Quantitative Determination of Trypsin Inhibitory Activity in Complex Matrices

Abstract

A quantitative assay using azocasein was developed to measure trypsin inhibitory activity in emulsions and other complex systems that are refractory to analysis. The method was tested for reproducibility on pure protein solutions as well as protein-containing material rich in fats and sugars, with special attention to emulsions. In the clean situation, the overall relative standard deviation was less than 6% while for the more complex systems it was less than 16%. The procedure proved robust against deliberate variations of temperature, incubation time and substrate concentration.

A Kunitz trypsin inhibitor from chickpea (<i>Cicer arietinum</i> L.) that exerts an antimicrobial effect on Fusarium oxysporum f.sp. ciceris

“…The method we adopted here does not require ampholytes, so it is a cheaper and more convenient method. In the present study, a comparison on the trypsin inhibitor activity was carried out using different natural and synthetic substrates where, the TIA results were in conformity to Spelbrink et al [16]. Earlier reports also suggest the use of both natural and synthetic substrates in determination of antitryptic activity as natural substrates are more difficult to displace from the active site to form the enzyme inhibitor complex while synthetic substrate are accurate in determining the inhibitor content of the materials [17] thereby providing a value with physiological relevance [16].…”

Effects of Electrospun Potato Protein–Maltodextrin Mixtures and Thermal Glycation on Trypsin Inhibitor Activity

“…The activity of an inhibitor of an enzyme is quantified by its inhibitory activity (IA). IA, expressed as the amount of trypsin inactivated by a certain quantity of sample, was calculated according to Equation (1) [ 38 ]. The amount of potato protein in the well of the microtiter plate of the dilutions from each sample was plotted against the absorption at 450 nm.…”

“…The amount of potato protein in the well of the microtiter plate of the dilutions from each sample was plotted against the absorption at 450 nm. A premise for the calculation of IA from the slope of the resulting line is that the decrease in absorbance is linear with the amount of trypsin ( Figure 2 A) [ 38 ]. This method was confirmed by a positive control, without potato protein ( Figure 2 B).…”

“…The assay for determining the trypsin inhibitor activity was performed according to Spelbrink et al [ 38 ]. In brief, 30 g/L of azocasein stock solution was always freshly prepared on the day of analysis.…”

“…The amount of trypsin that was inactivated by a certain amount of the sample is called the inhibition activity (IA). Its calculation was derived from Spelbrink et al [ 38 ], and is shown in Equation (1). …”