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O.P. Srivastava, K. Srivastava; Characterization of Covalent Multimers of Crystallins in Water Insoluble Protein Fraction in Human Lenses . Invest. Ophthalmol. Vis. Sci. 2003;44(13):2344.
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Purpose: The purpose of the study was to identify components of covalent multimers present in the water insoluble protein fraction during aging in human lenses. Methods: The water soluble (WS) and water insoluble (WI)-protein fractions were isolated from lenses of 25, 41, 52 and 72 year-old donors. Both WS- and WI-protein species were separated by two-dimensional gel electrophoresis and stained with Coomassie blue. The protein spots with Mr >40 kDa from WI-protein fractions were excised, digested with trypsin and analyzed by MALDI-TOF (Prospective Biosciences) and by ES-MS/MS (Micromass QTOF-2) Results: The 2D-gel profiles showed greater than thirteen distinct multimer spots (Mr >40 kDa) in the WI-protein fraction of 25 year-old lenses but their numbers, concentrations and complexity (as diffused and non-descript spots) increased in the older lenses. Among these, two spots of approximate Mr of 110 kDa (spot #6) and 115 kDa (spot #7) from 25-year old lenses were chosen for further mass spectrometric analysis. Spot #6 was found to be a covalent multimer of oxidized αA-crystallin (oxidized residues: M-1, and W-9[with one or two oxygen]) and filensin (a beaded filament structural protein). A similar MS/MS analysis of spot #7 provided amino acid sequences showing it to be a covalent multimer of fragments of αA, αB, ßB1, ßB2, γS and γD-crystallins. Conclusions: Covalent multimers existed as WI-proteins of human lenses at an early age of 25 years. One such 110 kDa-multimer contained covalent complex of oxidized αA-crystallin and filensin. An additional 115 kDa-multimer was a covalent complex of fragments of αA, αB, ßB1, ßB2, γS and γD-crystallins.
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