Mechanism of horseradish peroxidase inactivation by benzhydrazide
Mechanism of horseradish peroxidase inactivation by benzhydrazide
Mechanism of horseradish peroxidase inactivation by benzhydrazide
Create successful ePaper yourself
Turn your PDF publications into a flip-book with our unique Google optimized e-Paper software.
618 S. M. Aitken and others<br />
Figure 6 Absorption spectra <strong>of</strong> peaks (A) 1, (B) 2, (C) 4 and (D) 5 from chromatogram B in Figure 4<br />
Table 1<br />
k inact , K I ,IC 50 and K d values for the <strong>inactivation</strong> <strong>of</strong> HRPC <strong>by</strong> arylhydrazides<br />
K I and k inact determined as described in the Materials and methods section. Preincubation conditions for IC 50 determinations were 10 nM HRPC, 1 mM H 2 O 2 and (1–5) × 10 4 µMhydrazide<br />
in 100 mM sodium phosphate, pH 7.0, for 1 min at 25 ◦ C. IC 50 values are means from at least three experiments. MPO IC 50 values are from [10] and K d values are from [31]. -OH-BZH,<br />
-hydroxy<strong>benzhydrazide</strong>; -CH 3 -BZH, -methyl<strong>benzhydrazide</strong>; -NH 2 -BZH, -amino<strong>benzhydrazide</strong>; -Cl-BZH, -chloro<strong>benzhydrazide</strong>; -OH-NZH, -hydroxy<strong>benzhydrazide</strong>; -NO 2 -BZH, -nitro<strong>benzhydrazide</strong>;<br />
NICH, nicotinic hydrazide; 2-NZH, 2-naphthoichydrazide.<br />
Inhibitor k inact (min −1 ) K I (mM) IC HRPC<br />
50<br />
(mM) IC MPO<br />
50<br />
(mM) K d (mM) K I /K d<br />
2-NZH 0.14 + −<br />
0.01 0.014 + −<br />
0.006 0.0350 + −<br />
0.0008 0.0052 + −<br />
0.0001 2.7<br />
3-CH 3 -BZH 0.049 + − 0.006 0.7 + − 0.2 0.58 + − 0.03 0.108 + − 0.003 6.3<br />
BZH 0.035 + −<br />
0.003 0.08 + −<br />
0.03 1.00 + −<br />
0.05 0.042 0.120 + −<br />
0.002 0.66<br />
3-OH-BZH 0.073 + − 0.003 0.7 + − 0.1 0.8 + − 0.2 0.120 + − 1 5.6<br />
4-CH 3 -BZH 0.088 + − 0.004 0.45 + − 0.05 0.48 + − 0.03 0.143 + − 5 3.1<br />
4-NH 2 -BZH 0.45 + −<br />
0.02 1.4 + −<br />
0.2 2.2 + −<br />
0.4 0.002 0.23 + −<br />
0.02 6.1<br />
3-NH 2 -BZH 7 + − 2 60+ − 20 1.5 + − 0.1 0.25 + − 0.01 320<br />
3-Cl-BZH 0.025 + −<br />
0.003 1.4 + −<br />
0.5 2.4 + −<br />
0.1 0.27 + −<br />
0.01 5.2<br />
4-Cl-BZH 0.33 + −<br />
0.01 6.0 + −<br />
0.5 2.0 + −<br />
0.2 0.25 0.30 + −<br />
0.01 20<br />
4-OH-BZH 0.41 + − 0.01 0.28 + − 0.03 0.52 + − 0.02 0.018 0.54 + − 0.02 0.52<br />
2-OH-BZH > 10 5.7 + −<br />
0.2 0.63 + −<br />
0.02 54<br />
2-Cl-BZH 0.13 + − 0.02 7 + − 2 22+ − 1 2.05+ − 0.04 3.6<br />
2-CH 3 -BZH > 10 11 + − 2 2.76+ − 0.03 13<br />
4-NO 2 -BZH > 10 3.9 + −<br />
0.3 3 + −<br />
1 0.76<br />
2-NH 2 -BZH 0.47 + − 0.02 0.9 + − 0.1 1.1 + − 0.2 3.1 + − 0.2 0.27<br />
INH 0.025 + −<br />
0.002 1.4 + −<br />
0.4 3.3 + −<br />
0.2 3.36 + −<br />
0.04 0.42<br />
NICH 0.014 + − 0.003 0.6 + − 0.5 8.5 + − 0.1 4.6 + − 0.1 0.13<br />
3-NO 2 -BZH > 10 5.9 + − 0.3 9.11 + − 0.05 0.20<br />
2-NO 2 -BZH 0.03 + −<br />
0.02 9 + −<br />
10 61 + −<br />
4 90+ −<br />
30 0.11<br />
1-NZH 0.46 + − 0.01 0.94 + − 0.08 0.6 + − 0.1<br />
3-OH-2-NZH 0.05 + − 0.01 0.3 + − 0.2 0.25 + − 0.03<br />
PHZ undergoes both extensive auto-oxidation and haem-catalysed<br />
oxidation at physiological pH [20,29]. Auto-oxidation should not<br />
impede the development <strong>of</strong> hydrazides as potential therapeutics.<br />
However, recent results have demonstrated that the therapeutic<br />
effects <strong>of</strong> INH are due to its oxidation via the superoxide pathway<br />
rather than the <strong>peroxidase</strong> pathway <strong>of</strong> KatG [13,28]. Therefore,<br />
further work on hydrazide-dependent free radical generation<br />
catalysed <strong>by</strong> different <strong>peroxidase</strong>s is a necessary step in assessing<br />
the potential <strong>of</strong> these compounds as therapeutic agents.<br />
HRPC <strong>inactivation</strong> <strong>by</strong> H 2 O 2<br />
Approx. 0.12 µmol <strong>of</strong> O 2 was produced during the consumption <strong>of</strong><br />
0.43 µmol <strong>of</strong> H 2 O 2 (approx. 300 molar equivalents) <strong>by</strong> 1.5 nmol<br />
c○ 2003 Biochemical Society