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J. Keenan, D. F. Orr, B. K. Pierscionek; Crystallin Distribution Patterns in Porcine Eye Lenses. Invest. Ophthalmol. Vis. Sci. 2008;49(13):4092.
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The unique growth mode of the lens renders it an ideal tissue for studying developmental and ageing changes. This study has investigated protein distribution patterns in porcine lenses using fractionation that follows the lenticular growth mode.
Five month old porcine eye lenses (20), obtained from a local abattoir and used within 6 hours of death were fractionated into 8-10 concentric fractions by controlled dissolution in phosphate buffer in a manner previously applied to human, bovine and fish lenses1. Water-soluble proteins in all layers were separated into HMW, MMW and LMW fractions by size-exclusion HPLC and constituents of each class revealed by SDS gel electrophoresis. Water-insoluble proteins were also analysed by SDS gel electrophoresis. Protein proportions were calculated using integration and Bradford assay. Size-exclusion fractions were further separated by reverse-phase HPLC and the molecular masses of each peak determined by MALDI-TOF mass spectrometry.
Size-exclusion HPLC showed a series of peaks that changed in proportion across lens layers. SDS gel electrophoresis of peak fractions revealed bands characteristic of α-, β- and γ-crystallins for which accurate molecular masses were found by MALDI-TOF mass spectrometry. Protein proportions indicate the main crystallins present in all lens layers are β-crystallins. In outer layers there is a higher proportion of the βH-crystallin peak whereas in older layers the βL-crystallin peak dominates. Intermediate layers show gradual changes between these patterns. The proportion of α-crystallin in the soluble protein are highest in outer layers at around 20% and decreases towards the lens centre to about half this value. In contrast the proportion of γ-crystallin are highest in inner lens layers at around 30%. The proportion of water-insoluble protein, found to consist mainly of α-crystallin increased slightly in inner lens layers to a maximum of around 20%.
Porcine lenses are similar to lenses of other ungulates with lower levels of α-crystallin found in the soluble protein of deeper lens layers. The increase of γ-crystallin towards the lens centre corresponds to a harder lens consistency and supports previous findings of a high refractive index in the porcine lens centre2 .1. Pierscionek B, Augusteyn RC (1988) Current Eye Research 7(1): 11-232. Pierscionek BK, Belaidi A, Bruun HH (2005) Eye 19: 375-81
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