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(Circulation Research. 1996;78:635-642.)
© 1996 American Heart Association, Inc.

Articles

Removal of Arg¹⁴¹ From the Chain of Human Hemoglobin by Carboxypeptidases N and M

Bruno Michel Rajko Igi, Valérie Leray, Peter A. Deddish, Ervin G. Erdös

From the Departments of Pharmacology (B.M., P.A.D., E.G.E.) and Anesthesiology (V.L., E.G.E.), University of Illinois College of Medicine, and Cook County Hospital Department of Anesthesiology (R.I.), Chicago, Ill.

Correspondence to Ervin G. Erdös, MD, University of Illinois College of Medicine, Department of Pharmacology (M/C 868), 835 S Wolcott Ave, Chicago, IL 60612. E-mail egerdos@uicvm.uic.edu.

Abstract Both human plasma carboxypeptidase N (CPN) and membrane-bound carboxypeptidase M (CPM) released the C-terminal arginine (-Arg¹⁴¹) of the chain of human adult hemoglobin. An arginase contamination present in the hemoglobin preparation, which converted the released arginine to ornithine, was removed by gel filtration. CPM was about 20 times more efficient than CPN or its active subunit in hydrolyzing oxyhemoglobin and cleaved oxyhemoglobin twice as fast as deoxyhemoglobin. The hydrolysis of the peptide bond of -Arg¹⁴¹ accelerated the dissociation rate of the tetramer deoxy-des--Arg¹⁴¹ hemoglobin to dimers 2500-fold over that of deoxyhemoglobin, as measured by haptoglobin binding. Moreover, the dissociation of the deoxy-des--Arg¹⁴¹ hemoglobin tetramer to dimers was not affected by 2,3-diphosphoglyceric acid. Des--Arg¹⁴¹ hemoglobin had a higher oxygen affinity (P₅₀, 5.51 mm Hg; control, 19.94 mm Hg [P₅₀ is the partial pressure of oxygen that gives 50% of the saturation of hemoglobin]) and a lower apparent cooperativity (Hill coefficient: n, 1.02; control, 2.24) than unhydrolyzed hemoglobin. After hemoglobin was incubated in human plasma, its oxygen-binding parameters, the P₅₀, and the Hill coefficient decreased drastically due to cleavage by CPN. In the perfused rat heart, des--Arg¹⁴¹ hemoglobin was a more effective coronary vasoconstrictor than hemoglobin, possibly because it dissociated to dimers in the coronary vascular bed. A covalently cross-linked hemoglobin was less active than native hemoglobin. The coronary vasoconstriction was caused by multiple factors, including interference with vasodilation by nitric oxide and eicosanoids. Thus, the hydrolysis of hemoglobin by CPM and CPN demonstrated the contribution of the -Arg¹⁴¹ residue to sustaining the tetrameric structure of hemoglobin and its normal oxygen affinity and vasoactivity.

Key Words: blood substitutes • carboxypeptidases • cross-linked hemoglobin • oxygen affinity • coronary vasoconstriction

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