Hydroxyapatite column chromatography

The enzyme was purified to 277 fold with very high specific activity, 1387 unit/mg protein by hydroxyapatite column chromatography (Figure 3). 

K21. Kostner, G. M., and Holasek, A., The separation of human serum high density lipoproteins by hydroxyapatite column chromatography. Biochim. Biophys. Acta 488, 417-431 (1977). 

Figure 9. Radiochemical and protein analysis of column fractions after hydroxyapatite column chromatography of plasmin-treated (3-casein for various reaction times , total activity or specific activity, Fraction 1 M, total activity, Fraction 2 , specific activity, residual /3-casein O, total protein, Fraction 1. Values for total protein and total activity have been adjusted to correspond to trie same amount of total protein (21.92 mg) applied to each column (28).

An Fe-S protein has been found to be present also in the b /f complex of chloroplasts and cyanobacteria. This protein has been dissociated from the complex with Triton X-100 and hydroxyapatite column chromatography, and was shown to be associated with the 20000 Da subunit in chloroplasts [111] and the 22000 Da one in A. variabilis [128]. In all cases the dissociation resulted in an irreversible loss of activity the involvement of the Fe-S protein in electron transport was also proved by the inhibition by an antibody raised against the Triton isolated protein (but not by one against the SDS denatured subunit) [129,130]. An oxidoreduction potential of 0.290 V was measured in intact chloroplast membranes and in the complex or in the isolated homogeneous preparation the potential was pH independent below pH 8 [111]. 

After concentration, the solubilized enzyme was applied to a gel filtration column. The enzymes from pear and tomato were purified further (1.5-3 fold) by isoelectric focusing (20,25). Chloro-plastic hydroperoxide lyase was solubilized from tea leaves with Tween 20 and partially purified 8.5-fold with 34% recovery by hydroxyapatite column chromatography (33). Although attempts at... 

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. 

Interest in the nature of interactions between shortchain organic surfactants and large molecular weight macromolecules and ions with hydroxyapatite extends to several fields. In the area of carles prevention and control, surfactant adsorption plays an important role in the Initial states of plaque formation (1-5) and in the adhesion of tooth restorative materials ( ). Interaction of hydroxyapatite with polypeptides in human urine is important in human biology as hydroxyapatite has been found as a major or minor component in a majority of kidney stones ( 7). Hydroxyapatite is used in column chromatography as a material for separating proteins (8-9). The flotation separation of apatite from... 

Narita H, Takeda Y, Takagaki K, Nakamura T, Harata S, Endo M (1995) Identification of glycosaminoglycans using high-performance liquid chromatography on a hydroxyapatite column. Anal Biochem 232 133-136... 

Red-cell (dark adapted) Blepharisma japonicum were cultured in Pisa, in the dark, at 23 °C, in the presence of the Enterobacter aerogenes bacterium as food supply [7]. Blue-cell (light adapted) Blepharisma japonicum were produced by in vivo photoconversion of blepharismin into oxyblepharismin under a low intensity cold white lamp (below 10 W/m2). Blue cells were washed, collected by low speed centrifugation and resuspended in a 20-mM sodium cholate solution. The chromoprotein was obtained by FPLC chromatography of this preparation, on a hydroxyapatite column. The applied eluent was a phosphate buffer (pH 7.4), first 0.05 M and then 0.2 M. This ionic strength step affects the affinity of the biomolecules with the hydroxyapatite [8]. 

R Kasai, H Yamaguchi, O Tanaka. High performance liquid chromatography of glycosides on a new type of hydroxyapatite column. J Chromatogr 407 205 -210, 1987. 

Bemardi, G., Chromatography of nucleic acids on hydroxyapatite columns, Methods Enzymol., 21D, 95, 1971. 

Bernardi, G., Giro, M. G., and Gaillard, C. (1972). Chromatography of polypeptides and proteins on hydroxyapatite columns Some new developments. Biochim. Biophys. Acta 278, 409-420. 

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

The CCC fractions, HDL-LDL and VLDL-serum proteins, were each separately dialyzed against distilled water until the concentration of the potassium phosphate was decreased to that in the starting buffer used for the hydroxyapatite chromatography. These two fractions were concentrated separately by ultrafiltration. The concentrates of both fractions were chromatographed on the hydroxyapatite column. Fig. 4 shows the elution profile on hydroxyapatite obtained from the HDL-LDL fraction. A 1.4-mL volume of the concentrate was loaded onto a Bio-Gel HTP DNA-grade column (5.0 x 2.5 cm I.D.)... 

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. 

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... 

Starting from the fact that hexacoordinated ruthenium salts of the general formula [Ru(bpy)2L2]X2 or [Ru(phen)2L2]X2 are capable of forming specific interactions with double stranded DNA, Strekas suggested that the two (A) and (A) enantiomers could be separated by using column chromatography with double-stranded DNA immobilized on hydroxyapatite as the stationary phase. 

Sixteen glycoproteins have been separated from rat brain by a combination of hydroxyapatite and lectin affinity chromatography. A simple reproducible procedure for the fractionation of brain glycoproteins using hydroxyapatite-gel column chromatography has been developed. ... 

Adsorption chromatography usually employs microcrystalline hydroxyapatite columns that adsorb proteins vmder conditions above pH 5. Gradient or step-wise elution with increasing concentration of eluting agent in the mobile phase may be used to release the enzyme required. 

Identification and quantitative estimation of the components of nucleotide mixtures can be made by chromatographic techniques (which sometimes include the use of hydroxyapatite) (Figure 14.5). Nucleotide hydrolysis products can be separated by column chromatography and their amounts estimated by ultraviolet absorption, thus giving the overall base composition of the nucleic acid. Ultracentrifuging and gel electrophoresis based on polyacrylamide or agarose gels are also widely used. 

CCC is a very useful technique for the separation and fractionation of human semm lipoproteins. The HDL-LDL and VLDL-serum protein fractions were directly obtained from human serum using a solvent system composed of 16% PEG 1000-12.5% potassium phosphate at pH 9.2. The separated HDL-LDL and VLDL-serum protein fractions were loaded onto hydroxyapatite columns and separated into HDLs, LDLs, VLDLs, and serum proteins, respectively. The combination of CCC and hydroxyapatite chromatography is a usefid method for the separation of the three main classes of lipoproteins from human seram, without prior ultracentrifugation. 

G. Bernardi, Chromatography of nucleic acids on hydroxyapatite column, in L. Grossman and K. Moldave (eds.). Methods in Enzymology, Vol. 21, Academic Press New York, pp 92-140, 1971. 

Hydroxyapatite columns have been shown to be helpiiil for the purification of proteins, especially when other types of chromatographic columns were not successful [12]. Although the separation mechanism of hydroxyapatite chromatography is not understood, it is known that it is not an ion-exchange mechanism. Applications of hydroxyapatite HPLC to the analysis of glycoproteins including... 

Figure 3 Hydroxyapatite chromatography of serum glycoproteins on a spherical hydroxyapatite column (0.75 x 10 cm, Toa Nenryo K.K., Tokyo), with guard column. The column was eluted at a flow rate of 0.8mL/min for 60 min with a linear gradient from 0.01 M sodium phosphate buffer, pH 6.8, containing 0.3 mM CaClg to 0.5 M sodium phosphate buffer, pH 6.8, containing 0.01 mM CaCl2. The abbreviations in Fig. 2 are used. The following abbreviations are used in the chromatogram of ceruloplasmin Apo, apoceruloplasmin I, carbohydrate variant type I II, carbohydrate variant type II. A sample up to 20 mg could be applied without any loss of resolution.

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