp Pro Val Asn Phe Lys Phe Leu Ala Gln Cys Val Leu Val Val Val Ala Ile His His Pro Gly Ser Leu Thr Pro Glu Val His Ala Ser Leu Asp Lys Phe 1 15 120 125 v Leu Cys Ala Val Ser Ser Val Leu Thr Ser Lys Tyr Arg (2) INFORMATION FOR SEQ ID NO: 3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 438 base pairs (B) TYPE: nucleic acid ( C ) STRANDEDNESS: s i ng l e (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) ( i i i ) HYPOTHETICAL: NO
( i i i ) ANTI -SENSE: NO
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..438 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:
Met Ser Phe Asp Pro His Glu Lys Gln Leu Ile Gly Asp Leu Trp His Lys Val Asp Val Ala His Cys Gly Gly Glu Ala Leu Ser Arg Met Leu Ile Val Tyr Pro Trp Lys Arg Arg Tyr Phe Glu Asn Phe Gly Asp Ile Ser Asn Ala Gln Ala Ile Met His Asn Glu Lys Val Gln Ala His Gly Lys Lys Val Leu Ala Ser Phe Gly Glu Ala Val Cys His Leu Asp Gly Ile Arg Ala His Phe Ala Asn Leu Ser Lys Leu His Cys Glu Lys Leu WO 95/1)7932 15 2 1 7 0 6 3 ~ PCT/GB9~/~1996 His Val Asp Pro Glu Asn Phe Lys Leu Leu Gly Asp Ile Ile Ile Ile Val Leu Ala Ala His Tyr Pro Lys Asp Phe Gly Leu Glu Cys His Ala Ala Tyr Gln Lys Leu Val Arg Gln Val Ala Ala Ala Leu Ala Ala Glu Tyr His (2) INFORMATION FOR SEQ ID NO: 4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 146 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:
Met Ser Phe Asp Pro His Glu Lys Gln Leu Ile Gly Asp Leu Trp His ys Val Asp Val Ala His Cys Gly Gly Glu Ala Leu Ser Arg Met Leu Ile Val Tyr Pro Trp Lys Arg Arg Tyr Phe Glu Asn Phe Gly Asp Ile Ser Asn Ala Gln Ala Ile Met His Asn Glu Lys Val Gln Ala His Gly Lys Lys Val Leu Ala Ser Phe Gly Glu Ala Val Cys His Leu Asp Gly le Arg Ala His Phe Ala Asn Leu Ser Lys Leu His Cys Glu Lys Leu is Val Asp Pro Glu Asn Phe Lys Leu Leu Gly Asp Ile Ile Ile Ile al Leu Ala Ala His Tyr Pro Lys Asp Phe Gly Leu Glu Cys His Ala WO 95/07932 ~ PCT/GB9~/(1199G
Ala 1T30r Gln Lys Leu Val Arg Gln Val Ala Ala Ala Leu Ala Ala Glu Tyr Hi s
WO 95/07932 PCT/GB9~/01996 SEQUENCE LISTING
(1) GENERAL INFORMATION:
~i) APPLICANT~
(A) NAME: Medical Research Counci~ .
(B) STREET: 20 Park Crescent `-(C) CITY: London (E) COUNTRY: United Kingdom (F) POSTAL CODE (ZIP): WlN 4AL
(G) TELEPHONE: (071) 636 5422 (H) TELEFAX: (071) 323 1331 (ii) TITLE OF INVENTION: Improvements in or Relating to Haemoglobin (I) (iii) NUMBER OF SEQUENCES: 4 (iv) COMPUTER READABLE FORM:
(A) MEDIUM TYP~: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1Ø Version #1.25 (EPO) (2) INFORMATION FOR SEQ ID NO: 1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 423 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(iii) ANTI-SENSE: NO
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1. 423 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:
ATG CTG TCT TCT GAC GAC AAA TGC AAC GTT AAA GCT GTr TGG TCT M A 48 Met Leu Ser Ser Asp Asp Lys Cys Asn Val Lys Ala Val Trp Ser Lys Val Ala Gly His Leu Glu Glu Tyr Gly Ala Glu Ala Leu Glu Arg Met Wo 9~/07932 2 17 a ~ ~ ~ PCT/GB94/01996 Phe Cys Ala Tyr Pro Gln Thr Lys Ile Tyr Phe Pro His Phe Asp Leu Ser His Gly Ser Ala Gln Ile Arg Ala His Gly Lys Lys Val Phe Ala Ala Leu His Glu Ala Val Asn His Ile Asp Asp Leu Pro Gly Ala Leu Cys Arg Leu Ser Glu Leu His Ala His Ser Leu Arg Val Asp Pro Val Asn Phe Lys Phe Leu Ala Gln Cys Val Leu Val Val Val Ala Ile His His Pro Gly Ser Leu Thr Pro Glu Val His Ala Ser Leu Asp Lys Phe Leu Cys Ala Val Ser Ser Val Leu Thr Ser Lys Tyr Arg (2) INFORMATION FOR SEQ ID NO: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 141 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii~ MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
Met Leu Ser Ser Asp Asp Lys Cys Asn Val Lys Ala Val Trp Ser Lys al Ala Gly His Leu Glu Glu Tyr Gly Ala Glu Ala Leu Glu Arg Met Phe Cys Ala Tyr Pro Gln Thr Lys Ile Tyr Phe Pro His Phe Asp Leu Ser His Gly Ser Ala Gln Ile Arg Ala His Gly Lys Lys Val Phe Ala Ala Leu His Glu Ala Val Asn His Ile Asp ASD Leu Pro Gly Ala Leu W0 25/07932 ..~ ;, PCT/GB9 1/01996 0 Cys Arg Leu Ser G15u Leu His Ala His Ser Leu Arg Val Asp Pro Val Asn Phe Lys Phe Leu Ala Gln Cys Val Leu Val Val Val Ala Ile His His Pro Gly Ser Leu Thr Pro Glu Val His Ala Ser Leu Asp Lys Phe 1 15 120 125 v Leu Cys Ala Val Ser Ser Val Leu Thr Ser Lys Tyr Arg (2) INFORMATION FOR SEQ ID NO: 3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 438 base pairs (B) TYPE: nucleic acid ( C ) STRANDEDNESS: s i ng l e (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) ( i i i ) HYPOTHETICAL: NO
( i i i ) ANTI -SENSE: NO
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..438 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:
Met Ser Phe Asp Pro His Glu Lys Gln Leu Ile Gly Asp Leu Trp His Lys Val Asp Val Ala His Cys Gly Gly Glu Ala Leu Ser Arg Met Leu Ile Val Tyr Pro Trp Lys Arg Arg Tyr Phe Glu Asn Phe Gly Asp Ile Ser Asn Ala Gln Ala Ile Met His Asn Glu Lys Val Gln Ala His Gly Lys Lys Val Leu Ala Ser Phe Gly Glu Ala Val Cys His Leu Asp Gly Ile Arg Ala His Phe Ala Asn Leu Ser Lys Leu His Cys Glu Lys Leu WO 95/1)7932 15 2 1 7 0 6 3 ~ PCT/GB9~/~1996 His Val Asp Pro Glu Asn Phe Lys Leu Leu Gly Asp Ile Ile Ile Ile Val Leu Ala Ala His Tyr Pro Lys Asp Phe Gly Leu Glu Cys His Ala Ala Tyr Gln Lys Leu Val Arg Gln Val Ala Ala Ala Leu Ala Ala Glu Tyr His (2) INFORMATION FOR SEQ ID NO: 4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 146 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:
Met Ser Phe Asp Pro His Glu Lys Gln Leu Ile Gly Asp Leu Trp His ys Val Asp Val Ala His Cys Gly Gly Glu Ala Leu Ser Arg Met Leu Ile Val Tyr Pro Trp Lys Arg Arg Tyr Phe Glu Asn Phe Gly Asp Ile Ser Asn Ala Gln Ala Ile Met His Asn Glu Lys Val Gln Ala His Gly Lys Lys Val Leu Ala Ser Phe Gly Glu Ala Val Cys His Leu Asp Gly le Arg Ala His Phe Ala Asn Leu Ser Lys Leu His Cys Glu Lys Leu is Val Asp Pro Glu Asn Phe Lys Leu Leu Gly Asp Ile Ile Ile Ile al Leu Ala Ala His Tyr Pro Lys Asp Phe Gly Leu Glu Cys His Ala WO 95/07932 ~ PCT/GB9~/(1199G
Ala 1T30r Gln Lys Leu Val Arg Gln Val Ala Ala Ala Leu Ala Ala Glu Tyr Hi s
Claims (14)
1. A .beta. globin modified so as to exhibit, when present as part of a haemoglobin molecule, an altered bicarbonate effect compared to the unmodified .beta. globin, the modification comprising alteration of at least two of the amino acid residues in the group consisting of residues 29-41.
2. A .beta. globin according to claim 1, which comprises substantially the amino acid sequence of human .beta. globin.
3. A .beta. globin according to claim 1 or 2, modified so as to exhibit an enhanced bicarbonate effect.
4. A .beta. globin according to any one of claims 1, 2 or 3, in which the naturally occurring amino acid residues have been replaced by the equivalent residues from crocodile .beta.
globin.
globin.
5. A .beta. globin according to any one of the preceding claims, comprising substitutions at residues 38 and 41.
6. A .beta. globin according to any one of the preceding claims, comprising substitutions at two or more of residues 29, 31, 33, 38, 39 and 41.
7. A .beta. globin according to any one of the preceding claims, comprising a lysine residue at position 108.
8. A haemoglobin molecule for use as an artificial blood substitute, comprising a .beta. globin in accordance with any one of the preceding claims.
9. A haemoglobin molecule according to claim 8, comprising a substantially human .alpha.
globin modified at one or more residues so as to optimise the oxygen-transport characteristics of the haemoglobin molecule.
globin modified at one or more residues so as to optimise the oxygen-transport characteristics of the haemoglobin molecule.
10. A haemoglobin molecule according to claim 8 or 9, comprising an .alpha. globin modified so as to exhibit an altered bicarbonate effect.
11. A haemoglobin molecule according to claim 8, 9 or 10 comprising an .alpha. globin with alterations at one or more of the following residues: 34, 35, 36, 37, 41, 100 and 103.
12. A haemoglobin molecule according to any one of claims 8-11, comprising an .alpha. globin with one or more of the following substitutions: Leu 34->Cys, Ser 35->Ala, Phe 36->Tyr, Thr 37->Gln, Thr 41->Ile, Leu 100->Phe, and His 103->Gln.
13. A haemoglobin molecule according to any one of claims 8-12, comprising one or more alterations to reduce the oxygen affinity of said haemoglobin molecule.
14. An artificial blood substitute, comprising a haemoglobin molecule in accordance with any one of claims 8 to 13.
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
GB939318974A GB9318974D0 (en) | 1993-09-14 | 1993-09-14 | Improvements in or relating to haemoglobin |
GB9318974.4 | 1993-09-14 | ||
GB9414772A GB9414772D0 (en) | 1994-07-22 | 1994-07-22 | Improvements in or relating to protein engineering |
GB9414772.5 | 1994-07-22 |
Publications (1)
Publication Number | Publication Date |
---|---|
CA2170638A1 true CA2170638A1 (en) | 1995-03-23 |
Family
ID=26303513
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CA002170638A Abandoned CA2170638A1 (en) | 1993-09-14 | 1994-09-13 | Improvements in or relating to haemoglobin |
Country Status (5)
Country | Link |
---|---|
US (1) | US5942488A (en) |
EP (1) | EP0719284A1 (en) |
JP (1) | JPH09506332A (en) |
CA (1) | CA2170638A1 (en) |
WO (1) | WO1995007932A1 (en) |
Families Citing this family (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPH11514237A (en) * | 1995-10-23 | 1999-12-07 | ライス ユニバーシティ | Hemoglobin variants that reduce heme loss |
US6812207B1 (en) | 1995-10-23 | 2004-11-02 | William Marsh Rice University | Hemoglobin mutants that reduce heme loss |
WO1998038211A2 (en) * | 1997-02-28 | 1998-09-03 | Somatogen, Inc. | Permuted hemoglobin-like proteins |
EP0979279B1 (en) * | 1997-05-02 | 2008-03-19 | Baxter Biotech Technology S.A.R.L. | Hemoglobin mutants with reduced nitric oxide scavenging |
US6610644B1 (en) * | 1998-07-08 | 2003-08-26 | The Procter & Gamble Company | Detergent compositions comprising aggolomerates of layered silicate and anionic surfactant |
-
1994
- 1994-09-13 US US08/619,708 patent/US5942488A/en not_active Expired - Fee Related
- 1994-09-13 CA CA002170638A patent/CA2170638A1/en not_active Abandoned
- 1994-09-13 WO PCT/GB1994/001996 patent/WO1995007932A1/en not_active Application Discontinuation
- 1994-09-13 JP JP7509053A patent/JPH09506332A/en active Pending
- 1994-09-13 EP EP94926314A patent/EP0719284A1/en not_active Withdrawn
Also Published As
Publication number | Publication date |
---|---|
US5942488A (en) | 1999-08-24 |
JPH09506332A (en) | 1997-06-24 |
EP0719284A1 (en) | 1996-07-03 |
WO1995007932A1 (en) | 1995-03-23 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
EP0358708B1 (en) | Blood substitutes | |
US6124114A (en) | Hemoglobins with intersubunit dislufide bonds | |
Perutz | Species adaptation in a protein molecule | |
US5739011A (en) | DNA for the production of multimeric hemoglobins | |
EP0857489A2 (en) | Production and use of multimeric hemoglobins | |
AU671728B2 (en) | Methods of production of analogues of human Cu/Zn superoxide dismutase | |
Martin de Llano et al. | Recombinant human sickle hemoglobin expressed in yeast. | |
Perutz | Species adaptation in a protein molecule | |
CA2170638A1 (en) | Improvements in or relating to haemoglobin | |
EP0173280B1 (en) | Plasmids containing lambda pl promoter, and engineered restriction site for convenient replacement of ribosomal binding site, hosts containing the plasmids and related methods | |
US6114505A (en) | Hemoglobin mutants that reduce heme loss | |
US5844090A (en) | Modified hemoglobin-like compounds | |
EP0826064B1 (en) | Low oxygen affinity mutant hemoglobin | |
CN101711255A (en) | Modified globin proteins with altered electron transport pathway | |
WO2005041898A2 (en) | Blood substitutes | |
EP0784983A2 (en) | Use of extracellular hemoglobin | |
EP3595702B1 (en) | Modified haemoglobin proteins | |
EP0561245A1 (en) | Blood substitutes comprising recombinant hemoglobin | |
US6812207B1 (en) | Hemoglobin mutants that reduce heme loss | |
AU715073C (en) | Hemoglobin mutants that reduce heme loss | |
Brantley Jr | The mechanism of autooxidation of myoglobin |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
FZDE | Discontinued |