Electrophoretic mobility of individual molecules of alkaline phosphatase

Abstract

The electrophoretic mobilities and catalytic rates of individual molecules of bovine intestinal alkaline phosphatase were determined in CHES and borate buffers of identical pH using a capillary electrophoresis based method. Both properties were found to be heterogeneous. In the presence of CHES, the mobility and rate were found to be −1.9 ± 0.2 × 10−9 m2 V−1 s−1 and 9.8 ± 7.4 × 104 min−1 ( N = 38), respectively. In the presence of borate, the mobility and rate were found to be −6.9 ± 0.5 × 10−9 m2 V−1 s−1 and 2.0 ± 1.3 × 104 min−1 ( N = 41), respectively. The means and variances for both properties were found to differ significantly between the two buffers. The difference in average mobility was attributed to an increase in negative charge caused by borate complexing with the carbohydrate moieties attached to the enzyme. The difference in variance was attributed to heterogeneous complexation with borate due to heterogeneity in the glycosylation. The differences in mean values for the catalytic rate were attributed to the inhibitory effect of borate and the difference in variance may suggest that the KI of this binding may also be heterogeneous.

Effect of Chemical Modification on the Distribution of Electrophoretic Mobilities of Individual Molecules of E. coli beta-Galactosidase

“…The Winzor model [2] used suggested that the variance in mobility of the unmodified enzyme could be attributed to relatively small difference in size and effective charge. This is consistent with other studies [13][14][15]. The model gave plausible results for the prediction of changes in size, shape, and charge required to accompany the changes in observed mobilities of -galactosidase caused by labelling.…”

“…Previous studies have suggested that the observed heterogeneity in electrophoretic mobility of individual enzyme molecules may result from relatively small differences in shape, size, and charge [13][14][15]. In order to assess this, -galactosidase was subjected to relatively small changes in its size, shape and charge through chemical modification.…”

“…The positions of the product peaks in the resultant electropherogram were used to calculate the distance travelled by the enzyme molecules during the 360 s separation period. From this, their electrophoretic mobilities were calculated [15].…”