The Effect of pH on Carbon Monoxide Binding to Menhaden Hemoglobin

The major hemoglobin fraction of menhaden (Breuoortia tyrrznnus) is a Root effect hemoglobin; at acid pH it exhibits low affinity behavior characteristic of the T state and at alkaline pH functional behavior characteristic of the R state. The rates of CO binding and dissociation have been measured by stopped flow and flash photolysis at low and high pH. At acid pH CO binding is slow and biphasic, with rate constants of 0.25 and 0.023 FM-’ s-‘; these were assigned as the T state binding rates of the two chains. CO dissociation is also biphasic. At alkaline pH CO binding and dissociation are homogeneous, with rate constants of 1.3 PM-’ s-’ and 0.028 s-‘, respectively; these were assigned as the R state rates for both chains. Full and partial flash photolysis data were collected over the pH range 6.0 to 8.5, and CO equilibrium data from 6.0 to 7.5. These were fit to the twostate model modified to include chain differences. The T and R binding rates, and the R dissociation rate, were assigned; L and the T dissociation rates were calculated. L varies from 4 x lo7 at pH 6.0 to 8 at pH 8.5; the T rates vary from 0.15 and 0.25 at pH 6.0 to 0.02 at pH 8.5. Both Hb and carbon monoxide hemoglobin (HbCO) have pH difference spectra in the Soret and visible regions. The Hb spectrum changes above pH 7.5 and the HbCO spectrum changes below pH 7.0. The transition from R to T state was observed at an Hb-HbCO isosbestic during flash photolysis of HbCO. The total absorbance change at this w-avelength rises, then falls with pH, with a maximum at pH 7.0. The time course of conformation change at pH 7.0 was followed after full and partial flash photolysis and fit to the extended two-state model with an L value of lo” and a 3-fold decrease in the rate of R to T transition for each additional bound CO. The magnitude of static and kinetic absorption changes was compared to the conformation state of Hb and HbCO calculated from the functional measurements. There is poor correlation; both spectral measurements indicate a too great effect of pH on the conformation of both derivatives. It is suggested that optical absorption measurements do not give a quantitative measure of quaternary conformation.