| Popis: |
The fluorescence resulting from the reaction of a protein with fluorescamine is an indication of the number of free amino groups in that protein, consequently glycated proteins which have fewer free amino groups will show less fluorescence. With the appropriate standards, the difference in fluorescence of glycated and non-glycated proteins can be translated into the number of glycated sites in a protein. Glucose (0·22 m final concentration) was incubated with bovine serum albumin (BSA) (0·1 mg/ml) in a total volume of 50 ml of 0·1 m phosphate buffer pH 7·2 at 37°C for a period of 35 days. In order to assess the effect of metal catalyzed free radical damage to the protein, the incubation was also carried out in the presence of a metal chelating agent EGTA (0·38 mg/ml). As expected, the fluorescence of both mixtures decreased with time, as more lysyl groups became glycated. Calculations based on the standard curve of t -BOC-lysine showed that approximately 18 mol of glucose were incorporated per mol of BSA during the incubation in the presence of EGTA and 23 mol in the absence of EGTA. This difference is explained by the metal catalyzed free radical fragmentation of BSA which exposes more N -terminal amino groups for reaction with glucose. |
