A multipass cuvette for laser photolysis studies and its uses in studying hemoglobin kinetics and equilibria

A simple multipass cuvette was constructed by cementing small first-surface mirrors to opposite optical faces of a standard cuvette, eliminating the need for complex alignment devices. The multipass cuvette could then be positioned to provide optical path lengths of approximately 5 and 7 cm for the observing beam directed perpendicularly to the laser photolytic pulse. Internal reflection losses in the cuvette elevated the baseline by 0.36 in absorbance for the seven-pass alignment. Heme proteins can easily be studied at 100 nM in this cuvette. Analysis of the concentration dependence of the rapid recombination phase following photolysis of HbCO allows KTD to be determined. Precise determination of this constant, however, requires that a large range of concentrations be studied, allowing the fraction of rapid phase to vary from 20 to 80%. Human HbCO at pH 7 cannot be effectively studied over this concentration range in ordinary cuvettes owing to the low concentrations required. By employing the multipass cuvette, we have been able to make very precise determinations of this constant and find at pH 7, 21 degrees C, a value for KTD of 0.66 microM. We also determined that the quantum yield for photolysis of HbCO dimers and tetramers must be very nearly the same. For HbCO in Tris buffer, pH 7.4, the R----T conformation change is some six to seven times slower than that in phosphate. We have developed a simple equation that allows both the rate constant for the conformational change and the KTD to be determined under these conditions. The KTD obtained is in excellent agreement with a reported value obtained by large-zone gel filtration.