Actions induced by urea and guanidine on xanthine dehydrogenase

Abstract

Urea (up to 8 M) and guanidine (up to 3 M) exercise a reversible inhibition on chicken liver XDH when working with the system: xanthine (1 X 10-4 M)-XDH-NAD (1 X 10-3 M), as long as the temperature at which both denaturants are operating is inferior to 30° C but when they operate at temperatures between 40° C and 55° C the enzyme does not recover its activity.

The percentages of inhibition provoked by the urea and the guanidine on the catalytic activity of the XDH increases with the time of preincubation of the enzyme and the inhibitor, as well as with the concentration of urea and guanidine and, if the concentration of guanidine is superior to 3 M, the inhibition is irreversible at any temperature.

The presence of the substrate xanthine or the electronic acceptor NAD does not protect the XDH from the actions induced by urea or guanidine. The NAD for its part, when it is previously kept in contact with the enzyme, unites with the proteic molecule and creates a stable and active complex.

Working with variable concentrations of xanthine and with NAD constant (1 X 10-3 M), the guanidine is revealed as a competitive inhibitor of the XDH (Ki = 2.15 X 10-2 M) and if the concentrations of NAD are varied and the xanthine is constant (1 X 10-4 M), the inhibition provoked by guanidine is of a non competitive nature: Kip = 5.1 X 10-2 M; Kii = 2.03 X 10-1 M.

The experiments described suggest that the action exercised by the urea and the guanidine on the XDH may be realized in two stages: in the first stage the changes induced by either would be minimal and reversible in character if no significant change had been produced in the structure of the enzyme molecule. In more severe operational conditions the joint effect of high concentrations of urea (8 M) or guanidine (3 M) and of the temperature (55° C) would provoke the irreversible denaturation of the XDH. The urea and the guanidine compete with the xanthine for the same active site of the enzyme molecule and are joined with a «locus» different from that of the NAD.