Factors influencing the absorption spectrum of vertebrate hemoglobin in solution

Abstract

An analysis of common methods of collection and preparation of solutions of hemoglobin shows that a number of techniques and materials produce a modified material, characterized by a relative increase in absorbance at 570 nm in the acidic methemoglobin form. The undesirable conditions include, most importantly, dialysis against distilled water, exposure to a large excess of oxidizing agent, oxidation of an inadequately dialyzed hemoglobin stock solution, failure to remove excess oxidant, exposure even briefly to a pH below 6.0, or freezing of methemoglobin solutions. The effect of 4 M urea on the spectrum of methemoglobin indicates that a common feature of these agents is protein “perturbation”. It is important that a spectrum of methemoglobin be taken at pH 6.0, since the admixture of a small proportion of the alkaline form of methemoglobin mimics this perturbation. The common practice of reporting a methemoglobin spectrum at pH 7.0 or 7.4 is to be discouraged.