Bovine hemoglobin pseudo-crosslinked with mono(3,5-dibromosalicyl)-fumarate.
Under oxygenated conditions bovine hemoglobin reacts with mono(3,5-dibromosalicyl)-fumarate which specifically acylates the EF5 lysines in the beta-cleft of the protein. The chemical modification introduces in the molecule a pseudo-crosslink which hinders the dissociation of the hemoglobin molecule into dimers. Retention time in circulation of the chemically modified bovine hemoglobin, measured in the rat, is increased fivefold with respect to untreated bovine hemoglobin. The oxygen affinity at 37 degrees C and at pH 7.4, has a P50 = 5.4 kPa and a value of n = 1.9. Under the same experimental conditions the oxygen affinity is not sensitive to anions and polyanions whereas it is sensitive to CO2. The Bohr effect is shifted toward the alkaline pH range by 0.5-1: the maximum number of protons released is 1.5/tetramer, similar to normal bovine hemoglobin (1.8 protons/tetramer). Analysis of the binding isotherms, using the two-state Monod-Wyman-Changeux model and fixing the value of the allosteric constant L = 10(5), shows that the oxygen affinity of the T structure is not modified, and that the low oxygen affinity of the system is due to a decrease of the oxygen affinity of the R structure. Analysis using the sequential Adair model shows a modification of the overall binding constants and suggests a redistribution of the intermediate species of oxygenation.
Fronticelli
C
C
Department of Biological Chemistry, Medical School, University of Maryland, Baltimore 21212.
Bucci
E
E
Razynska
A
A
Sznajder
J
J
Urbaitis
B
B
Gryczynski
Z
Z
D000818:Animals; D001241:Aspirin / Q000031:analogs & derivatives* / Q000737:chemistry; D002417:Cattle; D002499:Centrifugation, Density Gradient; D003432:Cross-Linking Reagents; D004563:Electrochemistry; D006207:Half-Life; D006454:Hemoglobins / Q000737:chemistry* / Q000378:metabolism / Q000493:pharmacokinetics; D015394:Molecular Structure; D010100:Oxygen / Q000737:chemistry; D010446:Peptide Fragments; D010449:Peptide Mapping; D051381:Rats
D003432:Cross-Linking Reagents; D006454:Hemoglobins; D010446:Peptide Fragments; C053936:hemoglobin XL99alpha; C021630:bis(3,5-dibromosalicyl)fumarate; D001241:Aspirin; D010100:Oxygen