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M.D. Argirova, B.J. Ortwerth; Immobilized Metal Ion Chromatography as a Tool to Study Native and Modified Lens Proteins . Invest. Ophthalmol. Vis. Sci. 2003;44(13):2347.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Immobilized metal affinity chromatography (IMAC) is gaining widespread popularity as an effective tool for protein separation and purification. However its analytical capacity has not been exploited for studies of lens proteins and their post-translational modifications. We report the chromatographic conditions and the results of application of IMAC for separation of total water-soluble calf lens protein. Methods: The protein was bound to Cu (II) ions on immobilized iminodiacetic acid support and eluted with a linear gradient from 0% to 100% 100 mM imidazole within 50 minutes. The proteins were detected by both UV absorption and fluorescence (ex.290/em.360 nm). Results: The chromatographic profile comprised three major and several minor peaks. The type of eluted proteins was determined on the basis of molecular mass and isoelectric point. The glycation of calf lens protein with glucose, fructose and ascorbic acid led to significant change in the chromatographic profile with a tendency of increasing the protein affinity toward immobilized Cu (II) with the advance of glycation process. The most significant changes were observed for peak 2, while the proteins in peak 1 were less affected by the glycation. Conclusions: The good binding of lens proteins to immobilized Cu (II) ion provides a new method for their fractionation and can be applied to study protein surface structure and metal-catalyzed oxidation reactions in lens proteins.
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