Modified hemoglobin and methods of making same
View Patent ↗The present invention provides a hemoglobin molecule (Hb) having six ± one PEG chains, wherein two of said PEG chains are bound to Cys-93 (β) of Hb, and the remaining PEG chains are bound to thiol groups introduced on ε-NH 2 of Hb. The present invention also provides a process for preparing a modified hemoglobin molecule (Hb), comprising the steps of: (a) reacting Hb with 8-15 fold excess of iminothiolane to form thiolated Hb; and (b) reacting the thiolated Hb with 16-30 fold excess of PEG functionalized with a maleimide moiety, to form the modified Hb.
1. A process for preparing a hemoglobin molecule (Hb) modified to have on average six ± one polyethylene glycol (PEG) chains, comprising the steps of:
(a) reacting Hb with 8-15 fold molar excess of iminothiolane to form thiolated Hb; and
(b) reacting the thiolated Hb with 16-30 fold molar excess of PEG functionalized with a maleimide moiety, to form the modified Hb having on average six ± one PEG chains.
2. The process of claim 1 , wherein Hb is reacted with 9-12 fold molar excess iminothiolane in step (a).
3. The process of claim 1 , wherein Hb is reacted with about 10 fold molar excess iminothiolane in step (a).
4. The process of claim 1 , wherein the thiolated Hb is reacted with 18-22 fold molar excess PEG functionalized with a maleimide moiety in step (b).
5. The process of claim 1 , wherein the thiolated Hb is reacted with about 20 fold molar excess PEG functionalized with a maleimide moiety in step (b).
6. The process of claim 1 , wherein Hb is reacted with 9-12 fold molar excess iminothiolane in step (a), and the thiolated Hb is reacted with 18-22 fold molar excess PEG functionalized with a maleimide moiety in step (b).
7. The process of claim 1 , wherein Hb is reacted with 9-12 fold molar excess iminothiolane in step (a), and the thiolated Hb is reacted with about 20 fold molar excess PEG functionalized with a maleimide moiety in step (b).
8. The process of claim 1 , wherein Hb is reacted with about 10 fold molar excess iminothiolane in step (a), and the thiolated Hb is reacted with 18-22 fold molar excess PEG functionalized with a maleimide moiety in step (b).
9. The process of claim 1 , wherein Hb is reacted with about 10 fold molar excess iminothiolane in step (a), and the thiolated Hb is reacted with about 20 fold molar excess PEG functionalized with a maleimide moiety in step (b).
10. The process of claim 1 , wherein PEG has a molecular weight of 3000-10,000 daltons.
11. The process of claim 10 , wherein the modified Hb does not produce hypertension in a subject.
12. The process of claim 1 , wherein PEG has a molecular weight of about 5,000 daltons.
13. The process of claim 12 , wherein the modified Hb does not produce hypertension in a subject.
14. The process of claim 1 , wherein the modified Hb does not produce hypertension in a subject.