SÜNE ZARARININ TESPİTİ İÇİN FLOROMETRİK YÖNTEM GELİŞTİRİLMESİ
Özet
Sunn Pest (Eurygaster spp.) is one of the most common wheat bugs in our country, causing excessive losses of crop yield and decrease in technological properties of wheats. During the feeding of Sunn Pest from wheat grain, bug salivates into the grain which contains digestive secretions of bug. Especially the proteolytic enzymes cause the degradation of the gluten proteins and decreasing in bread making quality. Therefore, detection of bug damage in wheat flour samples has critical importance. The methods widely used in the detection of bug damage are time consuming and require specialists. For this reason, rapid, easy and in-situ detection of bug damage in wheats has vital importance.
The aim of this study is the development of peptide substrate for sunn pest proteases using the repeated sequences of high molecular weight glutenin subunits which enzymes cleavage and analyzing the substrate in a fluorometric assay system. For this purpose, firstly sunn pest protease was extracted from flour samples and extraction conditions were determined for the highest protease activity. Then, sunn pest protease was partially purified to examine of peptide-protease interactions more deeply. Afterwards, optimum conditions for peptide-sunn protease interaction were determined. The 15-amino acid long PGQGQQGYYPTSPQQ peptide sequence, which is a highly affected by proteases, was chosen as the potential substrate. The peptide, which sunn pest proteases act on, was labeled with a FRET flourophore pair.
For the quantitative determination of the sunn pest protease, the fluorescent peptide substrate and the extract solution obtained from bug damaged flour were incubated at optimum pH and temperature conditions. The increase in fluorescence intensity of the substrate was measured by fluorescence spectrophotometer. Enzyme activity values were calculated from the increase in fluorescence intensity per minute. Finally, it was found that the reaction rates were increased in accordance with varied protease concentration in the extract solution.
The determination the performance parameters of the developed analysis method, the enzyme activity values were calculated by extracts obtained from flour samples with different Zeleny / modified Zeleny difference values. Afterwards, the enzyme activity values were plotted against the Zeleny difference values to obtain the calibration curve. It was found that the results of the developed fluorometric analyze method were linear between the range of -16 to 2 mL Zeleny difference value and the sensitivity in this range was 0.14 mL. The lower detection limit of the analysis method was -2.71 mL Zeleny difference value. In addition, a different flour sample was analyzed by fluorometric method in order to determine the accuracy of the developed analysis method. The Zeleny difference value was determined by using the calibration graph with the reaction rate obtained from the flour sample and compared with the experimentally obtained value of the flour sample. When comparing these two Zeleny difference values, the error rate was found to be 3.1%.
As a result of the study, analysis time for the developed assay was determined as 80 minutes including sample preparation as 70 minutes and fluorescence measurement as 10 minutes. The results show that, the selected fluorogenic peptide sequence is an important substrate for the detection of sunn pest protease activity. A rapid and easy method of analysis has been developed for the detection of bug damage with this identified peptide substrate.
