Exploring the activity and specificity of gold nanoparticle-bound trypsin by capillary electrophoresis with laser-induced fluorescence detection

This paper describes the use of micellar electrokinetic chromatography (MEKC) and capillary zone electrophoresis (CZE) in conjunction with laser-induced fluorescence (LIF) detection for investigating the specificity of biocatalysis by trypsin when it is conjugated to gold nanoparticles (GNPs). In the presence of sodium dodecyl sulfate (SDS), adsorption of the tryptic fragments on GNP-trypsin and on the capillary wall is reduced. As a result, the sensitivity and resolution of electropherograms of the tryptic fragments from bovine serum albumin (BSA) is improved. MEKC-LIF measurements show clearly that the specificity of GNP-trypsin differs from that of free trypsin and that the tryptic digest of GNP-BSA is significantly different from the GNP-tryptic digest of BSA. We have used CZE-LIF to observe differences in the biocatalytic activity of trypsin and GNP-trypsin. Changes in the electropherograms provide information of the progress of digestion and indicate that the activity of GNP-trypsin is lower than that of free trypsin. The results of this study suggest that changes in the conformations and steric effects contribute to the loss of activity and changes in specificity of trypsin adsorbed on GNPs.