Investigation of changes in proteolytic activity of polyacrylic acid-trypsin conjugated and free trypsin using fluorescence lifetime distribution

Polymer conjugates of proteins are synthesized to increase their stability and expand their application area. A range of functional groups, which can be used in the covalent conjugation of enzymes, including amino, hydroxyl, carboxyl and phenolic groups. There are many materials, including synthetic organic polymers, biopolymers, hydrogels, inorganic supports, and smart polymers, to be used to immobilize enzymes, and good activity retention, and enhanced thermo-stability are often observed. The physical structure and chemical composition of support can also influence the microenvironment of the immobilized species and consequently their biological properties. Fluorescence lifetime distributions may be useful tool to monitor the changes of enzyme activity by the conjugation with polymers. Fluorescent groups embedded in different regions of proteins have different chemical environments and exhibit varying fluorescence lifetime profiles specific to the protein they are involved in. The differences of the changes of the distributions by free and conjugated protease activity may provide useful information about the hydrolysis process of the protein substrate and the activity and stability of "Polymer-Protase" conjugate. Fluorescence lifetime distribution profiles obtained from protein substrates modified with excimers that are extremely sensitive to protein denaturation can be used to more precisely determine the proteolytic differences between the free natural states of proteases and polymer conjugates of proteases. In this study; the differences between the proteolytic activities of free trypsin enzyme and polyacrylic acid conjugated typsin (PAA-Tripsin) were examined with the fluorescence lifetime distribution changes of Bovine Serum Albumin (BSA), which was gained the ability to exhibit excimer emission around the maximum 464 nm, by modifying with N-(1-Pyrenyl) maleimide (PM) at basic pH (pH 9). Because the conjugation reaction caused decreasing in enzyme activity, a significant change in excimer emission could not be observed in the resultant fluorescence spectrum, however, large changes in fluorescence lifetime distributions were obtained. These results showed that the analysis of fluorescence lifetime distribution can be used as an extremely sensitive method for determination and distinguishing of proteases from each other, according to the differences of their hydrolytic activities.