Temperature and domain size dependence of sickle cell hemoglobin polymer melting in high concentration phosphate buffer

Abstract

Deoxygenated sickle cell hemoglobin (Hb S) in 1.8 M phosphate buffer, and carbon monoxide (CO) saturated buffer were rapidly mixed using a stopped-flow apparatus. The binding of the CO to the Hb S polymers and the polymer melting was measured by time resolved optical spectroscopy. Polymer melting was associated with decreased turbidity, and CO binding to deoxy-Hb S was monitored by observation of changes in the absorption profile. The reaction temperature was varied from 20°C to 35°C. Polymer domain size at 20°C was also varied. The data for mixtures involving normal adult hemoglobin (Hb A) fit well to a single exponential process whereas it was necessary to include a second process when fitting data involving Hb S. The overall Hb S–CO reaction rate decreased with increasing temperature from 20°C to 35°C, and increased with decreasing domain size. In comparison, Hb A–CO reaction rates increased uniformly with increasing temperature. Two competing reaction channels in the Hb S–CO reaction are proposed, one involving CO binding directly to the polymer and the other involving CO only binding to Hb molecules in the solution phase. The temperature dependence of the contribution of each pathway is discussed.