Sephadex gel chromatography

Figure 3.19. Fractonation of casein by Sephadex gel chromatography. (From Nakai et al. 1972. Reprinted with permission of the American Dairy Science Association.)...

Polymerization of benzylpenicillin has been described by several authors (Batchelor et al. 1967 Stewart 1967 a de Weck et al. 1968 Butcher and Stewart 1969, 1970 Dewdney et al. 1971 Smith et al. 1971 Smith and Marshall 1971 Bungaard and Larsen 1977). Polymerization probably involves hydrolysis of the beta-lactam ring to form penicilloic acid, followed by acylation of the exposed thiazolidine nitrogen by another intact penicillin molecule or a benzyl-penicillenic acid derivative (Stewart 1973 Smith and Marshall 1971 Schneider and de Weck, 1970) (Fig. 8) Such polymers have been isolated by Sephadex gel chromatography (Smith et al. 1971) or by high-pressure liquid chromatography (Bundgaard 1977 b). Polymers have been isolated also from other semisynthetic penicillins such as hetacillin and carbenicillin (Smith et al. 1971), methicillin and pheneticillin (Butcher and Stewart 1970). 

Mechanism of Inhibition, Additional tests indicated that inhibition by conqpound 1 was occurring via a suicide-like mechanism (1-4 7) Sephadex gel chromatography of the inactivated enzyme did not restore activity demonstrating irreversibility of the inhibition. The inhibition was shown to be active site-directed by co-preincubating the AChE with 1 mM of its substrate ACh and 40 iM of compound 1. The observed protection (>90%) against inactivation indicated inhibitor specificity for the active site. 

Sodium taurocholate [145-42-6] M 555.7. Purified by recrystn and gel chromatography using Sephadex LH-20. 

Bromelain (anti-inflammatory Ananase from pineapple) [37189-34-7] Mr 33 000, [EC 3.4.33.4]. This protease has been purified via the acetone powder, G-75 Sephadex gel filtration and Bio-Rex 70 ion-exchange chromatography and has Aj 20.1 at 280nm. The protease from pineapple hydrolyses benzoyl glycine ethyl ester with a Km (app) of 210mM and kcat of 0.36 sec. [Murachi Methods Enzymol 19 273 1970 Balls et al. nd Eng Chem 33 950 1941.]... 

Gel chromatography on Sephadex GlOO (2.8x50cm) of polysaccharide fraction I (Sample 2). The polysaccharide fraction was dissolved in a phosphate buffer at pH 6. After centrifugation, the supernatant was applied to the column at a flow rate of 0.8 ml/min. The elution was performed with a phosphate buffer and fractions of 10 ml each were collected. The refraction of each fraction was measured interferometrically. Fractions with coincident peaks were collected and analyzed for content of galacrturonic acid, neutral sugars and protein. 

Fractionations. Following lyophilization of the polar leachate fractions, separations by exclusion chromatography suggest three major molecular weight regions, labelled peak 1A, peak 1B and peak 2 (Figure 3). Retention times on a G—15 Sephadex gel indicate molecular weights of peaks 1A and 1B compounds to be between 600 and 1000. 

Figure 3. Exclusion chromatography of polar leachate through G-15 Sephadex gel. Retention time at flow rate of 4.8 ml/min in K50 Pharmacia column.

The highest degree of purification was achieved with extracellular endopectate lyase from the anaerobic bacterium Clostridium fel-sineum.Mi A 225-fold purified preparation, homogeneous in disc electrophoresis, was obtained by precipitation with ethanol, and chromatography on CM-cellulose and on Sephadex G-200. Its molecular weight (determined by gel chromatography) was 105,000. 

Dialysis for 6 months at RT of M Fe(N03)3 solution against bidistilled water of pH 5. This gives a mixture of polymeric particles and uniform, rod-like crystals of goethite. The goethite can be separated from the polymer by gel chromatography using a Sephadex 200 substrate (Van der Woude and de Bruyn, 1984). 

Dextrans of low molecular weight have also been fractionated by gel chromatography. Bremner and coworkers,67 in preparing (by alkaline degradation) dextrans of low molecular weight suitable for incorporation in the clinically important iron-dextran complex, fractionated their product on Sephadex G-50, with (O M sodium chloride as the eluant. Separate fractions, ranging in Mn (as determined by osmometry) from 1510 to 4860, were obtained in this way. 

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