Influence of Macromolecular Crowding on the Oxidation of ABTS by Hydrogen Peroxide Catalyzed by HRP

Abstract

The interior of the living cell is highly concentrated and structured with molecules having different shapes and sizes. However, almost all experimental biochemical data have been obtained working in dilute solutions that do not reflect in vivo conditions. In this paper, we study in vitro the effect of macromolecular crowding on the reaction rates of the oxidation of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) by hydrogen peroxide (H 2 O 2 ) catalyzed by Horseradish Peroxidase (HRP), by adding Dextrans of various molecular weights to the reaction solutions as crowding agents. The results indicate that the volume occupied by the crowding agent, regardless its size, plays an important role in the rate of this reaction. Both Michaelis-Menten parameters, ‫ݒ‬ ௫ and ‫ܭ‬ , decrease when the Dextran concentration in the sample increases, which might be due to a crowding-induced effect in the catalytic constant, ݇ ௧ , of this enzymatic reaction. Thus, our results suggest that there is an activation control of the enzymatic reaction in this particular system. In our opinion, this work could facilitate the understanding of biomolecules behavior in vivo and be useful for biotechnology in vitro applications, since HRP is widely used in the development of biosensors.

Unusual Aspects of Charge Regulation in Flexible Weak Polyelectrolytes

“…Finally, because of its impact in the diffusion and conformational properties of the biomacromolecules, MCr can affect the kinetic properties of enzymes [ 239 , 240 , 248 , 265 , 266 , 267 , 268 , 269 , 270 ]. In their pioneering work, Ogston and Laurent predicted an increase in the chemical potential of the protein when a crowding agent was added to the solution [ 271 , 272 ].…”

DNA Processor Modules Enabled Pattern Recognition of Extracellular Vesicles for Facile Cancer Diagnosis†

“…To achieve a different visible output for SKBR3 EVs and LNCaP EVs, two kinds of enzyme substrates, 2,2’‐azino‐bis (3‐ethylbenzothiazoline‐6‐sulphonic acid) (ABTS) and p ‐nitrophenyl phosphate (pNPP), were chosen. HRP initiated the oxidation of colorless ABTS into green‐colored oxidized ABTS (ABTS ·+ ) in the presence of H 2 O 2 , [ 45 ] while AP could catalyze the hydrolysis of colorless substrate pNPP to a yellow‐colored product p ‐nitrophenol (pNP). [ 46 ] Compared to normal EVs, color changes from SKBR3 and LNCaP EVs were obvious (from colorless to dark green and yellow).…”

Quantum-chemical study of the electronic structure - antioxidant action relationship of some chemical compounds

“…Este de remarcat faptul că datele privind detaliile posibilului mecanism de acțiune antioxidantă a compușilor fenolici care captează radicalul cationic stabil ABTS •+ sunt practic absente în sursele cunoscute din literatură. Evident, situația dată este legată în principal de diferite dificultăți experimentale care însoțesc testul ABTS •+ [14,15].…”