Human insoluble lens protein I. Separation and partial characterization of polypeptides

Abstract

A number of new methods are described for fractionating the polypeptides of the water insoluble protein from human lenses. In most cases, three major fractions can be obtained: a high molecular weight (HMW) polypeptide fraction containing components greater than 100 000 daltons plus a 43 000 dalton fraction, an intermediate sized fraction containing components from 27 000 to 20 000 daltons and a low molecular weight fraction containing an 11 000 dalton component. Under certain conditions, the 11 000, 43 000 and > 100 000 dalton fraction can be obtained pure on the basis of SDS gel electrophoresis. Marked charge heterogeneity was observed in all samples investigated. The HMW polypeptide fraction is the major component containing 50% of the H 2O insoluble protein with 30% represented by the 27 000–20 000 dalton fraction and 20% by the 11 000 dalton component. Investigation of the urea soluble and urea insoluble components of the water insoluble fraction indicates a close similarity in chemical and physical characteristics. On the basis of similarity in chemical and physical properties and increasing insolubility, it is proposed that a precursor relationship may exist where by HMW protein → urea soluble → urea insoluble protein. The HMW polypeptide fraction may not be stabilized by intrachain disulfide bonds. Furthermore, the marked susceptibility of this fraction to tryptic digestion where essentially only low molecular weight components rapidly appear, suggests that there may be few if any non-reducible crosslinks stabilizing the overall HMW polypeptide structure. The absence of any free NH 2-terminal residue in the H 2O insoluble fraction suggests that unmodified γ-crystallin may not be present.