Wierzchowski Jacek, Pietrzak Monika, Szelag Malgorzata, Wroczyński Piotr
Department of Biophysics, University of Warmia and Mazury, 4 Oczapowskiego Street, PL-10-719 Olsztyn, Poland.
Arch Oral Biol. 2008 May;53(5):423-8. doi: 10.1016/j.archoralbio.2007.11.004. Epub 2007 Dec 21.
We have applied fluorimetric method to monitor aldehyde dehydrogenase (ALDH*) activity in human saliva samples to study inactivation, reactivation and inhibition of the enzyme.
Saliva samples were collected to buffer stock solution, containing various thiols, and assayed in the presence of the fluorogenic substrate 6-dimethylamino-2-naphthaldehyde and NAD(+). Fluorescence of the produced 6-dimethylamino-2-naphthalene carboxylate was used to measure the reaction rate.
Kinetic parameters for the highly fluorogenic substrate, 6-dimethylamino-2-naphthaldehyde were measured, with apparent K(m) of 7.9 microM at pH 7.3. The apparent K(m) for NAD(+) was 1.2 microM. The observed ALDH activity is unstable in the absence of thiols, but can be stabilized by 1mM glutathione, and inactivated enzyme can be re-activated within 10 min by treatment of 0.5 mM DTT. Two-assay procedure was applied to measure degree of inactivation of ALDH in saliva samples. It was found that degree of ALDH inactivation in fresh samples, stabilized by glutathione, is between 0% and 90%, with average value ca. 40%. Caffeine and theophylline were shown to be moderate inhibitors of salivary ALDH.
Oxidation of the salivary ALDH in fresh saliva may be reliably measured using fluorimetric two-assay procedure. Preliminary statistics indicate that in most individuals this enzyme is partially inactive. Inhibition of the salivary ALDH by caffeine may have consequences for nutrition safety.
