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

Escherichia coli β-galactosidase was labelled with 1 mM fluorescein 5-carbamoylmethylthiopropanoic N-hydroxysuccinimidyl ester for 1 and 3 min. The samples were separated by capillary electrophoresis and peak areas compared to standards of label after attachment of BSA in order to determine the concentration of attached label. Enzyme concentration in the samples was determined by single molecule counting. The average number of labels attached to each molecule of enzyme was found to be 3.1 and 4.5. The distribution of single enzyme molecule electrophoretic mobilities for the unlabelled enzyme and that labelled for 1 and 3 minutes were measured using capillary electrophoresis. The average mobilities were found determined to be -1.99x10 − 8 m 2 V − 1 s − 1 ± 1.3x10 − 9 m 2 V − 1 s − 1 (N = 39), -2.16 x10 − 8 m 2 V − 1 s − 1 ± 1.9x10 − 9 m 2 V − 1 s − 1 (N = 46), and − 2.18 x10 − 8 m 2 V − 1 s − 1 ± 2.1x10 − 9 (N = 39) respectively. A protein electrophoresis model was applied and predicted that the differences in average mobilities could be explained through relatively minor changes in overall charge, Stokes radius, and shape. This difference was similar to the range in mobilities observed in the unlabelled protein. This is consistent with the electrophoretic heterogeneity of the unmodified enzyme being caused by relatively small differences in charge, size, and shape of the individual molecules in the population.