Fluorescence correlation spectroscopy as a tool to investigate the directionality of proteolysis.

Enzymatic proteolysis or protein digestion is the fragmentation of protein into smaller peptide units under the action of peptidase enzymes. In this contribution, the directionality of proteolysis has been studied using fluorescence correlation spectroscopy (FCS), taking human serum albumin (HSA) as the model protein and papain, chymotrypsin and trypsin as the model enzymes. Domain-I of HSA has been tagged with tetramethylrhodamine-5-maleimide (TMR) and domain-III with p-nitrophenylcoumarin ester (NPCE) separately and subjected to proteolysis. Following the change in hydrodynamic radius, as monitored by FCS, it has been confirmed that under similar experimental conditions the order of efficiency of digestion is papain > trypsin > chymotrypsin. More interestingly, a faster decrease of hydrodynamic radius was observed when the fluorescence from domain-I was monitored in FCS, compared to that of domain-III. This observation clearly indicates that all these enzymes prefer to start cleaving HSA from domain-I. We assign this preference to the hydrophilic natures of the enzyme active site and domain-I surface. The dependence of the proteolysis on temperature and enzyme concentration has also been studied for papain using the same approach. Reverse-phase HPLC results are found to be in line with the FCS results and validates the applicability of our proposed method.