br Materials and methods br Results

Materials and methods
Results and discussion
Conclusions In this study, for the first time to the best of our knowledge, the stability of pure hsALDH has been studied under various conditions. The enzyme exhibited low storage stability both at 4°C and 25°C, which could be improved to a good extent by storing the enzyme in the presence of 10% glycerol. The enzyme was also observed to have very low thermal stability and unfolds at higher temperatures. Further it is unstable in the presence of denaturants like urea and GnHCl which unfold the enzyme. Salt (NaCl) has an activating effect on the enzyme, resulting from perhaps due to some conformational changes in the enzyme which facilitates the catalysis process. Therefore, low concentrations of NaCl are beneficial for the activity and perhaps the stability of the enzyme. Saliva is a complex fluid containing a mixture of many components which might help in stabilizing and protecting the enzyme under physiological conditions. Under such conditions, the activity and stability of hsALDH would be higher than that observed under in vitro conditions. This study will enable future researchers working with hsALDH to realize that this enzyme under in vitro conditions has very low stability and hence should be used quickly after purification. Also, more strategies are needed to be developed to stabilize the enzyme under in vitro conditions for its use in research and for therapeutic purposes. Further, certain additives like glycerol and NaCl can improve the activity/stability of the enzyme. Therefore, a stabilizing agent is required to use the enzyme in in vitro studies.
Conflict of interest
Acknowledgements Research facilities provided by the Aligarh Muslim University is gratefully acknowledged. A.A. Laskar and M.F. Alam are thankful to the Department of Biotechnology, Govt. of India for financial assistance in the form of Senior Research Fellowship.
Introduction Aldehyde dehydrogenases (ALDH; EC 1.2.1.3) are short-chain dehydrogenases/reductases (SDR) superfamily containing NAD(P)+-dependent enzymes that catalyse the irreversible dehydrogenation of a wide range of endogenous and exogenous aldehydes to their corresponding less toxic carboxylic acids [1–3]. ALDHs are widely distributed in prokaryotic and eukaryotic chir99021 and play important roles in detoxification of toxic and reactive aliphatic and aromatic aldehydes formed during the metabolism of alcohols, amino acids, carbohydrates, lipids, biogenic amines, vitamins and steroids [4]. Currently, there are 19 known members of the ALDH superfamily [5,6]. ALDHs functional and physiological properties have been studied extensively and are involved in the maintenance of cellular homeostasis, modulate cell proliferation, differentiation, survival and cellular response to oxidative stress [1,7,8]. ALDHs play essential role in the metabolic pathways that are critical for cell development and response to environmental changes [9]. ALDHs are homo-biopolymers composed of two or four polypeptides of 50–55kDa, and made up of N-terminal NAD+-binding domain, a catalytic domain and an oligomerisation domain [10,11]. Aldehyde dehydrogenases kinetic mechanism is literarily an ordered sequential kinetic mechanism with NAD(P)+ binding first, followed by the aldehyde [12–14]. In some cases, it is random kinetic mechanism with preference for initial binding of NAD(P)+[15]. The ternary complex forms thio-hemiacetal intermediate which is transformed to thioester by giving its hydride ion to NAD(P)+. Eventually, the thioester is hydrolysed by a water molecule to carboxylic acid. The sequential dissociation of carboxylic acid and NADH, which is the rate-limiting step, ends the reaction [14,16]. ALDHs exhibit additional, non-enzymatic functions, the non-catalytic binding properties for endobiotics, some hormones and other small molecules [1,17]. It is 'housekeeping' functions linked with detoxification. This is associated with the ubiquitous, ample and constitutively expressed properties of the enzyme. These ligand binding properties might be connected to protective function through the sequestration of metabolites. They conceivably serve to prevent the accumulation or minimize potentially toxic free endobiotics and xenobiotics or involved in the uptake and transport of hydrophobic non-substrate prior to its detoxification. Catalytic and ligand complexing properties (ligandin) are important for detoxification mechanism [1] and there is connection in both [17]. Although ALDH catalytic mechanisms of detoxification have been investigated extensively, however, relatively little is known about its non-catalytic binding function.