Sephacryl S-200 HR Gel Filtration Research Articles

Purification and Characterization of Phytase from Bran of Triticum aestivum L.cv. Nourin #61.

Two phytase isozymes (PHY1 and PHY2) were purified homogeneously from bran of Triticum aestivum L. cv. Nourin #61 by (NH4)2SO4 fractionation, methanol fractionation, Sephacryl S-200 HR gel filtration chromatography, DEAE-TOYOPEARL chromatography, CM-TOYOPEARL chromatography and second Sephacryl S-200 HR gel filtration chromatography. Molecular weights of the two enzymes were 71,000 and 66,000 by gel filtration, and 68,000 and 66,000 by SDS-PAGE, respectively. Optimum pH and temperatures were 6.0 and 45°C for PHY1, and 5.5 and 50°C for PHY2. The activity of both phytases was stable at pHs between pH 4.0-7.0 and below 40°C. The Km values for myo-inositol hexakisphosphate (IHP) were 0.48 μM for PHY1 and 0.77 μM for PHY2. The Ki values for Pi were 2.69 mM for PHY1 and 6.59 mM for PHY2. Both phytases showed relatively high specificity for IHP.

Purification and properties of beta-N-Acetylhexosaminidase from cabbage.

beta-N-Acetylhexosaminidase was purified from the extract of cabbage by sequential steps of ammonium sulfate fractionation, chromatofocusing, DEAE-Sepharose CL-6B ion exchange chromatography and Sephacryl S-200 HR gel filtration. By these steps, the purity of the enzyme increased by 256 fold with a recovery of 8%. The purified enzyme was homogeneous as examined by native PAGE. It showed an optimal pH of 4, an optimal temperature of 60 degrees C and a Km of 0.94 mM for hydrolysis of pNp-beta-GlcNAc. The molecular mass of the enzyme determined from filtration through Sephacryl S-200 was 150 kDa. Three subunits with molecular mass of 64, 57 and 51 kDa were observed as determined by SDS-PAGE. NBS (0.025 mM), DEPC (3 mM) and WRK (30 mM) significantly inhibited the activity of the enzyme. The enzyme also showed activity toward pNp-beta-GalNAc, N,N'-diacetylchitobiose, N,N',N"-triacetylchitotriose and N,N',N",N"'-tetraacetyl chitotetraose but showed no activity toward pNp-alpha-GlcNAc, chitin and ethylene glycol chitin.

Purification and properties of chitinase from cabbage stems with roots.

Chitinase has been purified from the extract of cabbage stems with roots through successive steps of ammonium sulfate fractionation, Sephadex G-75 gel filtration, chromatofocusing and Sephacryl S-200 HR gel filtration. By these steps, the purity of the enzyme increased by 63 fold and the recovery of the enzyme activity was 18%. The purified enzyme was homogeneous when analyzed by SDS-PAGE. It showed an optimal pH of 6 and optimal temperature of 60 degrees C for hydrolysis of ethylene glycol chitin (EGC). The molecular mass of the enzyme was 41 kDa, as determined by SDS-PAGE. Heavy metal ions (1.5 mM) Ag+, Hg2+ and Fe2+, and chemical modification agents NAI (1 mM), NBS (0.5 mM) and CHD (0.5 mM) significantly or completely inhibited the activity of the enzyme. Substrate EGC at high concentrations also inhibited the activity. BSA (0.05%), Triton X-100 (0.5%) and glycerol (50%) provided significant protection of the enzyme from freezing inactivation.

Platelet-activating factor (PAF) concentration and PAF acetylhydrolase activity in cerebrospinal fluid of patients with subarachnoid hemorrhage.

The authors studied the sequential changes in platelet-activating factor (PAF) and PAF acetylhydrolase in the cerebrospinal fluid (CSF) of patients with subarachnoid hemorrhage (SAH). Levels of PAF in CSF showed a gradual increase after the onset of SAH, with a subsequent decrease. The PAF concentration between 5 and 9 days after SAH was greater in patients with cerebral infarction due to vasospasm than in patients without infarction. Conversely, PAF acetylhydrolase activity decreased gradually after SAH, then increased. The enzyme activity for the same period was smaller in patients with cerebral infarction than in patients without infarction. The distribution of the days of maximum PAF concentration and minimum PAF acetylhydrolase activity did not differ significantly between the two groups. The CSF as a source of PAF acetylhydrolase activity gave an apparent Michaelis constant value of 90.8 microM and a maximum velocity of 0.2 nmol/min/mg. The optimum pH level for the PAF acetylhydrolase activity obtained from CSF was 6.5. The enzyme activity of CSF increased, depending on the incubation temperature, ranging from 25 degrees to 45 degrees C. Ethylene-glycol tetra-acetic acid (1 mM) was found to inhibit PAF acetylhydrolase activity in CSF obtained from patients with SAH. Unaltered PAF acetylhydrolase activity was inhibited by adding an aliquot of CSF and minimum PAF acetylhydrolase activity decreased following SAH. Two peaks of inhibitory activity were detected on Sephacryl S-200 HR gel filtration: one was eluted in void volume and the other with an apparent molecular mass of 13 kD. The inhibitory activity was very labile and was lost completely within 3 days of incubation at 4 degrees C. The regulation of the PAF concentration in the CSF of SAH patients is discussed.

A new endo-beta-galactosidase acting on the Gal beta 1-3Gal linkage of the proteoglycan linkage region.

A new type of endo-beta-galactosidase acting on the linkage region of peptidochondroitin sulfate was isolated from the mid-gut gland of the mollusk Patinopecten. The purification procedure included ammonium sulfate precipitation, Sephacryl S-200HR gel filtration, DEAE-Sephacel chromatography, and TSKgel Phenyl-5PW RP high performance liquid chromatography. The purified enzyme was free from exoglycosidases, sulfatases, and phosphatases. The specificity of the enzyme was as follows. 1) It acted on the internal galactoside linkage of sugar chains; 2) it specifically hydrolyzed the galactosylgalactose (Gal beta 1-3Gal) linkage, but not the galactosylxylose (Gal beta 1-4Xyl) linkage in the linkage region of peptidoglycans; 3) the enzyme activity was unaffected by the type of glycosaminoglycan, chondroitin sulfate, dermatan sulfate or heparan sulfate used as a substrate; 4) keratan sulfate and some oligosaccharides from glycolipid were not degraded by the enzyme. These properties of the endo-beta-galactosidase characterize it as a new endo-beta-galactosidase with unique specificity.