Hemocyanins in spiders

Summary

• 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 (F_deoxy/F_oxy 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 (ΔlogP ₅₀/ΔpH=−1.2; Fig. 7). Cooperativity is maximal at about pH 8.0 (n ₅₀=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,K ₁ andK ₂₄, were determined by means of a modified Scatchard plot (Edsall et al., 1954);K ₁=0.0036 mm Hg⁻¹=0.0022×10⁶ M⁻¹;K ₂₄=2.69 mm Hg⁻¹=1.636×10⁶ M⁻¹. With [T ₀]≫[R ₀],K ₁ representsK _Tass , whereasK ₂₄ ([T ₀]≪[R ₀]) 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, ΔF ₁, is 3.9 kcal/site (25°C).