Single-Step Separation of Lactate Dehydrogenase Using Thiophilic Chromatography

1998 ◽  
Vol 63 (6) ◽  
pp. 851-856
Author(s):  
Milena Kmínková ◽  
Jiří Kučera
Keyword(s):  
Lactate Dehydrogenase ◽  
Proteolytic Activity ◽  
Cyprinus Carpio ◽  
Hydrophobic Interaction ◽  
Specific Activity ◽  
Hydrophobic Interaction Chromatography ◽  
Single Step ◽  
Hydrophobic Adsorption ◽  
Negative Adsorption ◽  
Final Purification

Lactate dehydrogenase (EC 1.1.1.27) from carp (Cyprinus carpio) hepatopancreas was purified by the single-step chromatography on thiophilic sorbent. Hydrophobic negative sorption was used as negative adsorption step for final purification. Final specific activity was 8.88 U/mg protein and the yield 84%. The enzyme is not adsorbed during hydrophobic interaction chromatography, but proteolytic activity is adsorbed completely on hydrophobic sorbent. Thus hydrophobic adsorption can be used as a prepurification step.

Purification of Glycerol Kinase by "Dye-Ligand" Chromatography and Hydrophobic Interaction Chromatography on Bead-Cellulose Derivatives

1993 ◽  
Vol 58 (2) ◽  
pp. 445-451 ◽  
Author(s):  
Vladimír Žúbor ◽  
Albert Breier ◽  
Marta Horváthová ◽  
Dagmar Hagarová ◽  
Peter Gemeiner ◽  
...  
Keyword(s):  
Hydrophobic Interaction ◽  
Specific Activity ◽  
Hydrophobic Interaction Chromatography ◽  
Glycerol Kinase ◽  
Enzymic Activity ◽  
Cellulose Derivatives ◽  
Long Term Storage ◽  
Bead Cellulose ◽  
Term Storage

The crude extract of cytosole enzymes was obtained from homogenized cells of Saccharomyces cerevisiae by partition. The enzyme was then isolated from the lower aqueous phase displaying higher glycerol kinase activity by dye-ligand chromatography on Cibacron Blue (CB) or Remazol Brilliant Blue R (RB)-derivatized bead-cellulose, ATP being the eluent. The specific activity of glycerol kinase rised more than 10 and 7-times after affinity dye-ligand chromatography and hydrophobic interaction chromatography, respectively. Glycerol kinase obtained by the latter method was purified by CB-bead cellulose. The final preparation maintained its enzymic activity without noticeable losses during a long-term storage at 4 °C in dark.

Poly(β-L-malate) hydrolase from Plasmodia of Physarum polycephalum

1995 ◽  
Vol 41 (13) ◽  
pp. 192-199 ◽  
Author(s):  
Christian Korherr ◽  
Michael Roth ◽  
Eggehard Holler
Keyword(s):  
Hydrophobic Interaction ◽  
Malic Acid ◽  
Physarum Polycephalum ◽  
Culture Medium ◽  
Specific Activity ◽  
Hydrophobic Interaction Chromatography ◽  
Reduced Form ◽  
Serine Esterase ◽  
Hydroxybutyric Acid ◽  
Ph Optimum

A 68-kDa extracellular glycoprotein from Physarum polycephalum that hydrolyses specifically poly(β-L-malic acid) by removing monomers of L-malic acid in an exolytic manner has been purified and characterized. The enzyme was purified 1740-fold from the culture medium by ammonium sulfate precipitation, hydrophobic interaction chromatography on butyl-Toyopearl, and gel permeation chromatography on Superdex 200 to a specific activity of 9.0 μmol∙min−1∙mg−1. The hydrolase was also purified from the cytosol, which contained 1 mg in 43 g cells in contrast to 1 mg extracellular enzyme in 28 L of culture medium. The pH optimum was pH 3.5 as a result of the effect of an acidic side chain on Vmax and the preferred binding of poly(β-L-malate) in the ionized form. Intracellular hydrolase was only marginally active on [14C]poly(β-L-malate) that had been injected into plasmodia. Poly(L-aspartate), poly(L-glutamate), poly(vinyl sulfate), and poly(acrylate) were neither bound nor degraded by the hydrolase. Poly(β-hydroxybutyric acid), which was considered the reduced form of poly(β-L-malate), was not a substrate. The enzyme is neither a metallo- nor a serine-esterase, and is distinct from poly(3-hydroxybutyric acid) depolymerases. It is related to a glucosidase with respect to hydrophobic interaction chromatography, the pH-activity dependence, and its inhibition with mercuribenzoate, N-bromosuccinimide, and D-gluconolactone, but not the use of the substrates.Key words: poly(β-L-malate), polymalatase, Physarum polycephalum, biodegradative polymer.

Purification of Myristoylated and Nonmyristoylated Neuronal Calcium Sensor-1 Using Single-Step Hydrophobic Interaction Chromatography

2000 ◽  
Vol 20 (1) ◽  
pp. 66-72 ◽  
Author(s):  
Jon R. Fisher ◽  
Yogendra Sharma ◽  
Sheila Iuliano ◽  
Robert A. Piccioti ◽  
Dimitri Krylov ◽  
...  
Keyword(s):  
Hydrophobic Interaction ◽  
Hydrophobic Interaction Chromatography ◽  
Single Step ◽  
Calcium Sensor ◽  
Neuronal Calcium Sensor

Purification of Tissue Activator Using Affinity Adsorption on Fibrin and Hydrophobic Interaction Chromatography. Effect of Fibrin on the Enzymatic Properties of the Activator

1975 ◽  
Author(s):  
P. Wallén ◽  
B. Wiman
Keyword(s):  
Ammonium Sulfate ◽  
Hydrophobic Interaction ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Hydrophobic Interaction Chromatography ◽  
Potassium Acetate ◽  
Heart Tissue ◽  
Isoelectric Precipitation ◽  
Affinity Adsorption ◽  
Rate Of Activation

A method for purification of pig heart tissue activator is described. The method is based on affinity adsorption on fibrin and on hydrophobic interaction chromatography. Acetone dry powder from pig heart tissue was extracted with potassium acetate buffer and fractionated with ammonium sulfate essentially as described by Bachmann et al. (Biochemistry 3, 1578, 1964). A fraction obtained between 14 and 66% saturation with ammonium sulfate was dissolved in phosphate buffer pH 7.0 and adsorbed with fibrin prepared from fibrinogen and thrombin free from plasminogen (about 0.7 g fibrin was used for a preparation prepared from 1 kg heart tissue). The fibrin was collected after adsorption for 2 hours and eluted with 2 M KSCN at pH 7. Practically all activity was precipitated by isoelectric precipitation at pH 4.2. Finally the activator was subjected to hydrophobic interaction chromatography on phenylethyl-Sepharose. The activity was eluted with a KSCN-gradient. The specific activity of the active fraction was about 23,000 Ploug units/mg. The over all yield was about 25%. One strong and three weak fractions were obtained on SDS-polyacrylamide gel electrophoresis. In pure systems tissue activator activates plasminogen at a very low rate. In presence of fibrin the rate of activation increases markedly. Even fibrinogen effects the activation but to a much smaller extent.

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