Uncertainty analysis of trypsin inhibitor activity determination using microtiter plate method

Abstract

Measurement of proteinase inhibitor activity of legume seeds is widely used since some of these exert toxic and antinutritional effects. Standard method assay for trypsin inhibitor activity (TIA) determination is based on the hydrolysis of benzoyl-DL-arginine-p-nitroanilide (BAPA) by trypsin and includes spectrophotometric measurement of the reaction products. Assaying of trypsin inhibitor activity is usualy performed using reaction mixture volumes of 4-10 ml, however, it was shown that microtitration method can also be used for TIA determination. Although the results obtained by microtitration method were consistent with those obtained by French Association for Normalization reference method (AFNOR), it was noted that any experimental error in absorbance values was increasingly magnified as the sample size was diminshed. The aim of this study was to analyse measurement uncertanty of TIA determination using microtiter plate method. Assaying procedure was conducted at 37 °C on sample (soybean variety Vojvoñanka), positive and negative control, which were set in four replicats in each row of the microtiter plate, with preincubation time of 10 minutes and incubation time of 30 minutes. The trypsin inhibitor activity was expresed in number of trypsin units inhibited (TUI) per miligram of sample, taking into account the fact that one trypsin unit is defined as an increase of 0.01 absorbance units at 405 nm. Large number of experiments was conducted and uncertainty analysis was obtained using uncertainty budget. Values of calculated trypsin inhibitor activity were approximately 70 TUI/mg and estimated expanded measurement uncertainty (with coverage factor k=2) was aproxitemately 15%.