Enzymatic reactions for the determination of sugars in food samples using the differential pH technique.

All ATP coupled reactions, when performed at neutral or moderately alkaline pH, produce an acidification of the reaction mixture. The detection of small pH changes--0.1 mpH (1 mpH = 10(-3) pH)--in a constant buffering capacity solution makes it possible to quantify, over a wide concentration range (1-1500 mmol L(-1)), various analytes with very high precision and accuracy. Glucose, fructose, glycerol and gluconic acid can be analysed in less than 1 min with a single step reaction. Wine samples were analysed using the hexokinase reaction for glucose + fructose (sugars undergoing fermentation) and compared against an established method, showing excellent performance over the whole range of concentrations (R = 0.9994). Increased sensitivity in some applications can be obtained by cycling reactions, e.g. a kinase reaction followed by a phosphatase reaction. in a one step analysis, as required for lactulose assay in milk, a useful indicator of heat treatment damage. A sensitivity well below 0.1 mmol L(-1) in the original milk sample has been demonstrated.

Hydrophobic properties of NAD glycohydrolase from neurospora crassa conidia and interaction with dioxane.

NAD glycohydrolase (NADase, EC 3.2.2.5) from Neurospora crassa conidia shows marked hydrophobic properties which are related to the self inhibition of the enzyme. Both aliphatic amines and carboxylic acids are able to inhibit noncompetitively the catalytic activity of the enzyme and the inhibition depends on the non-polar moiety of the substances. Also dioxane is an inhibitor of NAD glycohydrolase even though it apparently increases the specific activity of the enzyme. This effect can be explained by the fact that NADase is present as a dimer when the enzyme is concentrated or at high temperature, and dioxane binds the enzyme breaking the hydrophobic bonds in the dimeric enzyme and yielding the most active monomeric form which is only slightly inhibited by the organic solvent.

Purification of NAD glycohydrolase from Neurospora crassa conidia by a polyclonal immunoadsorbent.

NAD glycohydrolase from Neurospora crassa conidia was purified by affinity chromatography on a column of polyclonal antibodies bound to an agarose matrix. The procedure was easy, non-denaturating and suitable for repetitive use of the gel. The enzyme obtained appeared homogeneous by sodiumdodecyl sulphate-polyacrylamide gel electrophoresis.

Automatic computation of enzyme kinetics by HPLC.

Enzyme activity can be easily measured by HPLC using traces of the product itself as an internal standard. Our procedure involved the development of an equation using the experimental data obtained in the kinetic assay. The entire procedure can thus be automated and a computer program is presented here for facilitating the assay and saving time. The determination of the activity of NAD kinase is reported as an example.

High-performance liquid chromatography determination of enzyme activities in the presence of small amounts of product.

Enzymes can be assayed by HPLC by calculating the amount of substrate(s) left over, or product formed, through the peak area ratios with a suitable internal standard. However, sometimes the substrates used are contaminated with small amounts of products and this can lead to errors in the determination of the enzyme activity. A method for a HPLC test of such enzymes, which prevents eventual errors, uses the ratio substrate/product at time zero as internal standard and the kinetics can be followed with the aid of a simple mathematical equation. This approach was applied to the determination of the activities of papain, urokinase, NAD glycohydrolase, and pyruvate kinase samples and it was compared with the data obtained by the internal standard method, giving reproducible results in all cases.

Preparation of immobilized NAD glycohydrolase from Neurospora crassa conidia by hydrophobic interaction--characteristics of the enzyme derivative.

NAD glycohydrolase from Neurospora crassa conidia has been immobilized by hydrophobic interaction on Sepharose 4B beads coated with propyl residues through CNBr activation. The bond resulted stable under a wide range of conditions (ionic strength, temperature, pH). As a result of immobilization the pH optimum for catalytic activity shifted by about 0.2 pH unit in the acidic direction, to lie between 7.5 and 7.3. The stability of the enzymatic activity was largely enhanced by effect of immobilization but the Km value towards NAD+ was increased compared with that of the free enzyme (1 X 10(-3) and 2 X 10(-4) M respectively).

Colorimetric determination of 1-(4'-nitrophenyl)-2-aminopropane-1,3-diol with 2,4,6-trinitrobenzenesulfonic acid in the presence of chloramphenicol.

A colorimetric method based on the interaction between the chloramphenicol degradation product 1-(4'-nitrophenyl)-2-aminopropane-1,3-diol and the 2,4,6-trinitrobenzenesulfonic acid reagent was developed. Analytical solutions were reacted with the reagent at pH 9.1 for 20 min at room temperature, and the resulting color was measured at 340 nm. A linear relationship between absorbance and concentration occurred within the 5--25-micrograms/ml range under the conditions studied. Replicate analyses were in good agreement. An average recovery of 99.4 +/- 0.4% was obtained for the synthetic mixtures.