Calcium phosphate protein chromatography

Hydroxylapatite column chromatography relies on the selective adsorption of the protein onto the surface of calcium phosphate. The final stage of purification, on a high-performance DEAE column, is carried out just prior to crystallization of the protein. 

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. 

H18. Hjerten, S., Calcium phosphate chromatography of normal human serum and of electrophoretically isolated serum proteins. Biochim. Biophys. Acta 31, 216-235 (1959). 

Til. Tiselius, A., Hjerten, S., and Levin, O., Protein chromatography on calcium phosphate columns. Arch. Biochem. Biophys. 65, 132-155 (1956). 

A type of column chromatography that uses a calcium phosphate gel called hydroxyapatite has been especially useful in nucleic acid research. Because hydroxyapatite binds to double-stranded nucleic acid molecules more tenaciously than to single-stranded molecules, double-stranded DNA (dsDNA) can be effectively separated from single-stranded DNA (ssDNA), RNA, and protein contaminants... 

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... 

Hydroxyapatite (HA) chromatography is a typical I didn t know what else to do method and thus most purifiers use it as the last step. HA chromatography is an inexpensive and easy method that can process large amounts of protein. However, the purification factors are often bad (2 to 5) and the yields moderate (40 to 60%). With acidic proteins, HA sometimes exhibits stunning purification effects. This is especially true for preparations that are prepurified via lEC, because HA separates acidic proteins by principles other than an ion exchanger. HA is a calcium phosphate mineral that binds proteins via two mechanisms (Figure 5.5). 

Cystalline hydroxylapatite [Cai0 (P04)6 (OH)2] is an adsorbent used to separate mixtures of proteins or nucleic acids. The mechanism of adsorption is not fully understood but is thought to involve both the calcium ions and phosphate ions on the surface and to involve dipole-dipole interactions and possibly electrostatic attractions. One of the most important applications of hydroxylapatite chromatography is the separation of single-stranded from double-stranded DNA. Both forms of DNA bind at low phosphate buffer concentrations but as the buffer concentration is increased further, double-stranded DNA is released. The affinity... 

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