PANTETHINE INHIBITS THE FORMATION OF HIGH-T-C PROTEIN AGGREGATES IN GAMMA-B CRYSTALLIN SOLUTIONS

Purpose. Solutions of the bovine lens protein gamma B (or yII) crystal lin at neutral pH in the absence of reducing agents, undergo a slow, p artial conversion to a new protein species, gamma IIH. This species is an aggregate composed of an intermolecular, disulfide-crosslinked dim er (approximate to 32% of total protein by weight) and loosely associa ted dimers (approximate to 66%). gamma IIH has a phase separation temp erature (T-ph), at least 40 degrees C higher than that of native gamma II crystallin at any given protein concentration. In this paper we de monstrate that pantethine, a derivative of coenzyme A, inhibits the fo rmation of gamma IIH. Methods. gamma II crystallin solutions were incu bated at pH 7.1 and room temperature with increasing amounts of pantet hine. The T-ph Of the solutions was monitored as a function of incubat ion time. Corresponding to each T-ph measurement, aliquots of each sol ution were analyzed by cation-exchange HPLC to determine the amount of gamma IIH formed. Results. Incubation of yII crystallin with increasi ng amounts of pantethine lowers T-ph and suppresses the formation of g amma IIH. With pantethine to protein mole ratios of 0.66, 1 and 2, the T-ph of gamma II crystallin is lowered from 8 degrees C in the native protein, to 2 degrees C, -3 degrees C and -4.3 degrees C respectively , at a protein concentration of approximate to 200 mg/ml. The amount o f gamma IIH accumulated decreases from similar to 25% in the native pr otein to 10%, 1% and 0% respectively in these pantethine-treated prote in solutions. For complete suppression of the rise in T-ph and inhibit ion of gamma IIH formation, a 2:1 mole ratio of pantethine to protein is required. Conclusions. We suggest that pantethine reacts with two c ysteine residues of gamma II crystallin by forming a mixed disulfide, and effectively suppresses protein aggregation and lowers T-ph. This i s due to the strong polar character of pantethine which reduces the ne t attractive interactions between the protein molecules.