The anaerobic reaction of bovine hemoglobin with divinyl sulfone: structural changes and functional consequences.
The anaerobic reaction of bovine hemoglobin with divinyl sulfone results in a non-cross-linked intramolecularly-modified new derivative. This chemically-modified hemoglobin is homogeneous with respect to its molecular mass (64 kDa) and electrophoretic properties. The absence of any 32 kDa band from its SDS-PAGE pattern proves the lack of intramolecular cross-linkage, while a single-peak high-performance gel-permeation chromatogram demonstrates the absence of intermolecular cross-linkage. The oxygen binding properties determined at 37 degrees C, 0.15 M Cl- and pH 7.4 display a P50 of 52 mmHg and a Hill coefficient n of 1.9. Under the same experimental conditions the oxygen affinity is not sensitive to chloride anions, suggesting that the covelant modification is in the beta-cleft. The maximum number of Bohr protons released is 0.8/tetramer, which is half that of normal bovine hemoglobin. The retention time in circulation, measured in rats, is similar to that of native bovine hemoglobin. Using a high molar ratio of divinyl sulfone to modified hemoglobin, it is feasible to effect anaerobic intermolecular cross-linkage. The polymerized material, isolated from a 24 h reaction, is a mixture of modified intermolecularly-crosslinked hemoglobins with a molecular mass range from 130 to approx. 500 kDa. The oxygen-transport characteristics of the polymerized material are similar to those of the modified non-cross-linked derivative, whereas its retention time in rats is increased three-fold with respect to native bovine hemoglobin.
Artificial Cells and Organs Research Centre, Faculty of Medicine, McGill University, Montreal, Québec, Canada.
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Ilan
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Morton
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Chang
D000494:Allosteric Regulation; D000693:Anaerobiosis; D000818:Animals; D002417:Cattle; D006454:Hemoglobins / Q000737:chemistry* / Q000378:metabolism / Q000493:pharmacokinetics; D008297:Male; D008657:Metabolic Clearance Rate; D010100:Oxygen / Q000378:metabolism; D051381:Rats; D013450:Sulfones / Q000737:chemistry* / Q000378:metabolism / Q000493:pharmacokinetics
D006454:Hemoglobins; D013450:Sulfones; C081606:bovine hemoglobin-divinyl sulfone; C081607:polymerized bovine hemoglobin-divinyl sulfone; C009873:divinyl sulfone; D010100:Oxygen