Chromatography on hydroxyapatite

Hydroxyapatite occurs naturally as a mineral in phosphate rock and also constitutes the mineral portion of bone. It may also be used to fractionate protein by chromatography. 

Hydroxyapatite is prepared by mixing a solution of sodium phosphate (Na2HP04) with calcium chloride (CaCl2). A white precipitate known as brushite is formed. Brushite is then converted to hydroxyapatite by heating to 100°C in the presence of ammonia  

The underlying mechanism by which this substance binds and fractionates proteins is poorly understood. Protein adsorption is believed to involve interaction with both calcium and phosphate 

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Various chromatographic techniques may be utilized to purify urokinase further. Commonly employed methods include anion-exchange (DEAE-based) chromatography, gel filtration on Sephadex G-100 and chromatography on hydroxyapatite columns. Urokinase is a relatively stable molecule. It remains active subsequent to incubation at 60 °C for several hours, or brief incubation at pHs as low as 1.0 or as high as 10.0. 

Gorbunoff, M.J. (1990) Protein chromatography on hydroxyapatite columns. Methods Enzymd. 182, 329-339. 

Separate ss cDNA from duplex DNA RNA and RNA by chromatography on hydroxyapatite columns (Section 8.1.1.5) at 62°C in HAP buffer (0.12 M sodium phosphate, pH 7.5,1 mM EDTA and 0.2% SDS ss DNA will elute with several washes of HAP buffer). Wash the ss DNA again in the Centricon-30 with TRES. Remove the duplex from the column with > 0.4 M sodium phosphate. 

Three histone-specific acetyltransferases have been partially purified and characterized from rat thymus nuclei (225). The enzymes were extracted from rat thymus nuclei by sonication in the presence of 1M ammonium sulfate and separated into two active fractions (A and B) by DEAE-cellulose chromatography. Fraction B was further separated into two active fractions (Bi and B2) by gel filtration on Sephadex G-200. Each fraction was then purified further by chromatography on hydroxyapatite. The molecular weights, determined by Sephadex G-200 and by sucrose density gradient centrifugation, were 99,000, 110,000, and 92,000 for enzymes A, Bi, and B2, respectively. All three enzymes required acetyl CoA as acetate donor, and the activity of the enzymes was inhibited by p-chloromercuribenzoate. Acetyltransferase A preferentially acetylated histone I (FI) and also poly-L-lysine. Acetyltransferase Bi and B2 preferred histone H4 (other names IV, F2al) and did not acet-ylate poly-L-lysine and histone H3 (III, F3). In addition to c-N-acetyl-lysine, two other unidentified amino acid derivatives were obtained from a digest of histone H4 acetylated by the two B enzymes. 

D-Mannanase (j3-D-mannosidases) purified from the germinated seeds of Trifolium repens by a procedure that included chromatography on hydroxyapatite, gel filtration on acrylamide/agarose (Ultragel 5/4) and preparative polyacrylamide gel electrophoresis. The final purification has been completely resolved into two ]3-D-mannanases (I and II) with distinct specificities (see p. 468). 

After recombination of the inducing protein with the constituents of the aqueous phase in the presence of 6 M urea the inducing activity was again completely inhibited. This suggested the presence of an inhibitor. RNA could be separated from the inhibitory fraction without loss of activity by chromatography on hydroxyapatite (Bom et al, 1972b). 

Kurzban GP, Strobel HW (1986) Purification of flavin mononucleotide-dependent and flavin-adenine dinucleotide-dependent reduced nicotinamide-adenine dinucleotide phosphate-cytochrome P-450 reductase by high-performance liquid chromatography on hydroxyapatite. J Chromatogr 358 296-301... 

Ion-exchange, hydrophobic-interaction chromatography chromatography on hydroxyapatite and different types of affinity and pseudo-affinity chromatography are the most frequently used methods for isolation of nondenatured, physiologically active proteins. 

An arabinanase has been isolated from culture fluids of B. subtilis by fractional precipitation, repeated chromatography on hydroxyapatite, and gel filtration. The purified endo-enzyme hydrolysed arabinans to arabinose and arabinobiose, but was inactive towards phenyl a-L-arabinofuranoside, 4-nitrophenyl -D-galacto-pyranoside, arabinoxylan, and gum arable. 

The dextransucrase activity (mol. wt. 9.4 x 10 , pH optimum 5.5) from supernatants of cell-free cultures of Streptococcus mutans has been purified (1500-fold) by fractional precipitation, chromatography on hydroxyapatite, and isoelectric focusing (p/4.0). Electrophoresis on polyacrylamide gel showed that the preparation contained two enzymes having appreciably different specific activities. The dextransucrase possesses a broad temperature optimum (34—42 C), is active over a narrow range of pH, and is competitively inhibited by D-fructose. 

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