İbRahim Ethem Özyiğit

2-thioxanthone thioacetic acid (TXSCH₂COOH, T), which has a fluorometric character, was used for new fluorometric system upon Fe(II) analysis in biological samples as the main target. T-BSA binary complex was firstly consisted with non-covalent interactions between T and BSA at the equilibrium concentration as 1.77 × 10^-4.M. T-BSA binary complex emission was increased at the ratio of 24.40% due to stabilization property of BSA (pH:7), compared with T emission intensity. Fluorescence emission spectroscopy was used for the all measurements because of an economic, a sensitive and a practical method compared with other spectroscopic analysis. T-BSA-Fe(II) triple complex was also obtained by adding Fe(II) ion to T-BSA binary complex solution. Its characterization was performed to be investigated with optimum excitation wavelength, buffer concentration, pH and temperature as 297 nm, 10^-3 M Tris HCl (10^-2M NaCI), pH:7.2 at 25 °C, respectively. The results of Fe(II) analysis in serum showed a certain response in fluorometric T-BSA-Fe(II) triple complex measurement system as 50.42 ± 5.8 µg/dL. The analyses of our fluorometric triple complex system were compared with the reference electrochemiluminescence method and similar results were obtained. Fluorometric measurements of T-BSA-Fe(II) triple complex, its characterization and Fe(II) analysis in this system have not been investigated in literature gives originality to our study.

Electroanalytic, photometric or fluorometric methods may provide information about the presence of proteolysis in a related sample, but they cannot accurately identify which protease belongs to the proteolytic activity. In other words, they cannot distinguish the proteases according to the differences in their activities. Previous studies on the chymotrypsin and trypsin have shown that the activities of proteases can be distinguished from each other by fluorescence lifetime distributions. In this study, it is aimed to show the sensitivity of the distributional model on the proteolytic activities of the two proteases. For this purpose, the proteolytic activities have been reduced by making covalent conjugation with polyacrylic acid (PAAc) and the effects of the low activities were examined on Bovine Serum Albumin (BSA) which excimer emission character was incorporated into its structure by modification with N-(1-pyrenyl)maleimide. The time-resolved spectrofluorometer was used to collect fluorescence decay data at lambda(excimer) = 464 nm, which were analyzed by using Exponential Series Method (ESM) to obtain the changes of lifetime distributions. The results showed a significant decline in the activities. Despite the very low activities, the changes of fluorescence lifetime distributions exhibited remarkable specific differences that proved the sensitivity of ESM analysis.

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.