Calcium phosphate elution from

Sample preparation is rather involved. A sample of urine or fecal matter is obtained and treated with calcium phosphate to precipitate the plutonium from solution. This mixture is then centrifuged, and the solids that separate are dissolved in 8 M nitric acid and heated to convert the plutonium to the +4 oxidation state. This nitric acid solution is passed through an anion exchange column, and the plutonium is eluted from the column with a hydrochloric-hydroiodic acid solution. The solution is evaporated to dryness, and the sample is redissolved in a sodium sulfate solution and electroplated onto a stainless steel planchette. The alpha particles emitted from this electroplated material are measured by the alpha spectroscopy system, and the quantity of radioactive plutonium ingested is calculated. Approximately 2000 samples per year are prepared for alpha spectroscopy analysis. The work is performed in a clean room environment like that described in Workplace Scene 1.2. 

The first demonstration of transhydrogenase activity in Pseudomonas fluoreacens by Colowick et al. (1) was carried out with a crude extract obtained from cells grown on citrate as the sole carbon source. This extract could be fractionated further by acetone precipitation followed by calcium phosphate adsorption and subsequent elution with potassium phosphate. A second acetone fractionation and calcium phosphate adsorption gave a total purification of about 200-fold. This preparation was devoid of dehydrogenase activity using glutamate, isocitrate, lactate,... 

Another example is in the separation of sulfate or phosphate from various cations. Samuelson devised a method for sulfur in pyrites that is based on the retention of iron(m) on a cation-exchange resin. The sulfuric add that passes through the colunm can be determined by the usual gravimetric method as barium sulfate. In a similar way, phosphate in phosphate rock can be determined by retention of calcium, magnesium, iron, and aluminum on a cation-exchange resin followed by deterlnination of the phosphate as magnesium pyrophosphate. The metal ions can be eluted from the column with 4 M hydrochloric acid. 

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. 

As a fractionation method, I developed chromatography of nucleic acids on hydroxyapatite, a calcium phosphate which had been used by Tiselius et al. (1956) for the fractionation of proteins. Previous observations (Bernardi and Cook, 1960a,b,c) that hydroxyapatite was particularly good as a chromatographic substrate for fractionating of phospholipoproteins characterized by different phosphorylation levels convinced me to try it on DNA. The main discovery was that hydroxyapatite could fractionate single- from double-stranded DNA (Fig. 1.4 left panel), the former being eluted by a lower phosphate... 

Peng P et al (2008) Concurrent elution of calcium phosphate and macromolecules from alginate/chitosan hydrogel coatings. Biointerphases 3(4) 105-116... 

The enzyme can be precipitated from the various culture filtrates with ammonium sulfate at 70 to 80% saturation. Clostridium perfringens hya-luronidase has been purified by (a) adsorption on calcium phosphate (163) (6) adsorption on kaolin C7, elution with disodium phosphate, and fractional precipitation of the eluate with ammonium sulfate (62) and (c) ethanol fractionation (17). The isoelectric point of this enzyme is pH 4.75 (62). Streptococcal, staphylococcal, and Clostridium welcMi filtrates have been purified by adsorption on ferric hydroxide (166) and by column chromatography and elution with 0.2 M disodium phosphate (167). Highly purified streptococcal hyaluronidase has been obtained by fractional precipitation first with ammonium sulfate and then with ethanol and methanol at different hydrogen ion concentrations (22). Pneumococcal hyaluronidase has been isolated from the autolyzed organisms (127,128, 158) as well as from the culture filtrate (105,185,194). 

Ichihara et reported that urocanate is oxidized by an enzyme system found in liver and the bacterium, Psevdommas aeruginosa. The enzymes from both sources have been considerably purified by isoelectric precipitation, ammonium sulfate fractionation, and adsorption and elution from calcium phosphate gel. The d ree of enrichment cannot be determined from the published data. The oxidative activity, usually, is markedly increased by addition of EDTA. 

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.)... 

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