ИСТИНА

Anionic tobacco peroxidase (TOP) isolated earlier in our laboratory exhibits a number of in-teresting properties. TOP has a better storage and operational stability than horseradish peroxidase isoenzyme C (HRP C) commonly used for bioanalytical purposes, and is more active than HRP C to-wards a number of peroxidase substrates. In particular, TOP is extremely active towards luminol in the absence of chemiluminescence enhancers. Hence, TOP can be used for development of chemilumines-cence enzyme immunoassays of superb sensitivity. In addition, TOP molecular size even less than that of HRP C and thus, permits a higher number of enzyme molecules to be conjugated to an antibody molecule particularly in comparison to alkaline phosphatase. Fungal (class II) and plant (class III) peroxidases, including TOP, have two conserved sites of Ca2+ binding, one in each of the domains sandwiching the heme. It is known that calcium ions are im-portant structural elements responsible for the stability and catalytic properties of peroxidases. In par-ticular, calcium removal resulted in a 40-50 % decrease of activity of HRP C and peanut peroxidase. Furthermore, calcium-depleted HRP С is much less stable upon thermal inactivation. It was also dem-onstrated that peroxidase properties can be modulated by exogenous Ca2+ concentration. For example, addition of CaCl2 to the enzyme mixture resulted in an increase of thermal stability of recombinant Phlebia radiata manganese peroxidase. Similar effect of Ca2+ addition was described for HRP C un-folding induced by guanidine chloride. The oxidation of veratryl alcohol by soybean peroxidase was about 2.5 times more effective in the presence of 200 mM CaCl2. In contrast, addition of CaCl2 to HRP A1 resulted in a dramatically decrease of catalytic activity. Modulation of TOP properties by CaCl2 was also previously demonstrated. For instance, it was shown that enzymatic activity of native TOP towards veratryl alcohol is strongly dependent on the presence of Ca2+ [1]. Addition of calcium ions at low pH had protective effect on the inactivation of the enzyme [1]. At the same time, a strong suppressing effect of CaCl2 on the rate constant for direct and even mediated electron transfer catalyzed by native TOP was observed [2]. In the present study the effect of Ca2+ concentration on the refolding yield, thermal stability, and activity of recombinant TOP (rTOP) was investigated. It was shown that at pH values above 4 the addition of CaCl2 reduces the activity of rTOP towards different aromatic substrates, such as phenol, ABTS, and guaiacol. In the presence of Ca2+ TOP catalytic activity in chemiluminescent reaction of luminol oxidation is likewise reduced. Refolding yield of rTOP yield also decreases with increasing CaCl2 concentration. Simultaneously, at low CaCl2 concentrations the enzyme is less susceptible to thermal denaturation, i.e. Ca2+ exerts a protective function. The obtained data allow us to suggest Ca2+ binding to a low-affinity site that leads to conformational changes and altered activity and stability of rTOP. This work was supported by the Russian Foundation for Basic Research (project no. 14-04-32011-mol-a).