Horseradish peroxidase, assayed with o-phenylenediamine,
is irreversibly inactivated when incubated in
phosphate buffer, 100 mmol/L, at pH 5. The inactivation
depends on both duration of incubation and phosphate
concentration. Phosphate was the most potent inactivator
and citrate the least potent of a series of buffers tested.
The inactivation is not attributable to ionic strength per se
or to Na+ or K+. The observed inactivation did not occur
at high concentrations (2500 nmol/L, 0.1 g/L) of enzyme;
however, this “protective” effect could not be reproduced
by adding bovine serum albumin or a surfactant (Tween
20) to lower concentrations of enzyme. The inactivation
was independent of commercial source of the enzyme or
the kind of chromogenic assay used. On the basis of this
information, we optimized the assay so that it gave
eightfold greater absorbance values than those reported
by others. The improved assay was sensitive to as little as
0.4 pmol/L (16 ng/L) of peroxidase, and was linear over
the range of 0.4 to 5 pmol/L (16-200 ng/L).