Heterogeneous inhibition of horseradish peroxidase activity by cadmium Jacqueline Keyhani a, * , Ezzatollah Keyhani a,b , Nahid Einollahi c , Dariush Minai-Tehrani b , Sekineh Zarchipour b a Laboratory for Life Sciences, Saadat Abade, Sarve Sharghi, 34, 19979 Tehran, Iran b Institute of Biochemistry and Biophysics, University of Tehran, Tehran, Iran c Tehran University of Medical Sciences and Health Services, Faculty of Allied Health Sciences, Tehran, Iran Received 19 November 2002; received in revised form 17 February 2003; accepted 20 February 2003 Abstract Inhibition of horseradish peroxidase (HRP) activity by cadmium was studied under steady-state kinetic conditions after preincubation of the enzyme with millimolar concentrations of Cd 2+ for various periods of time. The H 2 O 2 -mediated oxidation of o-dianisidine by HRP was used to assess the enzymatic activity. Cd 2+ was found to be either a noncompetitive inhibitor of HRP or a mixed inhibitor of HRP depending both on the duration of incubation with HRP and on Cd 2+ concentration. Furthermore, for the same inhibition type, K i values dropped as incubation time increased. These results suggested that Cd 2+ would slowly bind to the enzyme and progressively induce conformational changes. Spectrophotometric analysis showed that indeed Cd 2+ altered the heme Soret absorption band on binding HRP and exhibited a K d which decreased as the incubation time of HRP with Cd 2+ increased. Hill plots suggested a cooperative binding of up to three Cd 2+ ions per molecule of HRP. Thus, Cd 2+ binding to HRP resulted in progressive inhibition of enzymatic activity with a change in the inhibition type as the number of Cd 2+ ions per HRP molecule increased. Results also illustrated the potential danger of long-term exposure to heavy metals, even for enzymes with low affinity for them. D 2003 Elsevier Science B.V. All rights reserved. Keywords: Cadmium; Enzyme inhibition; Cooperativity; Horseradish peroxidase; O-dianisidine 1. Introduction Cadmium is a widely distributed metallic pollutant of our environment, which can be absorbed into biological systems through direct uptake as well as by accumulation in food chains. It has toxic effects on all living systems, whether human, animal, plant or bacterium [1–6]. Among its re- ported toxic effects we note the following. It is a potent animal and human carcinogen [1,2]; it causes skeletal deformation [4] and decrease in bone strength [5]; it induces DNA strand breakage in vivo [6] and in vitro [7,8]; it is responsible for a decrease in the biomass production in plants [9]. Peroxidases are important detoxifying enzymes serving to rid cells of excess H 2 O 2 under normal and stress con- ditions [10–17], including contamination by toxic levels of heavy metals [18,19]. However, extreme stress con- ditions may affect the activity of the detoxification en- zymes themselves. Although peroxidases remain active in the presence of a number of metal ions, including cad- mium, as part of their detoxifying role [20,21], recent reports have also indicated their inhibition by metal ions [21–23]. In this study, the effect of increasing amounts of Cd 2+ ion on peroxidase activity was investigated in vitro using horseradish peroxidase (HRP), one of the best character- ized peroxidases. The H 2 O 2 -dependent oxidation of o- dianisidine was inhibited in a time- and concentration- dependent manner by Cd 2+ ions. The type of inhibition observed was either noncompetitive or mixed, depending on the Cd 2+ ion concentration and on the time of incuba- 0304-4165/03/$ - see front matter D 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S0304-4165(03)00053-9 * Corresponding author. Tel.: +98-21-695-6974; fax: +98-21-640- 4680. E-mail address: keyhanie@ibb.ut.ac.ir (J. Keyhani). www.bba-direct.com Biochimica et Biophysica Acta 1621 (2003) 140 – 148