title: Enhanced molecular volume of conservatively pegylated Hb: (SP-PEG5K)6-HbA is non-hypertensive.
abstract: 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.
authors.affiliation: Department of Medicine, Albert Einstein College of Medicine, Bronx, NY, USA.
authors.forename: Seetharama A
authors.initials: SA
authors.lastname: Acharya
authors.forename: Joel M
authors.initials: JM
authors.lastname: Friedman
authors.forename: Belur N
authors.initials: BN
authors.lastname: Manjula
authors.forename: Marcos
authors.initials: M
authors.lastname: Intaglietta
authors.forename: Amy G
authors.initials: AG
authors.lastname: Tsai
authors.forename: Robert M
authors.initials: RM
authors.lastname: Winslow
authors.forename: Ashok
authors.initials: A
authors.lastname: Malavalli
authors.forename: Kim
authors.initials: K
authors.lastname: Vandegriff
authors.forename: Paul K
authors.initials: PK
authors.lastname: Smith
mesh_terms: 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
chemical_list: D001802:Blood Substitutes; D006454:Hemoglobins; C109960:PEG-hemoglobin; D011092:Polyethylene Glycols; D006441:Hemoglobin A