May 2008
Volume 49, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2008
Characterization of Crystallin Complexes in High Molecular Weight Proteins of Aging Normal and Cataractous Human Lenses by Blue-Native Gel Electrophoresis
Author Affiliations & Notes
  • K. Srivastava
    Department of Vision Sciences, University of Alabama at Birmingham, Birmingham, Alabama
  • J. M. Chaves
    Department of Vision Sciences, University of Alabama at Birmingham, Birmingham, Alabama
  • O. P. Srivastava
    Department of Vision Sciences, University of Alabama at Birmingham, Birmingham, Alabama
  • Footnotes
    Commercial Relationships  K. Srivastava, None; J.M. Chaves, None; O.P. Srivastava, None.
  • Footnotes
    Support  NIH-EY06400
Investigative Ophthalmology & Visual Science May 2008, Vol.49, 4101. doi:https://doi.org/
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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)

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Abstract

Purpose: : To examine non-covalent crystallin complexes in water soluble-high molecular weight (WS-HMW) proteins of normal and cataractous human lenses.

Methods: : 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.

Results: : 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.

Conclusions: : 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.

Keywords: crystallins • cataract • aging 
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