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KMID : 1007519960050020099
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Food Science and Biotechnology
1996 Volume.5 No. 2 p.99 ~ p.103
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Probing and Enhancing Dimer Formation of Bovine β - Lactoglobulin A
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Lee, Sam Pin
Ha, Young Duck/Batt, Carl A
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Abstract
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The ability of bovine β-lactoglobulin A (BLG A) to form dimers has been probed by the selective substitution of a histidine residue which resides at the dimer interface. Histidine 146 residue of BLG A was modified with arginine or cysteine (H146R, H146C) by site-directed mutagenesis. Recombinant BLG (rBLG) protein was overproduced in E. coli as an inclusion body and purified using denaturation, renaturation, refolding and gel filtration. Both BLG A and H146C rBLG protein showed the similar tryptic digestion patterns indicating that H146C rBLG refolded into a native-like conformation. The conformation of rBLG proteins were not significantly different from BLG A protein by a comparison of the intrinsic fluorescence spectrum. An additional thiol group of H146C and H 146R rBLG protein was confirmed by DTNB method and was exclusively exposed with increasing pH. The dimer formation of both BLG A and rBLG proteins via chemical or air oxidation was determined on non-reducing SDS-PAGE. H146R rBLG protein perturbs the formation of dimers. In contrast, the substitution of a cysteine residue for a histidine 146 residue allows the oxidation-driven formation of covalent dimers.
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