A Comparison of Rates of Heme Exchange: Site-Specifically Cross-Linked Versus Polymerized Human Hemoglobins

Abstract

The stability of the heme-globin interaction of chemically modified human hemoglobin (Hb) was tested by measuring rates of heme loss from methemoglobin. Heme transfer from methemoglobin to human serum albumin was measured by rapid-scanning spectrophotometry, and the resulting absorption matrices were analyzed by singular value decomposition. Unmodified human HbA₀, hemoglobin cross-linked between β subunits with either 2-nor-2-formylpyridoxal 5′-phosphate or 3,5-(dibromosalicyl)fumarate (DBBF), hemoglobin cross-linked between α subunits with DBBF, and pyridoxalated hemoglobin polymerized with either glycolaldehyde or glutaraldehyde were tested. Initial rates were evaluated by fitting the time courses to a biexponential equation using a matrix least squares curve-fitting algorithm. Reaction rates fell into two classes: (1) HbA₀, and the site-specifically cross-linked hemoglobins, with biphasic rates of heme loss of 0.02 and 0.004 min^-1, and (2) polymerized hemoglobins, with 10–20-fold higher rates at 0.5 and 0.03 min^-1. The total fitted amplitudes of the reaction depended upon the specific modification: ββ-cross-linked Hbs < αα-cross-linked Hb > glycolaldehyde polymerized Hb < glutaraldehyde polymerized Hb < HbA₀.