Calcium phosphate gels and

Adsorptive Properties. Under certain conditions of pH and low ionic strength, certain proteins will be adsorbed by various substances. Calcium phosphate gel and alumina Cy gel, for example, are frequently used to adsorb specific proteins from heterogeneous mixtures. The adsorbed proteins can frequently be released from the insoluble support either by altering the pH or increasing the ionic strength. Thus, purification can be obtained by removing either the extraneous unwanted proteins or the desired protein with the gel. 

Wang et al. 146) reported in 1956 the isolation of succinate dehydrogenase from heart muscle preparations treated with succinate and cyanide. The enzyme was extracted with 20% aqueous ethanol at pH 9.0. The final product, after adsorption on calcium phosphate gel and ammonium sulfate fractionation, was stated to be electrophoretically homogeneous and to contain 1 mole of flavin and 4 g-atoms of iron per 140,000-... 

Lactate dehydrogenase is found in the cytoplasm and can be liberated into solution when the cells are broken with a homogenizer or by osmotic shock. The procedure introduced by Straub (5) consisted of extraction of the enzyme, adsorbing onto calcium phosphate gel, and fractionating in a series of acetone and ammonium sulfate steps. The method of Jecsai 106) gives crystalline M4 enzyme from pig skeletal muscle simply by repeated ammonium sulfate fractionation. Although much refinement was achieved with these methods, and multienzyme isolation procedures were... 

Itagaki and Hager have crystallized cytochrome 6-562 from E. coli 172). Purification involved chromatography on calcium phosphate gel and ammonium sulfate fractionation 172,173). Both the oxidized and reduced forms of the cytochrome were crystallized. 

An enzyme catalyzing the formation of CHjOH-FH4 has been observed in pigeon liver 53). This enzjune was partially purified from acetone powder extracts by adsorption and elution from calcium phosphate gel, and by salting-out at 55-75 % saturation of (NH4)2S04. The assay of this enzyme could be performed by a shift in the ultraviolet absorption of FH4 (Xm x = 298 m/i) to that of the formaldehyde adduct (X uix = 290 nut). Osborn et al. 63) report that only a single product is formed by the enzymic reaction. Blakley found a change in the absorption maximum from 298 m/t to 294 m/t on the mere addition of formaldehyde (10 M) to FH4. 

Purification of the enzyme from extracts of E. coli was carried out by precipitating nucleic acids with MnCU, adsorbing the enzyme on calcium phosphate gel and eluting with phosphate buffer, and then fractionation with (NH4)sS04 between 0.32-0.45 saturation. This procedure produced roughly a tenfold enrichment of enzyme activity. The reductase has its pH optimum at about 8.5. Michaelis constants at pH 8.0 were determined to be 5.5 X 10 M for shikimic acid and 3.1 X 10 M for TPN. (Because of the scarcity of dehydroshikimic acid, the reaction was followed only in the reverse direction.)... 

Macmillan and coworkers51 105 106 purified pectinesterase produced by Clostridium multifermentans by using practically all of the available methods and materials (calcium phosphate gel, DEAE-cellulose, DEAE-, QEAE-, CM-, and SE-Sephadex, Sephadexes G-75, G-100, G-150, and G-200, Sepharose 4B, and zonal centrifugation). However, they could not separate pectinesterase from exo-pectate lyase, and, hence, they postulated that either (a) a complex of the two enzymes having an apparent molecular weight of 400,000 exists, or (b) the two enzymes are identical in their molecular species. On the basis of the mode of action of this pectinesterase in comparison with that of those from tomatoes and from Fusarium ox-ysporum, the existence of a complex of pectinesterase and exopectate lyase in Clostridium multifermentans appears to be the more probable. 

From a theoretical point of view, the gel layer is a barrier that reduces further hydrolysis of the silicate network, and is supposed to be more stable than the glass matrix, thus reducing the overall rate of corrosion. However, gel exfoliation may momentarily re-activate corrosion, at least locally. No clear trend was observed for the presence of the crystalline secondary phases identified at the surface of the corroded HT samples. The most abundant minerals are aluminosilicates, calcium phosphates, Fe- and Mg-rich minerals, and zeolites their role in the scavenging or release of metals remains ambiguous, although many mineral phases identified bear traces of metals. 

Eigel, W. N. and Randolph, H. E. 1974. Preparation of whole 7-casein by treatment with calcium phosphate gel. J. Dairy Sci. 57, 1444-1447. 

The combined extracts, which represented a 22-fold purification from the prostate, were dialyzed for 24 hours against distilled water at room temperature. The material inside the cellophane bag separated into a precipitate, which accounted for 60-70% of the total protein, and a clear supernatant containing over 85% of the activity and representing a 41-fold purification. This supernatant was then mixed with calcium phosphate gel at pH 7.5 and filtered. The filter cake was eluted with 0.02 M sodium citrate at pH 7.0, and the cake was washed with distilled water. The combined eluate, which showed an 81-fold purification, was concentrated by lyophilization. The enzyme solution was... 

The dialyzed enzyme solution was now subjected to a repetition of the preceding procedures admixture of suflBcient calcium phosphate gel to adsorb protein but leave the enzyme in solution centrifugation and addition of more gel to the supernatant to adsorb the enzyme elution of the enzyme from the gel with a mixture of 0.15 M acetate and 0.015 M citrate at pH 4.5 addition of solid ammonium sulfate to the eluate to 55% saturation and precipitation of the enzyme. At this stage, the purifications ranged from 650- to 1100-fold with a recovery of approximately 20-30% of the activity present in the crude red cell hemolysate. Solution of this precipitate, dialysis treatment with solid ammonium sulfate and collection of the precipitate appearing between 40 and 55% saturation yielded a preparation that represented a 1500-fold purification. The preparations were stable when left sedimented in the ammonium sulfate solution. 

A much purer preparation of acid phosphatase from horse erythrocytes was obtained by Ito et al. (12) by adding the DEAE-chromatography procedure to the method of Tsuboi and Hudson (T2). Since this procedure may be applicable to human erythrocytes, it will be mentioned briefly. One liter of horse erythrocytes was washed and lysed by the addition of 4 liters of distilled water. One liter of calcium phosphate gel suspension was added to the hemolysate to remove most of the nonenzymatic protein, and the mixture was centrifuged. Five liters of the gel suspension were added to the supernatant, resulting in the adsorption of the enzyme. The enzyme was eluted with citrate-acetate buffer, pH 4.5, and solid ammonium sulfate was added to the eluate up to 60% saturation. The precipitate was collected, dissolved in 40 ml of water, dialyzed against water at 5°C for 10 hours, and again subjected to calcium phosphate gel adsorption, elution, and precipitation with solid ammonium sulfate to 60% saturation. 

Hydroxyapatite. A calcium phosphate gel used, in the case of nucleic acids, to selectively absorb duplex DNA-RNA from a mixture of single-stranded and duplex nucleic acids. 

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