Potassium phosphate fractionation

Preparation of luciferase. Organisms were freeze-dried, powdered, and washed with ethyl acetate to destroy the majority of catalase activity. The washed residue was extracted with 50 mM potassium phosphate buffer, pH 6. The extract was fractionated by ammonium... 

Figure 14 Fractionation of 40-60-base oligodeoxyadenylates. Column 0.41 x 5 cm column packed with cross-linked and methylated PEI on Hypersil , 3 p. Eluent 50 mM potassium phosphate, 15% acetonitrile, pH 5.9 with a gradient from 200-500 mM ammonium sulfate. Flow rate 0.5ml/min. Oligomers of deoxyadenylic acid were fractionated up to a degree of polymerization of 60.180 (Reproduced with permission of Academic Press from Drager, R. R. and Regnier, F. E., Anal. Biodiem., 145, 47, 1985.)...

Figure 15.3 (a) Heat absorption in solutions of native RNase A (trace 1) and RNase A kept in 10% buffered formalin for 2 days (trace 2) and 6 days (trace 3) at pH 7.4 and 23°C. All samples were dialyzed against 75 mM potassium phosphate buffer (pH 7.4) prior to DSC. (b) Dependence of Td of the dialyzed RNase A samples on time of incubation in 10% buffered formalin at pH 7.4 and 23°C. (c) Heat absorption of solutions of formalin-treated RNase A fractions isolated by size-exclusion gel chromatography monomer (trace 1), dimmer (trace 2), and a mixture of oligomers with >5 cross-linked proteins (trace 3). Protein concentrations were 0.5 mg/mL. The thermal denaturation transition temperature (Td) is defined as the temperature of the maximum in the excess heat absorption trace associated with the protein s endothermic denaturation transition. See Rait et al.10 for details. 

Purified LCCs (10 mg dry weight) were incubated at 50°C for 3 h with 50 units of each enzyme alone and in specified combinations in a total volume of 1.0 mL. Reaction mixtures contained a final concentration of 20 mM potassium phosphate buffer, pH 6.0. Enzyme digests were then fractionated on columns of Sephadex G-50 or Sephadex G-10 as described above. 

Apparent first-order rate constants for peroxynitrite decomposition in various buffers versus pH. When peroxynitrite is fully protonated at acidic pH, the decomposition rate is constant. The breakpoint in the curve identifies the pK, of peroxynitrite since a larger fraction present as an anion slows the rate of decomposition. In 50 mM potassium phosphate, the apparent pK, is at 6.8 and is not affected by temperatute (Koppenol, 1993). The rate of decomposition is not affected hy DMSO, mannitol, or ethanol. As shown in Fig. 28, many buffers can slightly accelerate the decomposition of peroxynitrite and the rate of decomposition reaches a maximum at high buffer concentrations. When these maximal rates are plotted as a function of pH, peroxynitrite exhibits a second pK, of approximately 8.0. 

Fig. 3. Sephadex G-100 chromatography on DNase I, inhibitor II, and mixture containing the two proteins. (A) DNase I only, (B) inhibitor II only, (C) and (D) both components with different molar excess of inhibitor, (E) equimolar amounts of inhibitor and enzyme, (F) and (G) both components with a different excess of enzyme. Absorbance at 215 nm (solid line) was measured after 20-fold dilution with water using a similarly diluted blank of the elution buffer (0.5 M potassium phosphate, pH 7.6). Each chromatogram was analyzed for DNase activity ( ), inhibitor activity (O), and for the presence of DNase-inhibitor complex, in this figure represented as DNase I activity which was measured on samples of the fractions after adjustment of the pH to 3.5 with HC1 (V). [From Lindberg (34). Copyright 1967 by the American Chemical Society. Reprinted by permission of the copyright owner.]...

Figure 4. Affinity chromatography of diol dehydrase on the adenosyl form of II (37). About 1 unit of enzyme was applied to 1 mL of packed corrinoid gel in 0.1 M potassium phosphate buffer (pH 8.0) containing 2% 1,2-propanediol, in a total volume of 2 mL. Affinity chromatography was carried out as described in the text. Two-milliliter fractions...

Protein Production, Isolation, and Purification. The expression and purification of chicken lysozyme mutant proteins in yeast are performed as described by Malcolm et al. with the following modifications. The 50-ml minimal medium second seed yeast culture is used to inoculate a 2.8-liter Fembach flask containing 500 ml of 1% yeast extract/2% Bacto-peptone/ 8% glucose (w/v) medium and is then incubated for 7 - 9 days at 30°. Cells are harvested, washed twice with 60 ml of 0.5 M NaCl, and collected by centrifugation. The supernatants are pooled, diluted 5-fold with deionized water, and loaded onto a 20-ml column of CM Sepharose Fast Flow (Pharmacia, Piscataway, NJ) equilibrated with 0.1 M potassium phosphate, pH 6.24. The column is washed with the same buffer, and lysozyme is eluted with 0.5 M NaCl/0.1 M potassium phosphate, pH 6.24. Fractions are assayed by activity (decrease in A450 of Micrococcus lysodeikticus cell wall suspensions per minute). Fractions containing lysozyme are concentrated in Centricon-10 (Amicon, Danvers, MA) filter units, washed with 0.1 M potassium phosphate buffer, pH 6.24, and stored at 4°. The protein concentration is determined from e 1 = 26.4.15... 

Figure 5.9 Enzymatic activities of fractions following HPLC chromatography. A partially purified preparation was fractionated by ion-exchange HPLC (AX-300) with a mobile phase of 0.1 M potassium phosphate. Proteins were eluted with a gradient of sodium acetate. Column eluent was monitored at 280 nm. Fractions were collected, and each fraction was assayed for three different activities.

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,... 

Miyazaki et al. (1966, 1967 Miyazaki and Takemura 1966) have used column chromatography on DEAE-Sephadex A-25 to fractionate tRNA from Torulopsis utilis. They were able to purify an alanine, a valine and an isoleucine tRNA using 3 elution systems 1) Linear gradient of ammonium sulphate in 0.02 M potassium acetate, 1% dimethylformamide, pH 5.3 2) Linear gradient of KCl in 1 M potassium phosphate + 5% dimethylformamide, pH 6.1 or 6.2 at 30°C ... 

The performance of the XL cross-axis CPC, equipped with a pair of columns with a 165-mL capacity, was evaluated for purification of lactic acid dehydrogenase (LDH) from a crude bovine heart filtrate. Successful separation of the LDH fraction was achieved with 16% (w/w) PEG 1000-12.5%(w/w) potassium phosphate at pH 7.3. The separation was performed at 500 rpm at a flow rate of 1.0 mL/min using the potassium phosphate-rich lower phase as a mobile phase. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the LDH fractions showed no detectable contamination by other proteins. The enzymatic activity was also preserved in these fractions. 

The XLL cross-axis CPC, with a 250-mL capacity column, was used for the purification of recombinant enzymes such as purine nucleoside phosphorylase (PNP) and uridine phosphorylase (UrdPase) from a crude Escherichia coli lysate. The polymer-phase system used in these separations was 16% (w/w) PEG 1000-12.5% (w/w) potassium phosphate at pH 6.8. The separation was performed at 750 rpm at a flow rate of 0.5 mL/min using the upper phase as a mobile phase. About 1.0 mL of crude lysate, containing PNP in 10 mL of the above solvent system, was loaded into the multilayer coil. Purified PNP was harvested in 45-mL fractions. The SDS-PAGE analysis clearly demonstrated that PNP was highly purified in a one-step elution with the XLL cross-axis CPC. 

Fig. 3 Countercurrent chromatographic separation of HDL-LDL and VLDL-serum protein fractions from human serum with an aqueous polymer phase system. Column 2.6mm I.D. PTFE single-layer coil (x2) with 60-mL capacity sample 4 mL of human serum solvent system 16% PEG 1000-12.5% dibasic potassium phosphate at pH 9.2 mobile phase lower phase flow rate 0.5 mL/min revolution speed 500 rpm. SF = solvent front, UP = starting point of the reversed elution mode with the upper phase.

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.)... 

Fig. 4 Stepwise elution profile of HDLs and LDLs by hydroxyapatite chromatography. Column Bio-Gel HTP DNA-grade hydroxyapatite (5.0 x 2.5 cm I.D.) eluents 75 and 290 mM potassium phosphate buffers at pH 7.4 flow rate 1.0 mL/ min sample 1.4 mL concentrated HDL-LDL CCC fraction.

---