Hemocyanins in spiders

Abstract

1. Fluorescence (F) of the hemocyanin ofEurypelma californicum is strongly dependent on the degree of oxygenation (Fig. 2). Maximum excitation is found at 292 to 294 nm. There is only a small shift of maximum emission from 345 nm in oxygenated to 350 nm in deoxygenated hemocyanin, indicating that mainly tryptophan is responsible for oxygenation-dependent fluorescence (Fig. 3). Fluorescence enhancement depends linearly on the degree of deoxygenation (Fdeoxy/Foxy is about 16 at pH 7.4; Fig. 4). 2. Based on fluorescence quenching upon oxygenation, a fluorimetric-polarographic method for recording oxygen equilibrium curves was developed: With a favourable geometrical arrangement and low hemocyanin concentration, the error induced by reabsorption of emitted light is minimal (working range 0.02–0.2 mg/ml, corresponding to ca. 0.005–0.05 O.D. at 340 nm; Fig. 5). Data obtained by this method are in excellent agreement with data obtained by photometry (Figs. 6 and 7). 3. Oxygen affinity and cooperativity between oxygen binding sites ofEurypelma hemocyanin are strongly modified by protons: There is a very pronounced Bohr effect with a maximum between pH 8.0 and 8.4 (ΔlogP50/ΔpH=−1.2; Fig. 7). Cooperativity is maximal at about pH 8.0 (n50=7) and decreases towards low and high pH (Fig. 7). Oxygen affinity is independent of hemocyanin concentration, cooperativity, however, is slightly increased at high hemocyanin concentration. 4. Modification of oxygen affinity and cooperativity is interpreted in the framework of the Monod, Wyman and Changeux (1965) model. SinceK Tass andK Rass could not be estimated directly from the Hill plots, the intrinsic association constants of the first and the last oxygenation step,K1 andK24, were determined by means of a modified Scatchard plot (Edsall et al., 1954);K1=0.0036 mm Hg−1=0.0022×106 M−1;K24=2.69 mm Hg−1=1.636×106 M−1. With [T0]≫[R0],K1 representsK Tass , whereasK24 ([T0]≪[R0]) is equal toK Rass . From these constants, the MWC parameterc was calculated to be 0.00133 (c=K1/K24). The total free energy of interaction, ΔF1, is 3.9 kcal/site (25°C).