Enhanced molecular volume of conservatively pegylated Hb: (SP-PEG5K)6-HbA is non-hypertensive.
Recent studies have suggested that the "pressor effect" of acellular Hb is a consequence of perturbation of the macro-and microcirculatory system in multiple ways, and that PEGylation is an effective approach for controlling the same. In an attempt to confirm this concept, a new and simple thiolation mediated, maleimide chemistry-based conservative PEGylation protocol has been developed to conjugate multiple copies of PEG-chains to Hb. This approach combines the high reactivity of maleimides towards thiols with the propensity of iminothiolane to derivatize the epsilon-amino groups of proteins into reactive thiol groups, with conservation of their positive charge. One of the PEGylated products, namely (SP-PEG5K)6-HbA, that carries on an average six copies of PEG5000 chains per Hb, is non-hypertensive in hamster top load and in rat 50% exchange transfusion models. This hexa-PEGylated-Hb has (i) a hydrodynamic volume corresponding to that of an oligomerized Hb of 256kDa, (ii) a molecular radius of approximately 6.8 nm, (iii) high oxygen affinity, (iv) lowered Bohr effect, and (v) increased viscosity and colloidal osmotic pressure. These properties of (SP-PEG5K)6-HbA are consistent with the emerging new paradigms for the design of Hb based oxygen carriers and confirm the concept that the "pressor effect" of Hb is a multifactorial event. The thiolation mediated maleimide chemistry-based PEGylation protocol described here for the generation of (SP-PEG5K)6-Hb is simple, highly efficient, and is carried out under oxy conditions. The results demonstrate that a non-hypertensive PEG-Hb can be generated by conjugation of a lower number of PEG chains than previously reported.
Acharya
Seetharama A
SA
Department of Medicine, Albert Einstein College of Medicine, Bronx, NY, USA.
Friedman
Joel M
JM
Manjula
Belur N
BN
Intaglietta
Marcos
M
Tsai
Amy G
AG
Winslow
Robert M
RM
Malavalli
Ashok
A
Vandegriff
Kim
K
Smith
Paul K
PK
D000818:Animals; D001794:Blood Pressure / Q000187:drug effects; D001802:Blood Substitutes / Q000008:administration & dosage* / Q000138:chemical synthesis / Q000737:chemistry; D006224:Cricetinae; D006441:Hemoglobin A / Q000008:administration & dosage* / Q000737:chemistry; D006454:Hemoglobins / Q000008:administration & dosage* / Q000138:chemical synthesis / Q000737:chemistry; D006973:Hypertension / Q000503:physiopathology; D008297:Male; D011092:Polyethylene Glycols / Q000008:administration & dosage* / Q000138:chemical synthesis / Q000737:chemistry
D001802:Blood Substitutes; D006454:Hemoglobins; C109960:PEG-hemoglobin; D011092:Polyethylene Glycols; D006441:Hemoglobin A