A New Method For The Optical Analysis Of Trypsin Based On Cytochrome C Detection

Proteases, also known as proteolytic enzymes, are a unique class of enzymes playing important roles in a variety of physiological and pathological processes, such as protein digestion, blood clotting and apoptosis. As a unique protease, trypsin is produced by the pancreas and belongs to a kind of serine protease. It is specific for cleaving peptides mainly at the C-terminal side of arginine or lysine residues and plays a critical role in regulating pancreatic exocrine function. The level of trypsin in biological fluids can serve as a reliable and specific diagnostic biomarker for pancreatic function and its pathological changes. Therefore, the development of simple, sensitive, and convenient methods for the detection of trypsin is important for the diagnosis and therapeutics of these diseases, and for screening of protease inhibitors.Cytochrome c ?cyt c? is a well-known heme-containing electron transfer protein that plays significant roles in the mitochondrial respiratory chain. Cyt c can be used as trypsin's natrual substrate. When trypsin is present, cyt c will be hydrolyzed and releases heme-peptide fragment into solution, which has high catalytic role on the reaction between H₂O₂ and other substances.In this thesis, due to the importance of trypsin detection and its inhibitor screening, we develop two analytic methods for trypsin detection and its inhibitor screening.Part one. A sensitive and label-free trypsin colorimetric sensor was developed by employing cytochrome c ?cyt c? as a natural enzyme substrate and 3,3',5, 5'-tetramethylbenzidine ?TMB? as a chromogenic reagent.It was found that cyt c hardly catalyzes H₂O₂-mediated TMB oxidation to produce a blue solution. However, in the presence of trypsin, cyt c was hydrolyzed and released heme-peptide fragment. It would exhibit an intensive catalytic role on H₂O₂-mediated TMB oxidization and the solution changed into blue quickly. Based on the above experimental phenomenon, we develop a colorimetric method for trypsin detection. With the aid of a spectrophotometer, the absorbance at 652 nm was proportional to the concentration of trypsin in the range from 5 to 2000 ng/mL with a detection limit of 4.5 ng/mL. The detection process allows visually perceiving as low as 50.0 ng/mL trypsin with the naked eyes. The procedure has been successfully applied to the determination of trypsin in human urines and for inhibitor screening, demonstrating its potential application in clinic diagnosis and drug development.Part two. A sensitive and label-free trypsin fluorometric sensor was developed by employing cytochrome c ?cyt c? as a natural enzyme substrate and thiamine as a fluorogenic substrate.Thiamine has no fluorescence in reduction state. But, the oxidative products of thiamine have a strong fluorescence at 432 nm with excitation at 373 nm. In the presence of trypsin, cyt c was hydrolyzed and the released heme-peptide fragment would exhibit an intensive catalytic role on H₂O₂-mediated thiamine oxidization. The oxidative products of thiamine have a strong fluorescence at 432 nm. Based on the above experimental phenomenon, we develop a fluorometric method for trypsin detection. The flurescence at 432 nm was proportional to the concentration of trypsin in the range from 0.5 to 20.0 ?g/mL with a detection limit of 125.0 ng/mL. The procedure has been successfully applied to many inhibitors screening.In summary, we have successfully developed two cost-efficient methods for trypsin detection. The methods were simple, label-free, sensitive and could be used to the determination of trypsin in biological samples ?human urine? and for inhibitor screening.