| Description |
Organic chemists have developed an interest in designing and producing semisynthetic enzymes. In conjunction with our work in this area on Concanavalin A modification, we need to locate and determine which amino acids have been modified. The first efforts in locating specifically functionalzied amino acids on Con A are discussed. The paper concerns the work done on unmodified Con A to obtain reproducible and optimal conditions. The importance of separating the intact protein from the natural fragments, Al and A2, is covered. The best separation of intact Con A appears to be by TRIS-glucose gradient affinity chromatography. Also, less decomposition seems to occur in dry-stored Con A. Cyanogen bromide cleavage of Con A has yet to be fully optimized on intact protein. Immediate lyophilization or dialysis against water following the CNBr reaction or 5% gel filtration appear to be the less destructive to the protein than storage frozen at 4°C. Finally, a new RP-HPLC method for Con A and its fragment analysis have been developed. Two methods of the RP-HPLC analysis, varying a gradient elution of 0.1% TFA in water and 0.1% TFA in CH3CN, I provide relatively sensitive ways of determining Con A purity and analyzing for fragments. |
