Polymerization of Horseradish Peroxidase in the Presence of Inorganic Adsorbents

Abstract

Synthesis of polyglobular enzymes with covalently bound protein globules is an approach to modeling natural protein-protein interactions [1][2][3]. Glycoproteins [2][3] are mainly used in forming polyglobular enzymes, as their carbohydrate fragments can be readily activated and the effects of activation and polymerization can be minimized. Depending on the polymerization conditions (pH and concentrations of protein, oxidant, and cross-linking agent), it is possible to obtain intramolecular, intersubunit, and intermolecular cross-links producing soluble and insoluble polyglobular derivatives of proteins [2]. Apparently, the synthesis of polyglobular proteins and enzymes on a solid-liquid interface rather than in a solvent might decrease the degree of irregular association of monomers in the polymer and simplify the separation of the synthesis products. Methods for production of numerous precipitated and coprecipitated adsorbents are now available [4]. Presumably, it is possible to find sorbents with predominant binding of either monomeric or polymeric synthetic products.Horseradish peroxidase (HRP) is a promising and convenient object for studying specific features of protein polymerization [1]. This enzyme belongs to glycoproteins and is widely used in biotechnology (as an enzymatic label) and organic synthesis [5]. A disadvantage of HRP is that it is readily inactivated with an excess of hydrogen peroxide [6,7]. Peroxidases display maximum catalytic activity at relatively low concentrations of H 2 O 2 in the medium [2,8]. In the case of HRP at [ç 2 é 2 ] 0 > 1-3 mM, the concentration of catalytically inactive complex III increases [7]. HRP does not lose its activity at high concentrations of H 2 O 2 in the medium [2,9], when studied within an immune complex [9] or complexes formed by cross-linking to other HRP molecules [2].The goal of this work was to compare the efficiencies of binding of HRP and its polymers with inorganic adsorbents (precipitated and coprecipitated) and to develop a method for HRP polymerization in their presence. MATERIALS AND METHODSHRP (EC 1.11.1.7) with RZ = 2.9, produced by Biolar (Latvia); basic (LS 5/40) and neutral (L 40/250) aluminum hydroxide (Chemapol, Czech Republic); sodium metaperiodate and a set of reagents for PAGE (Reanal, Hungary); and 4-aminoantipyrine (Serva, Germany) were used in the work. The remaining reagents were from Reakhim (Russia). Hydroxyapatite was produced as described in [10].A PAN-20 (MIFIL, Belarus) ultrafiltration membrane with an exclusion limit of 20 kDa was used for washing and concentrating HRP polymers (HRPp).Precipitated and coprecipitated adsorbents were synthesized using the principles described in [11]. A thermostated reservoir equipped with a mechanical stirrer was filled with the calculated quantities of aqueous solutions of the components (metal chlorides and orthophosphoric acid). The solution was heated to 50°ë , and 1 or 2 M NaOH was added dropwise to the solution with a peristaltic pump, controlling pH in the medium for adsorbent synthesis ...

Polymerization of Horseradish Peroxidase by a Laccase‐Catalyzed Tyrosine Coupling Reaction

“…In an early report, Budnikova and Eryomin showed that the carbohydrate fragments of HRP could be utilized for the polymerization reaction of HRP by using sodium periodate as an oxidizing agent. However, recent reports on the polymerization of HRP utilizing the carbohydrate moiety followed chemical protocols, which required a one‐day reaction and the use of inorganic solvents . Other strategies of protein polymerization, including HRP, are the reversible addition‐fragmentation chain transfer (RAFT) polymerization and atom transfer radical polymerization (ATRP) .…”