Bioanalysis Using Porphyrin Derivatives

Enzymes hydrolyzing water-soluble short-chain triacylglycerols to corresponding fatty acids and glycerol are termed esterases (EC 3.1.1.1), and enzymes catalyzing the hydrolysis of carboxylic ester bonds at the lipid–water interface are termed lipases (EC 3.1.1.3). Lipases and esterasesare versatile biocatalysts and find potential application in organic synthesis. The activities of these enzymes usually are estimated using tributyrin and triolein as substrates, and the product of hydrolysis (i.e., the fatty acids released) is quantified. We have developed a colorimetric assay format for the determination of lipase activity, using sodium salt of tetra sulfonatophenyl porphyrin (Na–TSPP) as a chromogenic indicator that can also overcome the problem of the toxic nature of various indicators that are being used.

Protein identification in Polyacrylamide Gel Electrophoresis (PAGE) requires post-electrophoretic steps like fixing, staining and destaining of the gel, which are time consuming and cumbersome. On the other hand our method involves direct visualization of protein bands in PAGE using TSPP as a dye without the need for any post-electrophoretic steps, where separation and recovery of enzymes becomes much easier for further analysis. Activity staining was done to prove that the biochemical activity of the enzymes was preserved after electrophoresis. The use of tetraammonium TSPP a stain to analyse bacterial cells using fluorescent microscopy was also investigated. TSPP was effectively used to analyse two different bacteria; Pseudomonas aeruginosa and Bacillus cereus. The variation in brightness with varying concentrations of TSPP was studied. The patterns of variations for these bacteria were found to be the same, but with consistently higher brightness for Bacillus cereus. This allows for a possible method for distinguishing bacteria.