Salivary aldehyde dehydrogenase: activity towards aromatic aldehydes and comparison with recombinant ALDH3A1

Abstract

A series of aromatic aldehydes was examined as substrates for salivary aldehyde dehydrogenase (sALDH) and the recombinant ALDH3A1. Para-substituted benzaldehydes, cinnamic aldehyde and 2-naphthaldehydes were found to be excellent substrates, and kinetic parameters for both salivary and recombinant ALDH were nearly identical. It was demonstrated that for the fluorogenic naphthaldehydes the only produced reaction product after incubation in saliva is the carboxylate.

Scheme 1

Enzymatic oxidation of aryl aldehydes, catalyzed by ALDH.

Figure 1

HPLC elution profiles for oxidation of aromatic aldehydes with salivary ALDH, recorded with fluorimetric detector at 360 nm, with excitation at 315 nm. Reaction time was 5 (lowest curve), 10, 15 and 20 minutes (----).

Figure 2

Reaction progress curves, recorded fluorimetrically, for enzymatic oxidation of 2-naphthaldehde (left) and MONAL (right) with the recombinant ALDH3A1 as catalyst. Initial naphthaldehyde concentrations were 5, 2 and 1 μM. Concentration of NAD⁺ was 100 μM.

Figure 3

The Lineweaver-Burk plot for the enzymatic oxidation of 2 μM MONAL catalyzed by the recombinant ALDH3A1. Rates were obtained by numerical differentiation of the progress curve from Fig. 2. The fitted K_m is 0.16 μM.

Figure 4

SDS/PAGE of recombinant ALDH3A1 after purification: lane 1, PageRuler (Fermantas) molecular mass marker proteins (from bottom to top: 26, 34, 43, 55, 72, 95, 130, 170 kDa); lane 2, solution from the first elution; lane 3, solution from the second elution; lane 4, eluate after dialysis. The gel was stained with Coomassie Brilliant Blue.