APLIKASI REAKSI TRINDER UNTUK PENGUKURAN AKTIVITAS PEROKSIDASE YANG DIISOLASI DARI KULIT BIJI KEDELAI

• Main Author: Jayasaputra, Darmadi
• Format: Final Project
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/45424
• Institution: Institut Teknologi Bandung

Trinder reaction is based on two sequential enzymatics reaction, oxidation reaction which is produces H2O2 catalyzed by oxidase and reaction between 4-aminoantipyrine, phenol, and H2O2 to form adduct quinonimine (purple-redish colour) catalyzed by peroxidase and can be quantified at ?max = 510 nm. Trinder reaction can be applied to quantify the substrate concentration (phenol and H2O2) or to quantify the activity of the peroxidase. In Indonesia, many food can be processed from soybean, like tofu, tempe, and soymilk, but frequently the seed coat is wasted, whereas soybean seed coat is the source of peroxidase enzyme. The objective of this research was application of Trinder reaction to quantify the activity of soybean seed coat peroxidase. In this research, was determined the optimum substrate concentration (4-AAP and H2O2); effect of phenol addition; optimum pH, temperature, and incubation time. Purification was done by protein precipitation using ammonium sulphate. The amount of protein was determined using Lowry-method. Each the specific activity, KM, and Vmax calculated. The effect of inhibitor hydroxylamine HCl and phenylhydrazine to soybean seedcoat peroxidase activity was determined. The soybean seedcoat peroxidase was applied in quantitative assay of chlorogenic acid. The optimal Trinder reaction using soybean seed coat peroxidase, occurs at 24 mM 4-AAP, 1 mM H2O2, added with 1 mL phenol 40 mM, pH 5, at 25 o C, and the absorbance stable after 5 minutes. Purification by protein precipitation using ammonum sulphate (85%) increase the specific activity from 2.021 to 4.106. The KM/Vmax before and after purification consecutively 0.336/0.517 and 0.213/0.350. Hydroxylamine HCl inhibition results constant KM (0.449±0.012 mM) and decreasing Vmax (0.052; 0.039; dan 0.017), while Phenylhydrazine inhibition results constant Vmax (0,03525±0,00057) and increasing KM (0.055 mM; 0.121 mM; dan 0.285 mM). In quantitative assay of chlorogenic acid, the reaction optimum at 1 mL chlorogenic acid; 0.5 mL soybean seedcoat extract stock solution; 1 mL buffer pH 4, 100 ?L H2O2 1.5 mM; and 2 mL 4-AAP 10 mM (add to 5 mL) at 15 o C, measured the absorbance at ?max = 516 nm after 2 minutes. r-value of liniearity test = 0.856. The optimal Trinder reaction using soybean seed coat peroxidase, occurs at 1 mL soybean seed peroxidase (diluted 25x), 1 mL phospate buffer pH 5, 1 mL phenol 40 mM, 1 mL 4-AAP 24 mM, and 1mL H2O2 1 mM at 25 o C and measured the absorbance at ?max = 520 nm for 5 minutes. The protein precipitation using ammonium sulphate (85%) increases the specific activity up to ±2.032x. Hydroxylamine HCl inhibit soybean seed coat activity with non-competitive inhibition, while phenylhydrazine with competitive inhibition. In quantitative assay of chlorogenic acid, range chlorogenic concentration 0.005-0.02 mg/mL can be used, with linierity test r = 0.856.