Interaction of cupric ion with parvalbumin.

Cod parvalbumin, a calcium-binding protein, possesses a specific Zn2+ (or Cu2+) binding site per molecule. This work employed fluorescence energy transfer techniques to measure the distance between the Zn2+ (Cu2+) site and the stronger Ca(2+)-binding site in parvalbumin. Specifically, the distance between Tb3+ bound at the Ca2+ site and Co2+ bound to the Zn2+ (Cu2+) binding site was 10.3 +/- 0.9 A. Lastly, the effects of Cu2+ on the physico-chemical properties of parvalbumin were studied by measuring the accessibility of protein thiol groups to 5,5'-dithio bis(2-nitrobenzoic acid) and by its affinity for the fluorescent probe 4,4'-bis[1-(phenylamino)-8-naphthalene sulfonic acid] dipotassium salt. The thiol group accessibility decreased and the affinity to the fluorescent probe increased upon complexation of Cu2+ to the protein. It appears that the binding of Cu2+ converts parvalbumin to an apo-like state.

Functional implications resulting from disruption of the calcium-binding loop in bovine alpha-lactalbumin.

The strong calcium-binding site of alpha-lactalbumin comprises the carboxylate side chains of aspartic acid 82, 87, and 88 and the carbonyl oxygens of residues 79 and 84. A single methionine residue was selectively modified by controlled CNBr cleavage to yield homoserine at position 90. The CNBr-cleaved alpha-lactalbumin lost the ability to bind calcium strongly as monitored by intrinsic fluorescence, electrophoretic mobility, atomic absorption, and x-ray fluorescence. Remarkably, the modified protein was still competent in lactose biosynthesis, although activity was reduced to 1/40th that of the native form of the protein. Although the strong calcium-binding site was destroyed as a result of the cleavage of the calcium-binding loop, a secondary calcium site was retained that directly affects a rate enhancement of lactose biosynthesis when saturated, resulting in approximately a two- to threefold increase in rate at 1 mM CaCl2 with an activation equilibrium constant of 350 +/- 40 microM.