Abstract
Purpose. Solutions of the bovine lens protein γB (or γII) crystallin at neutral pH in the absence of reducing agents, undergo a slow, partial conversion to a new protein species, γIIH. This species is an aggregate composed of an intermolecular, disulfide-crosslinked dimer (≈32% of total protein by weight) and loosely associated dimers (≈66%). γIIH has a phase separation temperature (Tph), at least 40d`C higher than that of native γII crystallin at any given protein concentration. In this paper we demonstrate that pantethine, a derivative of coenzyme A, inhibits the formation of γIIH.
Methods. γII crystallin solutions were incubated at pH 7.1 and room temperature with increasing amounts of pantethine. The Tph of the solutions was monitored as a function of incubation time. Corresponding to each Tph measurement, aliquots of each solution were analyzed by cation-exchange HPLC to determine the amount of γIIH formed.
Results. Incubation of γII crystallin with increasing amounts of pantethine lowers Tph and suppresses the formation of γIIH. With pantethine to protein mole ratios of 0.66, 1 and 2, the Tph of γII crystallin is lowered from 8d`C in the native protein, to 2d`C, -3d`C and -4.3d`C respectively, at a protein concentration of ≈200 mg/ml. The amount of γIIH accumulated decreases from ∼ 25% in the native protein to 10%, 1% and 0% respectively in these pantethine-trcatcd protein solutions. For complete suppression of the rise in Tph and inhibition of γIIH formation, a 2:1 mole ratio of pantethine to protein is required.
Conclusions. We suggest that pantethine reacts with two cys-leine residues of γII crystallin by forming a mixed disulfide, and effectively suppresses protein aggregation and lowers Tph. This is due to the strong polar character of pantethine which reduces the net attractive interactions between the protein molecules.