5.3.1) is reported to be about pH 10 in horse, pH 9.8 in rat and pH 11 in Bacillus brevis. Those working with mammalian systems might favour an assay pH of about 7.2 which is believed to be around the physiological pH within the cell, but clearly this would be unphysiological for gastrointestinal enzymes, such, as pepsin and trypsin, or for lysosomal enzymes. Furthermore, the oxidation of ethanol by liver alcohol dehydrogenase (EC 1.1.1.1) is often followed at higher pH values because the equilibrium of the reaction greatly favours ethanol formation at neutral

pH. Naturally it would be appropriate to use physiological substrates for enzyme assays. However, many studies have used selleck chemicals unphysiological substrates for ease of manipulation and assay. For

example acetylthiocholine is frequently used to assay acetylcholinesterase (EC 3.1.1.7) because the thiocholine produced can be readily detected by reaction with sulfydryl reagent 5, http://www.selleckchem.com/products/ganetespib-sta-9090.html 5′-dithiobis-2-nitrobenzoate (Nbs2) releasing a yellow coloured compound whose formation can be followed spectrophotometrically at 412 nm (Ellman et al., 1961). Other examples include the use of 4-nitrophenyl phosphate to assay alkaline phosphatase (EC 3.1.3.1) (Schumann et al., 2011). The use of synthetic dyes as electron acceptors in oxidoreductase assays has been common and in some cases the physiological acceptor remains unknown. The demand for higher assay sensitivity and high-throughput procedures has resulted in the development of an increasing number of chromogenic and fluorogenic substrates (Goddard and Reymond, 2004 and Reymond et al., 2009). Clearly, in such cases a considerable amount of work would be necessary to show whether the enzyme behaves identically towards such substrates as it does towards its physiological substrates. It is often recommended that saturating substrate concentrations should be used (i.e. >10Km, for all substrates), as discussed Amino acid by Bisswanger

(2014). This, of course, assumes that the Km values have already been determined, at least approximately. Furthermore, this might not always be practicable because of factors such as solubility, the occurrence of high-substrate inhibition or a high absorbance of the assay mixture affecting the behaviour of optical assays ( Dixon et al., 1979 and McDonald and Tipton, 2002). It should also be remembered that any change in the assay conditions (e.g., pH, temperature, ionic strength) may affect the Km values. The buffers and ionic strengths and used in enzyme assays vary widely and are often far from physiological. It might be helpful if it were possible to recommend a simple standard buffer for use in all enzyme assays. Unfortunately, this goal appears to be unobtainable, because at least some enzymes are unhappy in one or other of the common buffers (see e.g., Boyce et al., 2004).