Quantification of Functional Actinidin in Whole Kiwifruit Extract Using the Selective Cysteine Proteinase Inhibitor E-64

Actinidin is a cysteine proteinase abundant in kiwifruit. It is of interest to the food industry as a meat tenderizer and an aid to digestion. The selective cysteine proteinase inhibitor E-64 allows the quantification of actinidin in whole extracts of commercial kiwifruit cultivars ‘Hayward’ (green) and ‘Zesy002’ (gold) . The quantity of E-64 required to inhibit actinidin is a direct measure of the amount of actinidin in the sample. Actinidin can be quantified by measuring the inhibition of digestion of the fluorescent protein substrate bodipy-casein or the small-molecule fluorescent substrate Bz-Phe-Arg-aminomethylcoumarin. Use of the protein substrate appears to overestimate the quantity of enzyme by around 15%. The synthesis of the fluorescent protein -casein labelled at a molar ratio of 5:1 (fluor: protein) greatly enhances the signal: noise ratio of substrate when it is digested with actinidin. The high quantity of actinidin in kiwifruit coupled with the selective nature of E-64 allows assay of extracts in a simple kinetic microplate assay. The method is particularly suitable for the comparison of Actinidia from different species since it reports the quantity of enzyme by mass and the relative activity of the enzyme against the chosen substrate. Gold kiwifruit are shown to have around half as much actinidin as green kiwifruit but the three-fold greater specific activity of green kiwifruit against these substrates results in an apparent six-fold greater actinidin activity of the green kiwifruit. This assay may simplify the reporting of actinidin activities which are variously reported as arbitrary fluorescence units, delta-optical density values and band intensities on polyacrylamide gels.