The use of dimethylsulfoxide as a solvent in enzyme inhibition studies: the case of aldose reductase

Abstract

Aldose reductase (AR) is an enzyme devoted to cell detoxification and at the same time is strongly involved in the aetiology of secondary diabetic complications and the amplification of inflammatory phenomena. AR is subjected to intense inhibition studies and dimethyl sulfoxide (DMSO) is often present in the assay mixture to keep the inhibitors in solution. DMSO was revealed to act as a weak but well detectable AR differential inhibitor, acting as a competitive inhibitor of the L-idose reduction, as a mixed type of non-competitive inhibitor of HNE reduction and being inactive towards 3-glutathionyl-4-hydroxynonanal transformation. A kinetic model of DMSO action with respect to differently acting inhibitors was analysed. Three AR inhibitors, namely the flavonoids neohesperidin dihydrochalcone, rutin and phloretin, were used to evaluate the effects of DMSO on the inhibition studies on the reduction of L-idose and HNE.

Keywords: Dimethyl sulfoxide; aldose reductase; aldose reductase differential inhibitors.

Figure 1.

Effect of DMSO on aldose reductase activity. Inhibition data of DMSO on AR are reported in double reciprocal plots. Panel A: AR activity was determined using different L-idose concentrations, both in the absence (circle) and in the presence of 40 mM (triangle), 100 mM (square), and 200 mM (diamond) DMSO. Panel B: AR activity was determined using different HNE concentrations, both in the absence (circle) and in the presence of 100 mM (triangle), 200 mM (square), and 300 mM (diamond) DMSO.

Figure 2.

Effect of DMSO on the inhibition of AR by NHDC. Panel A and B: AR activity was determined using different L-idose concentrations in the presence of 0 (circle), 50 (triangle), 100 (square), or 150 (diamond) µM NHDC, both in the absence (Panel A) and in the presence of 100 mM DMSO (Panel B). AR activity was determined using different HNE concentrations, both in the presence of 0 (circle), 50 (triangle), and 100 (square) µM NHDC both in the absence (Panel C) and in the presence of 200 mM DMSO (Panel D).

Figure 3.

Kinetic model of mutual exclusion inhibition by DMSO and a generic inhibitor on the transformation of a generic substrate. See the text for an explanation of the symbols.

Figure 4.

Simulation of the effect of DMSO on the inhibition of AR by NHDC, rutin, and phloretin. A computer-assisted plot was generated to simulate the effect of DMSO, described by Equation (1), on the inhibition of AR, using both L-idose (left Panels) and HNE (right Panels) as substrate. The kinetic parameters adopted in the simulation were as follows: i) V_MAX and K_M for L-idose as substrate were 0.011 mM/min and 2.3 mM, respectively; ii) V_MAX and K_M for HNE as substrate were 0.008 mM/min and 0.07 mM, respectively; iii) the inhibition kinetic parameters (i.e. K_i and K_i ^') referring to DMSO and to the three tested inhibitors are reported in Table 1. In each panel, curves 1, 2, and 3 were obtained both in the absence (solid line) and in the presence of 200 mM (dashed line) DMSO at the following inhibitor concentrations: NHDC 0, 50, and 100 µM, respectively (Panels A and B), rutin 0, 5, and 15 µM, respectively (Panels C and D), phloretin 0, 25, and 100 µM, respectively (Panels E and F).