Static and Kinetic Studies on Carp Muscle Parvalbumins

Abstract

Fluorescence titration and fluorescence stopped-flow studies were performed on carp muscle parvalbumin components 1, 2, 3, and 5 (the latter three components were modified with a SH-directed fluorescent reagent, dansyl-L-cysteine). Apparent binding constants (Kapp) of Ca²⁺ to these components decrease in the order of component 2 (K_app=2.8±0.9×10⁸ M^-1)>component 1 (K_app=1.25±0.25×10⁸ M^-1)>component 3=component 5 (K_app=4.0±0.5×10⁷ M^-1) in 30mM KCl, 50mM Na-cacodylate-HCl, pH 7.0 at 20°C. The rate constant of the conformational change of parvalbumin induced by Ca²⁺ binding or removal decreases in the order of component 2>component 1>component 5^>₌component 3; that is, component 2 undergoes the fastest conformational change and component 3 the slowest in response to the rapid free Ca²⁺ concentration ([Ca²⁺]) change in the protein solution. The fluorescence titration curves and [Ca²⁺]-dependences of the rate constants are analyzed by a simple two-state model, (partially unfolded state)κ₁_??_κ₂ (folded state). It is shown that the equilibrium constant K=κ₁/κ₂ depends on the second power of [Ca²⁺], the rate constant κ₁ on the first power of [Ca²⁺] and κ₂ on the inverse first power of [Ca²⁺], respectively