Three-step Purification Research Articles

Three-step purification of glucocorticoid receptors from rat liver.

The 90 000-Mr glucocorticoid receptor was purified to homogeneity according to sodium dodecylsulfate/polyacrylamide gel electrophoresis. An affinity column containing either dexamethasone-17 beta-carboxylic acid or dexamethasone-21-methanesulfonate bound to the matrix with the help of a disulfide bond is used in this study. Using this affinity matrix, in a single step, 8700-fold purification of glucocorticoid receptor from rat liver cytosol could be achieved. Following the method of activation and DNA-cellulose chromatography the 90 000-Mr receptor subunit was purified to homogeneity.

An improved three-step method for the purification of the third component of human complement

This paper describes a three-step purification method for the third component of human complement (C3) from plasma. The method consists of PEG precipitation, DEAE-cellulose chromatography and preparative isofocusing in a granulated dextran gel, with pH range 5–8. From this last step, a highly purified form of native C3 was obtained as indicated by immunoelectrophoresis and polyacrylamide gel electrophoresis analysis. The average final recovery was 20%.

Improved method for purification of enterotoxin from Clostridium perfringens type A.

The purification procedure of Clostridium perfringens type A enterotoxin has been improved. The cell sonic extract was precipitated twice with ammonium sulfate, first 40% saturated to concentrate the enterotoxin and then 15% saturated. The two precipitations were followed by gel filtration on Sephadex G-100. The enterotoxin appeared to be homogeneous on 7% polyacrylamide gel electrophoresis after this three-step purification procedure, with a recovery of 56% and a 12.3-fold purification. The solubility properties at different pH values, temperatures, and ammonium sulfate concentrations are also given as basis for the purification procedure.

Three-step purification of retinol-binding protein from rat serum

Rat serum retinol-binding protein has been purified to apparent homogeneity in high yield by a new procedure utilizing three simple steps: DEAE-cellulose chromatography at pH 6.0, Sephadex G-75 gel filtration in the presence of 3.0 M urea, and finally DEAE-cellulose chromatography at pH 8.3. The second step accomplished the dissociation and separation of retinol-binding protein from its complex with prealbumin; this represents a substantial improvement over published procedures, in which sample recycling and preparative polyacrylamide gel electrophoresis were necessary. The purified retinol-binding protein was characterized by molecular weight measurement, fluorescence spectra and immunological properties.

The identity of the acid and alkaline phosphatases of human seminal plasma.

Examination of human seminal plasma showed that there was no p-nitrophenylphosphatase activity maximum at alkaline pH values. A constant ratio of enzyme activities in 8 split ejaculates, identical temperature dependence and copurification through a three-step purification procedure led us to conclude that the alkaline phosphatase in human semen is identical to acid phosphatase.

Mouse C4 (Ss): Three-Step Purification of Its C4c Fragment and Production of a Monospecific Antiserum

We report here that a large fragment of mouse C4 (C4c) can be easily purified from serum with good recoveries, and used to produce a monospecific antiserum. The method of isolation of C4c is based on the observation that C4 is easily activated and fragmented in mouse serum. The fragments bind to the C4-binding protein (C4-bp), a newly described macromolecular component of the complement system. The high m.w. complexes were precipitated by dialysis of the serum against 0.1 M acetate buffer, pH 5.0, and purified by passage of the dissolved precipitate through a Sephadex G-200 column. The complexes were dissociated at high salt concentration, and the C4 fragments were isolated by passage of the mixture through a second Sephadex G-200 column. One of the C4 fragments (C4c) was used to immunize rabbits, and a monospecific antiserum was obtained, which showed a reaction of identity between C4 and Slp. Therefore the C4c fragment of C4 and the Slp protein are structurally related.

Streptolysin S: Improved purification and characterization

A simple and efficient procedure for the production and purification of streptolysin S has been developed. Maximal production of the hemolysin occurred when rapidly grown streptococci were harvested 1 h after reaching the stationary phase and incubated with the inducer oligoribonucleotide. Ammonium acetate at 0.1 m in 50 m m potassium phosphate buffer, pH 6.8, was found to effectively protect streptolysin S from thermal inactivation, and was used in the buffer throughout purification. The three-step purification procedure resulted in preparations with the highest specific activity (1.2 × 10 7 HU per mg protein) ever reported, in recoveries ranging from 35 to 45%. The apparent molecular weights of streptolysin S and the carrier oligonucleotide were estimated as 15,000 and 7,100, respectively. The peptide nature of the active principle was confirmed by studies of the effects of various hydrolytic enzymes on streptolysin S; only pronase, chymotrypsin and subtilisin were found to inactivate the hemolysin. Amino acid analyses indicated that the active peptide consisted of 32 amino acid residues.

Purification, characterization, and amino acid sequence of rat anaphylatoxin (C3a)

C3a anaphylatoxin derived from the third component of complement has been isolated from rat serum and its complete amino acid seuqence determined. A three-step purification procedure was employed that consisted of gel filtration on Sephadex G-100, followed by chromatography of the anaphylatoxin-containing pool on carboxymethylcellulose. A subsequent separation on DEAE-Sephadex resolved C3a from minor contaminating peptides. Biological studies have shown that purified rat anaphylatoxin is approximately twice as active as human or porcine C3a when tested for smooth-muscle contraction. In addition to the active form of rat anaphylatoxin, a serum carboxypeptidase B inactivated form of C3a (C3ades-Arg) was purified from rat serum and utilized in subsequent structural studies. Sequence analysis of rat C3a was facilitated by a long automated Edman degradation which established the first 55 residues of the anaphylatoxin. Overlapping peptides were generated by cyanogen bromide and trypsin, and the resultant fragments were sequenced by either automated or manual Edman procedures. The primary structure of rat C3a is 70% identical to the previously determined structures of human and porcine anaphylatoxin. Antisera raised to the purified rat peptide do not cross-react immunologically by Ouchterlony analysis with either human or porcine C3a.

Bovine thyroxine-binding globulin: Purification and comparison of molecular weight and amino acid composition with human thyroxine-binding globulin

Bovine and human thyroxine-binding globulin were purified from serum by a three-step purification procedure which comprised affinity chromatography consecutively on thyroxine- and Concanavalin A-Sepharose and finally preparative polyacrylamide gel electrophoresis. The molecular weights of the two proteins were similar (54 000) as well as their carbohydrate contents while some differences in amino acid composition were found. Rabbit antiserum against bovine thyroxine-binding globulin reacted with human thyroxine-binding globulin with no sign of spur formation.

55] Structures and activities of protease inhibitors of microbial origin

Compared with inhibitors obtained from animal and plant tissues, which are mostly of macromolecular nature, the enzyme inhibitors found in microbial culture filtrates are relatively small, and protease inhibitors are widely distributed in various species of actinomycetes which also produce a number of proteases. These inhibitors are generally resistant to being metabolized by animals. Assay methods of testing activities of various proteases can be applied to search for inhibitors in culture filtrates which are heated (100 °) for 3 min prior to testing. The assay methods employed at the time of discovery of each inhibitor, special fermentation conditions, and methods for extraction and purification of the inhibitors are described in this chapter. The three-step purification process is also described in this chapter. Physicochemical properties, biological properties and activity, and kinetic properties have also been discussed in this chapter. Several assay procedures have also been elaborated.