Photolysis method for determination of the tetramer-dimer dissociation constant of deoxyhemoglobin

A new method for determination of the tetramer-dimer dissociation constant Ku4.2 of deoxyhemoglobin is described. The method involves photolysis of hemoglobin solutions containing a few percent of bound CO (e.g. less than 3%). Under these conditions the nature of the observed CO rebinding is primarily determined by the properties of the dominant species, deoxyhemoglobin. The method makes use of the 30-fold difference in the rate constant describing CO binding to hemoglobin dimers and deoxyhemoglobin tetramers. Because of this large difference in rate constants CO rebinding is made significantly more rapid by the presence of even small concentrations of dimers. Treating this reaction as CO binding to a mixture of hemoglobin dimers and tetramers allows the determination of Ku4.2. Data is presented showing application of the method to human deoxyhemoglobin in the range from pH 9.5 to 11.2.