Papain-Catalyzed Hydrolysis of N α-Benzoyl-arginine- p-nitroanilide in an Aqueous-Organic Medium

Abstract

Enzyme-based biosensors have emerged as an effective alternative, providing simplicity, high sensitivity, and the capability to detect multiple residues. However, despite their widespread use, limited studies have examined how organic solvents inhibit these sensors. This study investigates the enzymatic reactions and structure of the selected model enzyme, papain, a protease derived from Carica papaya, in the presence of various organic solvents. Enzyme activity was monitored through the hydrolysis of N_α-benzoyl-arginine-p-nitroanilide (BAPNA), with the resulting yellow product, p-nitroaniline, measured at a wavelength of 430 nm. The experiments incorporated a 10% (v/v) concentration of dimethyl sulfoxide (DMSO) to ensure the solubility of BAPNA. Results showed that methanol and ethanol increased the K _m value while causing little change in V _max, which negatively impacted the enzyme's catalytic efficiency. In contrast, acetonitrile (ACN) behaved as a reversible mixed-competitive inhibitor of papain, exhibiting lower millimolar IC50 values. Furthermore, an emission maximum shift to lower wavelengths with increasing concentrations of ACN suggested that the tryptophan residues within the enzyme structure were slightly more buried. Molecular dynamics simulations of the BAPNA-papain complex in cosolvent environments containing water, DMSO, and ACN indicated that ACN could act as a mixed-competitive inhibitor alongside BAPNA and that solvent polarity could influence the binding of BAPNA to papain. These findings provide valuable insights for the application of organic solvents in biosensor technologies.

Figure 1

Concentration-dependent inhibition of papain was assessed for five model enzymatic assays. The substrate–velocity inhibition curves of papain activity at different concentrations of substrate BAPNA. (A) BAPNA was dissolved in various concentrations of DMSO. (B–E) The BAPNA were dissolved in fixed DMSO concentrations and the presence of various cosolvent concentrations: (B) methanol, (C) ethanol (D) acetonitrile, and (E) chloroform.

Figure 2

Effect of the presence of cosolvent on maximum absorbance for p-nitroaniline (A), acetonitrile (B), chloroform, and (C) the molar extinction coefficient of p-nitroaniline: acetonitrile (blue), ethanol (green), and methanol (orange). The asterisks represent significant differences at each condition (P < 0.05).

Figure 3

(A) Ribbon diagram of papain showing tryptophan residues and fluorescence spectra of papain in (B) methanol and (C) acetonitrile–mixed solvents.

Figure 4

Effect of enzyme concentration and incubation time on papain inhibition by (A) ethanol, (B) methanol, and (C) acetonitrile cosolvent systems. The remain activity of papain was assessed by comparing the enzyme activity in the presence of the specified organic solvent to that observed in its absence under the same conditions. All values are the averages of triplicate measurements and are expressed as mean ± SD from these experiments. Bars with different letters indicated statistical differences (p < 0.05, ANOVA test).

Figure 5

Dose–response inhibition curves of organic solvents against papain. IC50 values were calculated after fitting the curves by using the nonlinear regression function of GraphPad Prism7. IC50 value was calculated at a substrate concentration of 0.06 M.

Figure 6

Molecular modeling of the BAPNA–papain complex in co-organic solvents containing 40:10:50 (v/v) water, DMSO, and acetonitrile before and after 300 ns running in MD simulation, where papain, BAPNA, DMSO, acetonitrile, and water molecules were represented in pink, green, yellow, blue, and red color, respectively.

Figure 7

RMSD (a), R_g (b), and the distance between the center of mass of papain and BAPNA (c) observed in MD simulations of BAPNA–papain complex in co-organic solvents for 300 ns.

Figure 8

Binding position (a) and conformation (b) of BAPNA in papain and the active site of the BAPNA–papain complex (c) obtained from molecular dynamics simulations of the BAPNA (green)–papain (pink ribbon and light brown surface) complex solvated with co-organic solvents containing 40:10:50 (v/v) of water (red), DMSO (yellow), and acetonitrile (blue) at 0, 100, 200, and 300 ns.

Figure 9

Graphical snapshots represent solvent molecules within 8 Å of BAPNA during molecular dynamics simulations of the BAPNA (green)–papain (pink) complex solvated with co-organic solvents containing 40:10:50 (v/v) of water (red), DMSO (yellow), and acetonitrile (blue) at 0, 100, 200, and 300 ns.