Kinetic studies on the reactions of separated a, b and c subunits of Panulirus interruptus deoxy-hemocyanin with hydrogen peroxide.

The kinetics of the H2O2 oxidation of separated a, b and c subunits (75 kDa, 657 amino acids) of the arthropod Panulirus interruptus hemocyanin (Hc), in the deoxy Cu(I)2 state, have been studied at 25 degrees C, I = 0.100 M (NaCl). Solutions of oxyHc provide small equilibrium amounts of the deoxy reactant deoxyHc + O2<-->oxyHc (K approx. 10(5) M-1). The reaction is of interest because H2O2 is one of the few molecules which is able to access the active site and oxidise deoxyHc to the metHc Cu(II)2 state. The reaction was studied at pH values in the range 6.8-9.6. Traces of Ca2+ (and other 2+ ions) are controlled by addition of EDTA (5 mM). With or without EDTA hexamer forms are present at the lower pH values. At pH > 8.3 and with EDTA added, a and b (3% sequence difference) give monomer forms. The hexamers are however retained at the higher pH values if Ca2+ (10 mM) is added. As in the corresponding studies of deoxyHc with O2, rate constants for subunit c (42% sequence differences) show no variation with pH and the hexamer is retained over the whole pH-range explored. Rate constants for the reactions of H2O2 with the different monomer and hexamer deoxyHc forms of a, b, and c are in the range 1-75 M-1 s-1. A mild pseudo-catalase activity of metHc leading to reformation of oxyHc also contributes to the reaction.