HORSERADISH PEROXIDASE CATALYZED DEGRADATION OF BROMOPHENOL BLUE DYE

In this study, the oxidative destruction of bromophenol blue dye with hydrogen peroxide was carried out at pH 4.0-4.1 in the presence of a commercial horseradish peroxidase, as well as peroxidase isolated directly from horseradish roots (Armoracia rusticana). To determine peroxidase activity, a model reaction of the oxidation of phenol to quinone was used. With a dye concentration of 32.7 μM, the optimal concentration of hydrogen peroxide was 0.04 mM at peroxidase concentration of 1.15 nM. The optimal temperature of the enzymatic reaction was determined: at 23 °С for 10 min 90% of the dye was exposed to destruction. When the temperature rises to 50 °С, the reaction rate decreases, and the degree of destruction is 56% for the same time interval. It was shown that the initial rate of peroxidase oxidation of bromophenol blue follows Michaelis-Menten equation. The kinetic parameters of the enzymatic reaction were determined by linearizing Michaelis-Menten equation in Lineweaver-Burk coordinates. It was found that for the peroxidase oxidation reaction of bromophenol blue Michaelis constant and maximum rate were 42.7 μM and 57.5 μM·min–1, respectively. In this work, а high percentage of dye degradation was also achieved when using peroxidase isolated from horseradish roots. The experiments were conducted at a temperature of 30оС and pH 4.1. With the increase in the volume of the extract from 0.1 to 0.2 ml, the percentage decolorization increases from 75% to 90%. The results demonstrate the high degradation efficiency of bromophenol blue with the participation of the commercial horseradish peroxidase and peroxidase isolated from horseradish roots. Enzymatic oxidative degradation can be considered as an alternative to biodegradation.

Antioxidant Capacity and Cytotoxic Effects of Catechins and Resveratrol Oligomers Produced by Enzymatic Oxidation against T24 Human Urinary Bladder Cancer Cells

In this work the polymerization of catechin, epicatechin, and resveratrol was carried out through a peroxidase oxidation process in order to improve the biological activity of these phenolic compounds. The antioxidant activity of the oligomers was evaluated by their ability to scavenge reactive oxygen species (ROS) and their capacity to chelate metal ions Fe2+ and Cu2+. The antitumor effect of the oligomers was determined by their ability to induce toxicity in the T24 human bladder cancer cell line. By enzymatic peroxidase oxidation, it was possible to produce oligomers of catechin, epicatechin, and resveratrol with antioxidant capacity significantly higher than their preceding monomers. The ROS scavenging capacity of the oligomers was 20 times higher than that of the monomers, while the ability of the oligomers to chelate metal ions increased up to about 1000 times. Our data show the antitumor effect of the oligomers of catechin, epicatechin, and resveratrol in the T24 cell line, which was similar to that observed with cisplatin. Oligomers of catechin, epicatechin, and resveratrol have great potential to be used as therapeutic agents for the treatment of oxidative stress-related diseases and bladder cancer.

Effect of Cloxacillin on Bilirubin-albumin Binding

Cloxacillin is usually given to newborn infants with sepsis in particular for penicillinase-producing staphylococcus aureus. The effect of cloxacillin on bilirubin-albumin binding was investigated in vitro using the peroxidase oxidation method with human albumin (bilirubin 0.255 mM, albumin 0.45 mM, billalb 0.56, pH 7.4, temperature 3 0°C). Sulfisoxazole, a drug which is capable of displacing bilirubin from albumin was used as control. The displacement constant of cloxacillin was 4.86 x 1o5 Nr1, stronger than that of sulfisoxazole (1. 72 x 104 M-1). Both drugs were capable of displacing bilirubin as determined by their maximal displacement factors of 2.21 and 2.29, respectively. Since cloxacillin apparently increases the risk of bilirubin encephalopathy the use of this drug in jaundiced newborns with sepsis, especially in the premature infants, should be reconsidered.

Порушення процесів вільнорадикального окиснення і антиоксидантного захисту в печінці при експериментальному алергічному альвеоліті та їх корекція тіотриазоліном

ПОРУШЕННЯ ПРОЦЕСІВ ВІЛЬНОРАДИКАЛЬНОГО ОКИСНЕННЯ І АНТИОКСИДАНТНОГО ЗАХИСТУ В ПЕЧІНЦІ ПРИ ЕКСПЕРИМЕНТАЛЬНОМУ АЛЕРГІЧНОМУ АЛЬВЕОЛІТІ ТА ЇХ КОРЕКЦІЯ ТІОТРИАЗОЛІНОМ - У роботі показано, що експериментальний алергічний альвеоліт характеризується зростанням вмісту продуктів ліпопероксидаціі (дієнових кон’югатів і малонового діальдегіду) у всі періоди дослідження та активності ферментів супероксиддисмутази (СОД), каталази (КТ) і пероксидази (ПО) на 14 добу експерименту і суттєве зниження показників СОД, КТ і ПО на 34 і 44 доби цього модельного процесу захворювання та корегуючий вплив тіотриазоліну на дані показники.<br />НАРУШЕНИЕ ПРОЦЕССОВ СВОБОДНОРАДИКАЛЬНОГО ОКИСЛЕНИЯ И АНТИОКСИДАНТНОЙ ЗАЩИТЫ В ПЕЧЕНИ ПРИ ЭКСПЕРИМЕНТАЛЬНОМ АЛЛЕРГИЧЕСКОМ АЛЬВЕОЛИТЕ И КОРРЕКЦИЯ ИХТИОТРИАЗОЛИНОМ - В работе показано, что экспериментальный аллергический альвеолит характеризуется возростанием содержания продуктов липопероксидации (диеновых коньюгатов и малонового диальдегида) в печени во все периоды исследования и активности ферментов су- пероксиддисмутазы (СОД), каталазы (КТ) и пероксидазы (ПО), особенно на 14 сутки эксперимента и существенное снижение показателей СОД, КТ и ПО на 34 и 44 сутки этого модельного процесса заболевания и корригирующее влияние тио- триазолина на эти показатели.<br />DISTRUBANCES OF THE PROCESSES OF FREE RADICAL OXIDATION AND ANTIOXIDANT PROTECTION IN LIVER AT EXPERIMENTAL ALLERGIC ALVEOLITIS AND THEIR CORRECTION WITH THIOTRIAZOLIN - The paper shows that experimental allergic alveolitis is characterized by increase of content of lipid peroxidation products (dienic conjugates and malonic dialdehyde) in all periods of study and enzyme activity of superoxide dismutase (SOD), catalase (CT) and peroxidase (PO) on the 14th day of the experiment and significant decrease of SOD, CT and PO on 34th and 44th days of this model process of disease and corrective influence of thiotriazolin on these indicators.<br />Ключові слова: пероксидне окиснення ліпідів, антиоксидантна система, тіотриазолін.<br />Ключевые слова: пероксидное окисление липидов, антиоксидантная система, тиотриазолин.<br />Key words: peroxidase oxidation of iipids, antioxidant system, thiotriazoiin.

Catalytic surface radical in dye-decolorizing peroxidase: a computational, spectroscopic and site-directed mutagenesis study

We demonstrate that an exposed tryptophan is responsible for high-turnover oxidation by DyP, a representative of a new protein superfamily. Long-range electron transfer from surface tryptophan residues forming radicals appears as a general mechanism for peroxidase oxidation of bulky substrates.

Phenothiazines are slowly oxidizable substrates of horseradish peroxidase

Reactions of peroxidase oxidation of triftazine and thioproperazine have been investigated in the presence of horseradish peroxidase using steady state kinetic methods. It has been shown that phenothiazines are slowly oxidizable substrates for horseradish peroxidase. kcat and Km values have been determined in the range of pH from 4.5 to 7.5. The study of co-oxidation of phenothiazines and o-dianisidine (ODN) revealed that in the presence of aminazine and ODN in the reaction medium both substances follow sequential oxidation. ODN oxidation was not observed until full conversion of aminazine. At pH 4.5-5.5 thioproperazine bound to the enzyme-substrate complex and caused a nticompetitive inhibition of peroxidase. At pH>5.5 sequential substrate oxidation with preferential thioproperazine conversion occurred. In the range of pH from 4.5 to 7.5 triftazine did not influence ODN oxidation.