Melamine exhibited noncompetitive inhibition of horseradish peroxidase using the H2
/ABTS assay, with Vmax
= 14.63 ± 0.15μ
= 1.67 ± 0.06
mM, and Ki
= 9.5 ± 0.7
mM ( and ). A noncompetitive mechanism of inhibition implies that melamine binding does not compete with ABTS substrate binding but decreases the rate of catalytic turnover. No evidence of inhibition was observed in reactions in which cyanuric acid (1,3,5-triazine-2,4,6-triol) was substituted for melamine (data not shown). Melamine exhibited a mixed-model inhibition of lactoperoxidase (primarily competitive) using the H2
/KI iodoperoxidase assay ( and ) with Vmax
= 2.60 ± 0.10
= 2.9 ± 0.4
mM, and Ki
= 15 ± 5
mM. A mixed model of inhibition implies that melamine interferes with ABTS binding and also impairs the reaction velocity. Inhibition of horseradish peroxidase and lactoperoxidase by melamine was confirmed using the chemiluminescent peroxidase assay method (). Linear trends between luminescent intensity and melamine concentration were evident for both enzymes. Cyanuric acid failed to inhibit horseradish peroxidase and lactoperoxidase using the chemiluminescent assay (data not shown). Unfortunately, enzyme kinetic constants and inhibition constants could not be calculated using this method because the supplier declined to provide the identity and concentration of the chemiluminescent substrate.
Figure 3 (a) Plots of velocity (V) of oxidized ABTS formation versus ABTS concentration (ABTS) with melamine concentration ranged from 0.5–2mM. (b) Lineweaver-Burk plots of velocity (V) of oxidized ABTS formation versus ABTS concentration (ABTS) (more ...)
Enzyme kinetic parameters for horseradish peroxidase and lactoperoxidase.
Figure 4 (a) Michaelis-Menton plots of the velocity of the lactoperoxidase-catalyzed reaction versus KI concentration with 6–15mM melamine. (b) Lineweaver-Burk plots showing mixed model inhibition (primarily competitive) of lactoperoxidase by (more ...)
Plots of chemiluminescent intensity for reactions catalyzed by (a) horseradish peroxidase or (b) lactoperoxidase showing inhibition by melamine. Dotted lines depict 95% confidential bands for linear trends.
COX-1 and COX-2 are prostaglandin H synthases (EC 220.127.116.11) that convert arachidonic acid (AA) to prostaglandin H2
) in two steps [21
]. In the first step, the cyclooxygenase activity of COX acts as a dioxygenase to catalyze the incorporation of two moles of molecular oxygen to arachidonic acid (AA) to form prostaglandin G2
), a reactive 15-hydroperoxy-9,10-endoperoxide. COX acts as a peroxidase in the second step in which a cosubstrate molecule serves as an electron donor to reduce the PGG2
hydroperoxyl group which then becomes the hydroxyl group of prostaglandin H2
. We evaluated the effects of melamine on the peroxidase activity of COX-1 and COX-2 using 10-acetyl-3,7-dihydroxyphenoxazine (ADHP; Amplex Red) as the electron donor substrate. Melamine exhibited a significant concentration-dependent trend for COX-1 inhibition using the fluorescent assay method (). These data showed that COX-1 activity was inhibited in reactions containing 0.05–1.00
mM melamine. Inhibition of COX-2 was not apparent using this method.
Effects of melamine on peroxidase activity of (a) COX-1 and (b) COX-2.
Recent pharmacokinetic studies showed that melamine administered orally to Sprague-Dawley rats was absorbed almost completely (98.1% bioavailability) and then excreted rapidly (t1/2
194 ± 38
min), primarily via filtration through the kidneys [9
]. However, repeated exposure of lambs to high doses of melamine (2, 10, 30, or 100
mg/kg) or to 100
mg/kg melamine plus 100
mg/kg cyanuric acid for 60 days led to increasing melamine levels in the serum (167–267μ
g/kg max), liver (158–412μ
max), longissimus dorsi and gluteal muscles (227–374μ
max), and kidney (347–808μ
g/kg max) [22
]. The tissue levels of melamine observed in animals do not reach levels required for lactoperoxidase or COX-1 inhibition under the conditions described in his report, although it could be speculated that somewhat higher melamine levels might occur within the microenvironment of renal tubule cells. Nonetheless, our results show that melamine interferes with the catalytic activity of three of the four heme enzymes tested, demonstrating intermolecular interactions between melamine and HRP, LPO, and COX-1. Studies by Wang [15
] implicated interactions between melamine and another heme protein, myoglobin. Therefore, it will be important to determine whether other proteins present in plasma and/or urine may sequester melamine and/or cyanuric acid. Melamine- or cyanuric binding-proteins may inhibit crystal formation outside of the urinary tract and could influence the adsorption, transport, and retention of these compounds.