20] Double mixing methods for kinetic studies of ligand binding in partially liganded intermediates of hemoglobin

Abstract

This chapter focuses on the differences between the two methods of double mixing stopped-flow and single mixing, the type of reactions that can be studied by the double mixing method, experimental design, data treatment, and a brief discussion of the results obtained. Unless otherwise mentioned, all examples discussed in the chapter are for normal human hemoglobin (Hb). The double-mixing method , along with the micro peroxidase method for studying carbon mono oxide (CO) dissociation reactions and the high pressure liquid chromatography (HPLC) method for preparation of the valency hybrids, allows for the study of kinetics of CO association with, and dissociation from, partially liganded intermediates of Hb. All of these studies so far have been made by only two groups and, therefore, a certain degree of bias in perspective may have been unavoidable, particularly in formulating reaction schemes to fit the data. These studies have been made using a first-generation double mixing stopped-flow spectrophotometer with limited capabilities, such as a long dead time, use of large sample volumes for each kinetic shot, and some probability of leading-edge contamination at longer aging times. More information and precision can be obtained by using state-of-the-art double-mixing instruments.