A Review of: “HPLC of Biological Macromolecules (Methods and Applications) K.M. Goodring F.E. Regnier,Eds Marcel Dekker,New York, 1990; hardbound, 676 pages, $150”

An ionically unbound and thermostable polyphenol oxidase (PPO) was extracted from the leaf of Musa paradisiaca. The enzyme was purified 2.54-fold with a total yield of 9.5% by ammonium sulfate precipitation followed by Sephadex G-100 gel filtration chromatography. The purified enzyme exhibited a clear single band on native polyacrylamide gel electrophoresis (PAGE) and sodium dodecyl sulfate (SDS) PAGE. It was found to be monomeric protein with molecular mass of about 40 kD. The zymographic study using crude extract as enzyme source showed a very clear band around 40 kD and a faint band at around 15 kD, which might be isozymes. The enzyme was optimally active at pH 7.0 and 50°C temperature. The enzyme was active in wide range of pH (4.0–9.0) and temperature (30–90°C). From the thermal inactivation studies in the range 60–75°C, the half-life (t_1/2) values of the enzyme ranged from 17 to 77 min. The inactivation energy (Ea) value of PPO was estimated to be 91.3 kJ mol^?1. It showed higher specificity with catechol (K_m = 8 mM) as compared to 4-methylcatechol (K_m = 10 mM). Among metal ions and reagents tested, Cu²⁺, Fe²⁺, Hg²⁺, Mn²⁺, Ni²⁺, protocatechuic acid, and ferrulic acid enhanced the enzyme activity, while K⁺, Na⁺, Co²⁺, kojic acid, ascorbic acid, ethylenediamine tetraacetic acid (EDTA), sodium azide, β-mercaptoethanol, and L-cysteine inhibited the activity of the enzyme.     

A Review of: “Centrifugation in Biology and Medical Science,P. Sheeler,Wiley/Interscience,New York, 1981; hardbound, 269 pages, $35.00”

ABSTRACT

Peroxidases (E.C. 1.11.1.7., hydrogen donor oxidoreductase) utilize hydrogen peroxide or substituted peroxides for the oxidation of a large number of substrates. Peroxidases are widely distributed and have been isolated from many higher plants (1). The wide distribution of the enzyme suggests that it could be of great biological importance, but the physiological functions and metabolic control of these enzymes are still poorly understood. The simultaneous presence of amine oxidase and peroxidase in cell walls suggests that the peroxide generated on oxidation of the amines could be utilized by the peroxidase (2,3). Recently we have purified an amine oxidase from Hordeum vulgare (4) and we have attempted to purify the peroxidase in order to study in vitro the reconstituted coupled system. β-glucosidase (β-D-glucoside glucohydrolase E.C. 3.2.1.21.) is capable of transforming glucosides in glucose and the corresponding aglycone or disaccarides as cellobiose, sophorose, gentiobiose. This enzyme is widely distributed in plants, fungi, bacteria, yeasts and animals (5, 6). In the homogenate of Hordeum vulgare seedlings we also found β-glucosidase activity and also attempted to purify β- glucosidase. This enzyme copurified whit peroxidase up to the last step. We report here the isolation of peroxidase and β-glucosidase from Hordeum vulgare see-dlings: some molecular and kinetic properties are given.     

A Review of: “Methods of Protein Separation,N. Catsirapoolas,ed. Plenum Press,New York, 1975; 281 pages; hardbound; $27.50”

Tryporaastogotes of three strains of Trypanosoma cruzi were isolated from the blood of infected mice employing lymphoprep for separation of the red blood cells and a column of DEAE cellulose for removal of white cells and platelets. An average recovery of 45 to 5 8 percent of actively motile, infective organisms, free of contaminating blood cells was obtained. Protein and carbohydrate assays of the separated organisms revealed significant differences between the Tulahuen, a reticulotropic strain, and the House 510 and House 11, two myotropic strains of this parasitic species. The present procedure should provide sufficient parasites for physiological and biochemical studies; it has also served to indicate particular strain characteristics which may aid in a taxonomic classification of these organisms.     

A Review of: “Biological Recognition and Assembly,D. S. Eisenberg,J. A. Lake and C. F. Fox,Eds. Alan R. Liss,New York, 1980; hardbound, 355 pages, $52.00”

Two MMP-7-ase isoenzymes were purified 100-fold from rat muscle extract to apparent homogeneity, with an overall yield of 10%, using homogenization, ultracentrifugation, high-performance aqueous size-exclusion and high-performance anion exchange chromatography methods. When using a TSK G-2000SW column, the separation resulted in a 6-fold purification and 30% recovery of isoenzymes B and C. This concentrated enzyme extract was then passed through a TSK-DEAE-2SW column, using salt gradient at pH 7.5, with an additional 25-fold purification and 90% recovery of the isoenzymes. Two symmetrical enzyme peaks, representing isoenzymes B and C, were detected when performing purity tests of the active enzymes on the anion exchanger and reversed-phase HFLC columns. The procedures involved are extraction, ultracentri-fugation, chromatographies and enzyme assays and require less than five hours.     

A Review of: “Bioseparations, (Downstream Processing for Biotechnology) P.A. Belter,E.L. Cussler and W.S. Hu,Wiley Interscience,New York, 1988; hardbound, 368 pages, $39.95”

ABSTRACT

Covalent structural information on membrane proteins is not easily acquired since it is difficult to obtain pure membrane proteins in sufficient quantities. We have therefore examined the Bio-Rad 491 prep cell continuous elution electrophoresis apparatus as a method for providing the quantities of purified , alpha and beta subunits from (Na.K)-ATPase required for these studies. Twenty-four milligrams of crude (Na.K)-ATPase preparation was applied to the prep cell which consisted of a 7% Laemmli separating gel 4.5 cm in length. The prep cell was Y run under constant power and continuous cooling conditions. Those fractions containing the beta subunit were combined and further purified by wheat germ agglutinin affinity"' chromatography. Fractions containing the alpha subunit were combined and did not require further purification. The identity and the degree of purity of the proteins obtained using this approach was assessed utilizing SDS-PAGE, amino acid analysis 375 Copyright 1993 by Marcel Dekker, Inc. * and N-tertninal sequencing. This simple and fast method provides approximately 1.8 milligrams of each purified subunit from 24 milligrams of relatively crude microsomes. Recovery of the alpha and beta subunits from the crude (Na.K)-ATPase preparation was estimated to be 28% and 81%, respectively.