SOLUBILITY OF SICKLE HEMOGLOBIN MEASURED BY A KINETIC MICROMETHOD

We have developed a photolytic method to determine the concentration o f reactive hemes in a solution in the presence of a trace amount of CO . By measurement of the bimolecular rate of CO binding, and by calibra tion of the rate constant under equivalent conditions, the concentrati on of the reactive hemes can be determined. In a solution of sickle he moglobin, the molecules in the gel contribute negligibly to the recomb ination rate, allowing the concentration of the molecules in the solut ion phase to be determined. To optimize signal to noise, modulated exc itation methods were employed, although the method could also be used with pulse techniques and suitable signal averaging. Because the optic al method employs a microspectrophotometer, only a few microliters of concentrated Hb solution is required to reproduce the entire temperatu re dependence of the solubility previously determined by centrifugatio n using milliliter quantities of solutions of the same concentration. This should be especially useful for studies of site-directed mutants, and we present results obtained on one such HbS in which Leu 88 beta has been replaced by Ala. The free energy difference in the polymeriza tion of the Leu 88 beta double mutant is consistent with known differe nces in the amino acid hydrophobicities. The calibration required for these experiments also provides an excellent determination of the acti vation energy for binding the first CO to deoxy Hb.