Differential effects of haptoglobin and albumin on the oxygenation of arachidonic acid during prostaglandin biosynthesis

portions of the ligand solution, previously adjusted to the same pH as the reaction medium, were added successively. Changes in H+ concentration were detected with a microelectrode and pH-meter of sensitivity such that uptake or release of longatoms of H+ could be detected. The H+/haem ratio was calculated from reciprocal plots of ligand concentration against H+ uptake and extrapolation back to infinite ligand concentration to give the maximum number of H+ taken up. Table 1 shows that direct measurement of H+ uptake on ligand binding to metmyoglobin agrees with results obtained spectroscopically. No H+ movements are shown upon binding of azide and formate at pH 7, indicating that they both bind as their dissociated ions. Spectroscopic results show cyanide to be found in a mixed HCN/CNform, which agrees with the 0.8H+/haem ratio we obtain at saturation with cyanide. Lack of H+-movements at p H 7 upon cyanide binding to catalase agrees with the spectroscopic result that cyanide binds as HCN. Upon azide and formate binding only O.SH+/haem is taken up, whereas spectroscopic results indicate that 1 H+ should be taken up per haem. This may indicate that the two methods measure separate parameters, that is, direct H+ measurements respond to all the pK shifts on the haem protein, whereas spectroscopic changes are sensitive only to pK shifts around the haem environment. Purified cytochrome oxidase appears to react with HCN in the same way as catalase in that no H+ uptake occurs. Formate and azide appear to act more as myoglobin when H+ movements are measured, although these are anomalous with spectroscopic results that indicate that 1 H+ is taken up per ligand bound.