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K. Srivastava, J. M. Chaves, O. P. Srivastava; Characterization of Crystallin Complexes in High Molecular Weight Proteins of Aging Normal and Cataractous Human Lenses by Blue-Native Gel Electrophoresis. Invest. Ophthalmol. Vis. Sci. 2008;49(13):4101. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
To examine non-covalent crystallin complexes in water soluble-high molecular weight (WS-HMW) proteins of normal and cataractous human lenses.
The WS-HMW proteins of normal (four pooled 20 year-old lenses and four pooled 60-70 year-old lenses) and cataractous (four pooled 60-70 year-old lenses with nuclear cataract) human lenses were isolated by size-exclusion Agarose A 5 m column chromatography. The HMW protein preparations were analyzed by blue-native gel electrophoresis in the first dimension followed by SDS-PAGE in the second dimension. The protein bands of varying molecular weights that appeared in a single file on a SDS-PAGE gel were considered to exist as a complex. Individual bands of a complex were trypsin-digested, and the amino acid sequences of tryptic peptides were determined by ES-MS/MS method.
A protein complex in the WS-HMW protein fraction of 20 year-old lenses showed 20 bands, and the amino acid sequences of tryptic peptides of individual bands by ES-MS/MS method showed a complex of αA, αB, βA3, βA4, βB1, βB2, γS-crystallins and phakinin and filensin. A similar analysis identified a complex of 11 protein bands in the normal lenses of 50-70 year-old donors, which contained αA, αB, βA3, βA4, βB1, βB2, γS-crystallins, filensin and aldehyde dehydrogenase. A similar analysis of lenses with nuclear cataract showed the presence of a complex with 22 bands that contained αA, αB, βA3, βA4, βB1, βB2, γC, γD and γS-crystallins and aldehyde dehydrogenase.
The results suggested the existence of a complex of crystallins with phakinin and filensin in the WS-HMW proteins fraction of normal lenses from 20 year-old donors. An additional protein (aldehyde dehydrogenase) along with crystallins was detected in a WS-HMW protein complex of normal lenses from 50-70 year-old donors. A similar complex in the age-matched cataractous lenses contained additional γC and γD crystallins, suggesting their participation in the complex formation during nuclear cataract development.
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