Hexa-thiocarbamoyl Phenyl PEG5K Hb: Vasoactivity and Structure

A new hexaPEGylated hemoglobin, (TCP-PEG5K)(6)-Hb (TCP, thiocarbamoyl phenyl) has been developed using PEG-phenyl-isothiocyanate and its vasoactivity and structure has been investigated. Of the six PEG5K chains of (TCP-PEG5K)(6)-Hb, 4 are conjugated to the alpha-amino groups of Hb, and the other 2 chains are distributed on epsilon-amino groups, identified as Lys-40(alpha) (similar to 45%), Lys-56(alpha) (similar to 25%), and Lys-8(beta) (similar to 24%). The studies with hamster infused with a bolus of a 4 gm % solution of (TCP-PEG5K)(6)-Hb equivalent to 10% of their blood volume have established that this new hexaPEGylated Hb is vasoinactive. The viscosity and the colloidal osmotic pressure of (TCP-PEG5K)(6)-Hb at 4% is 1.9 cP and 69.7 mmHg, respectively. The molecular radius of (TCP-PEG5K)(6)-Hb is about 4.6 nm and is significantly smaller than hexaPEGylated Hbs developed using other direct and extension arm facilitated PEGylation platform. The presence of an outside the central cavity intramolecular crosslink, succinimidophenyl-PEG2K between Cys-93(beta, beta') in (TCP-PEG5K)(6)-beta beta-Hb strongly impacts its solution properties. These patterns of influence suggest that the inter-dimeric interactions in (TCP-PEG5K)(6)-Hb is weakened just as with other direct PEGylation platforms, and (SP-PEG5K)(6)-Hb generated by EAF-PEGylation is unique in not inducing this effect. A comparison of the properties of hexaPEGylated Hbs establishes that rigidity of the conjugation linkage between PEG and Hb plays a significant influence on the resultant dictating solution properties and/structure/conformation of PEG-Hb adduct.