Mario Pace

Polyphenol oxidases from latex of Hevea brasiliensis: purification and characterization

Polyphenol oxidase (PPO) was isolated from the B-serum obtained after repetitive freeze-thawing of the bottom fraction isolated from ultracentrifuged fresh latex. The B-serum was subjected to acetone precipitation and CM-Sepharose chromatography, affording two PPOs, PPO-I and PPO-II, which, upon SDS-PAGE, were 32 and 34 kDa, respectively. Both PPOs possessed the same pI (9.2), optimum pH (7) and optimum temperature (35-45 degrees C). They are stable up to 60 degrees C and active at broad pH ranges from 4-9. The K(m) values of PPO-I for dopamine, L-dopa and catechol as substrates are 2.08, 8.33 and 9.09 mM, while those for PPO-II are 2.12, 4.76 and 7.14 mM, respectively. Among various PPO inhibitors tested, 4-hexylresorcinol was the most potent. Anionic detergents were among the most effective activators of the enzymes, while cationic and nonionic detergents showed little and no effect on the PPO activities, respectively.

High-performance liquid chromatography for assaying NAD glycohydrolase from Neurospora crassa conidia

A rapid and sensitive high-performance liquid chromatographic technique was developed to determinate NAD glycohydrolase (EC 3.2.2.5.) activity from Neurospora crassa conidia. The separation of the assay substrate and products was achieved by isocratic reverse-phase chromatography and the peaks were detected by the absorbance at 259 nm. Quantities of NAD+ and nicotinamide as small as 10 pmol could be measured.

High-performance liquid chromatographic assay of glycosyltransferases using flavonoids as substrate

An HPLC method for the determination of glycosyltransferase activity, alternative to the radioactive assay, is proposed. The method is suitable for following the kinetics of consecutive enzymes that yield monoglucosides, diglucosides and triglucosides, as demonstrated with a pea seedling extract containing a mixture of three glucosyltransferases using flavonoids as substrate and UDP-glucose as carbohydrate donor. In this instance the HPLC determination of the three glucosides could be accomplished after separation of the aglycones by solid extraction on a Sep-Pak C18 microcolumn. After isolation of the enzyme catalysing the production of the monoglucoside of quercetin (isoquercitrin) or kaempferol (astragalin), the kinetics of the reaction were determined by HPLC, following both the increase of the product and the disappearance of the substrate. The increasing amounts of isoquercitrin and astragalin were consistent with the decrease in the amount of aglycone measured after direct injection of the reaction mixture into the HPLC system and its elution with a less polar solvent.

Analysis and purification of DNA restriction fragments by high-performance liquid chromatography

The purification and analysis of restriction fragments play a very important role in molecular biology but the traditional assay methods of DNA fragments, based on gel electrophoresis and caesium chloride gradient centrifugation, are time-consuming and difficult to quantify. High-performance liquid chromatography provides an alternative method which allows the direct quantitation of picogram amounts of eluents in short time. In the present work we report the separation of different restriction fragments, the purification of some fragments and the relationship between the length of double-stranded DNA fragments and peak areas.

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).

Comparison of the properties of human hemoglobin covalently bound to carboxyl dextrans with free and polymerized hemoglobin

Human stroma-free hemoglobin (SFH) was coupled in the oxy or deoxy conformations to carboxyldextrans through amide bonds. The complexes were analysed by gel permeation high performance chromatography, and their molecular mass distribution ranged from 90,000 to 300,000. Covalent coupling of SFH to carboxyldextrans determined an increase of the oxygen affinity when compared to free SFH. The P50 of the complex formed from carboxyldextrans and SFH in the oxy state was lower than that of the derivative obtained from SFH in the reduced state. On the other hand, glutaraldehyde cross-linked SFH still showed cooperativity when reacted in the deoxy state and in the presence of pyridoxal phosphate, and its oxygen affinity was similar to that of the free pyridoxylated SFH. These results lead to exclude the potential use of these dextran-SFH complexes as oxygen carriers.

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.

Improved Purification Protocol for Oxalate Oxidase from Barley Roots

Abstract A purification scheme is described for oxalate oxidase from barley roots. This procedure involves gel filtration on Bio-Gel A 0.5 m and affinity chromatography using oxalate, immobilized on Affi-Gel 10, as a ligand. The purified enzyme was homogeneous on SDS-gel electrophoresis, thin-layer electrophoresis and high-performance liquid chromatography (HPLC). The molecular weight of the enzyme was 150.000 by SDS-gel electrophoresis and the specific activity was 1.9 U/mg protein.

Characteristics of bioreactors made with urease and NAD glycohydrolase reversibly bound to immobilized antibodies

The characteristics of urease and NAD glycohydrolase, immobilized on different matrices through linkage with polyclonal antibodies, were studied for their use as enzyme reactors. The stability of the derivatives was enhanced by immobilization but some leakage of activity was found, due to the equilibrium between the free and bound enzyme. The release of the enzyme to the solution depends on the affinity constant of the antibodies and the concentration of the substrate circulating in the reactor. On the other hand the main advantage of this easy method of immobilization is the low cost replacement of the inactive enzyme with fresh catalyst.