Purpose: To measure the protein distribution patterns in single young porcine lenses. Methods: Twenty fresh porcine lenses from 5 to 6 months old animals were fractionated into 8-10 concentric fractions by controlled dissolution in phosphate buffer. Proportions of soluble and insoluble protein were determined by Bradford assay. Water-soluble proteins in all layers were separated into HMW, MMW, and LMW fractions by size-exclusion HPLC and constituents of each class further characterized by SDS gel electrophoresis, as were the water-insoluble proteins. Size-exclusion fractions were further separated by reverse-phase HPLC and the molecular masses of each peak determined by MALDI-TOF mass spectrometry. Results: The major soluble proteins in the porcine lens are beta-crystallins. They comprise around 45% of the total protein in the outer lens decreasing gradually to 35% in the central region. Soluble alpha-crystallins vary from 35% to 22% from outer to inner lens. The proportion of soluble gamma-crystallin levels, substantially lower than that of the other protein classes, increases gradually with progression into the lens center. Insoluble protein levels also increase from outer to inner lens layers. Conclusions: In the young porcine lens, there is relative constancy in the levels of all three crystallin classes in the outer lens with alpha- and beta-crystallins representing the predominant protein classes. The increase in gamma-crystallin in the inner lens may contribute to the refractive index gradient.
|Publication status||Published - Jul 2008|