Peptide bond hydrolysis of bovine serum albumin (BSA) by chymotrypsin and trypsin was investigated by employing time-resolved fluorescence spectroscopy. As a fluorescent cross-linking reagent, N-(1-pyrenyl) maleimide (PM) was attached to BSA, through all free amine groups of arginine, lysine, and/or single free thiol (Cys34). Time-resolved fluorescence spectroscopy was used to monitor fluorescence decays analyzed by exponential series method to obtain the changes in lifetime distributions. After the exposure of synthesized protein substrate PM-BSA to chymotrypsin and trypsin, it is observed that each protease produced a distinct change in the lifetime distribution profile, which was attributed to distinct chemical environments created by short peptide fragments in each hydrolysate. The persistence of excimer emission at longer lifetime regions for chymotrypsin, as opposed to trypsin, suggested the presence of small-scale hydrophobic clusters that might prevent some excimers from being completely quenched. It is most likely that the formation of these clusters is due to hydrophobic end groups of peptide fragments in chymotrypsin hydrolysate. A similar hydrophobic shield was not suggested for trypsin hydrolysis, as the end groups of peptide fragments would be either arginine or lysine. Overall, in case the target protein's 3D structure is known, the structural analysis of possible excimer formation presented here can be used as a tool to explain the differences in activity between two proteases, i.e. the peak's intensity and location in the profile. Furthermore, this structural evaluation might be helpful in obtaining the optimum experimental conditions in order to generate the highest amount of PM-BSA complexes.
Heterocyclic compounds containing atoms of the elements such as nitrogen, sulfur and oxygen as ring members are commonly used in various fields of industry as analytical reagents, ligands, dyestuffs, pharmaceutical substances and bioindicators.In this study 5-(Quinoline-2-ylidene)-1,3-diethyl-2-thiobarbituric acid have been synthesized via Knoevenagel condensation reaction , the product solutions were prepared 10 -3 -10 -5 M in CHCI 3 , THF, MeOH, DMF and DMSO. UV/VIS spectra recorded and then compared at each other. Absorbance of the solutions were measured at 200 -300 nm. The maximum absorbance value increased and a new absorption appeared at 400 -500 nm. The samples were excited at 337 nm in order to measure fluorescence. The maximum emission was observed in MeOH at 398 nm. These values increased by time; therefore, the samples were not stable in solution state.
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