Ray K. Brown

Separator isoelectric focusing: An improved method of protein analysis and purification

Abstract Addition of small amphoteric substances or separators to the carrier ampholytes used in isoelectric focusing improves the separation of protein components. The technique has been applied to partially oxidized human hemoglobin, goat anti-bovine pancreatic oxidized ribonuclease, human γ-globulin, and barley β-amylase. Each of these proteins is a complex mixture of species which can be resolved selectively and reproducibly by this method. Superior separation is provided by this technique when compared to focusing in commercially available narrow-range ampholytes. Addition of separators usually results in the flattening of the pH profile of the gel. Inspection of the protein patterns indicates that no new protein bands arise, although different patterns of existing components are obtained by choice and concentration of separator as well as by concentration of carrier ampholytes. This technique, thus, offers the advantage of making conventional isoelectric focusing more versatile.

Transport of pyrimidine nucleosides across human erythrocyte membranes.

Abstract The transport of pyrimidine nucleosides in human erythrocytes has been shown to occur by a facilitated diffusion mechanism. Transport studies of these nucleosides are not complicated by metabolic conversions as in the case of the action of phosphorylases and deaminases on purine nucleosides. The transport carrier K m and v max were K m = 3.5, 1.7, 1.3 and 0.7 mM and v max = 16.5, 6.9, 4.7 and 10.0 mM/min for cytidine, cytosine-β-arabinoside, thymidine and uridine, respectively. The initial rates for nucleoside exit competition experiments are in good agreement with those calculated using the above K m s and v max s. Some preliminary evidence that the pyrimidine and purine nucleosides are transported by a common system is also presented.

Carrier ampholyte distribution in isoelectric focusing

Abstract The spatial distributions of individual components of carrier ampholyte mixtures obtained by isoelectric focusing in cylindrical (6 mm × 75 mm) polyacrylamide gels (PAG) and in a thin-layer Sephadex gel (TLSG) were determined by ion-exchange chromatography of the eluates of gel slices. A ca. 1-mm slice of PAG separating the A and B forms of β-lactoglobulin (isoelectric points of 5.21 and 5.34, respectively) was found to contain at least 12–14 ampholyte components out of a total of at least sixty in the ampholyte mixture (LKB Ampholine, pH 3.5–10, Lot No. 17). Fifteen to twenty components were found in each 2-mm slice of a sequence of six slices of PAG subsequent to isoelectric focusing of sperm whale myoglobin with the same ampholyte mixture. An ampholyte mixture prepared by the copolymerization of triethylenetetramine with acrylic acid was focused in TLSG on 20 cm × 5 cm plates and the eluates obtained from ca. 5-mm slices were analyzed by ion-exchange chromatography. The results were compared with the caramelization pattern obtained by the Felgenhauer technique. The spatial distributions of individual ampholyte components were broad with half-band widths of ca. 1 cm. The effects of sample load, ampholyte concentration, and duration of focusing on spatial distribution were investigated in the PAG focusing of [ 14 C]histidine. The narrowest and most symmetrical distributions were obtained with small sample load and high ampholyte concentration.

Interaction of a peptide with antibody to oxidized ribonuclease

Abstract Peptide 105–124 from oxidized bovine pancreatic ribonuclease was iodinated with 125 I by the chloromine T. and by the iodine monochloride methods. Its association constant for a fraction of γ-globulin from goat antiserum to oxidized ribonuclease was 3.10 × 10 6 M −1 and 1.83 × 10 6 M −1 by the two methods respectively. Both technics gave a heterogeneity index of 0.98.

Analysis of carrier ampholytes by ion exchange chromatography

Commercially available and synthetic wide-range and short-range ampholytes used in the isoelectric focusing of proteins were analyzed by ion-exchange chromatography. A pH gradient over the pH range 3.8 to 11.0 was used to elute the ampholytes from a column of a sulfonated polystyrene resin. The wide-range ampholytes were resolved into some 60 to 70 ninhydrin-positive components. The recovery obtained with the method was quantitative. Analysis of the cathodic and anodic fractions of wide-range ampholytes focused concurrently with whale myoglobin on a polyacrylamide gel, suggested that no simple relationship exists between the pI of the ampholyte components and the pH at which they are eluted from the ion-exchange column.

Immunochemical studies of hemoglobin and the role of the sulfihydryl groups

Abstract Rabbit antibody was prepared to a derivative of human hemoglobin A 0 in which the two reactive sulfhydryl groups were substituted with N -ethylmaleimide. This antibody reacted completely with hemoglobins A 0 , A 1 , S 0 and a disubstituted parahydroxymercuribenzoate derivative of A 0 , suggesting that these sulfhydryl groups are in an immunogenically. silent region. Hemoglobins F 0 and A 2 and derivatives of A 0 with increasing amounts of parahydroxymercuribenzoate cross reacted with the antiserum. Globin, α-chains, and β-chains gave little reaction.

Studies of the carboxyl terminal antigenic region of oxidized ribonuclease

Abstract The antigenic reactivity of the carboxy terminal region of oxidized bovine pancreatic ribonuclease has been examined using natural and synthetic peptides. Only partial inhibition of the precipitin reaction between oxidized ribonuclease and its antiserum can be achieved with tryptic fragments of the antigen. The amount of inhibition is independent of peptide concentration with high amounts of peptide. Peptides labeled with 3-H acetic anhydride bind to antibody. Inhibition of this binding by unlabeled fragments was followed by gel filtration to separate bound and unbound peptides. A number of peptides containing seven or more amino acids inhibit binding. Smaller peptides give marginal inhibition. Although the unlabeled carboxy terminal eicosapeptide gives almost complete inhibition, the fragments do not. The inhibition is concentration independent at high peptide concentrations. Binding was also studied by equilibrium dialysis with peptides containing 20 and 12 amino acids. Both had similar binding constants and a heterogeneity index of 0·99–1·0. The smaller peptide bound to only one-third of the antibody. These data suggest that antibody to the carboxy terminal region is a mixture of antibodies directed toward different portions of the site.

The immunologic role of methionine and cysteine residues in ribonuclease

Abstract Antibodies were prepared in rabbits and in a goat to oxidized bovine pancreatic ribonuclease, an unfolded form in which the methionines have been changed to methionine sulfone and the cystines to cysteic acid. Rabbit antibodies were also made to ribonuclease which had been reduced and carboxymethylated, a process which leaves the methionines intact but converts the cystines to S-carboxymethyl-cysteines. The cross reactions of antigens containing intact methionine, methionine sulfone, or carboxymethyl methionine suggest that methionine and methionine sulfone play a minor immunologic role in this system. Derivatives containing cysteic acid, carboxymethyl cysteine, cysteine, or the p -hydroxymercuribenzoate derivative of cysteine cross react only partially with the antisera suggesting that cysteic acid and carboxymethyl cysteine are important in these immune systems.

The interaction of platelet proteins with three fibroblast-derived collagenases

Three collagenases were purified from the culture medium of human skin fibroblasts using ammonium sulfate fractionation, ion-exchange chromatography and gel filtration. The cationic collagenase had a molecular weight of 42,000; two anionic collagenases had molecular weights of 63,000 and 115,000. Preincubation of the individual collagenases with purified human and bovine platelet heparin binding proteins resulted in the inhibition of the two anionic activities, but only by bovine low heparin affinity platelet protein (beta-TG). Such inhibition was dose-dependent at the microgram level, was not antagonized by heparin, and persisted even when the collagenases had been transformed into their 53,000 and 105,000 forms through treatment with p-aminophenylmercuric acetate. Neither human nor bovine high heparin affinity platelet factors (PF-4) nor human low heparin affinity platelet protein (beta-TG) were inhibitory to any of the three collagenases studied. This suggests that the ability of platelet proteins to inhibit collagenase is specifically influenced by the ionic nature of the enzyme and this inhibition is specifically dependent upon the species and type of platelet protein.

The effect of organic solvents on rabbit antibody

Abstract 1. 1. The effects of urea and various organic solvents (ethanol, acetone, dioxane, tetrahydrofuran, tetrahydropyran) on anti-pneumococcus Type III γ-globulin were examined. Inhibition of the precipitin reaction was observed when the antibody was treated with urea and certain solvents simultaneously. The degree of inhibition by solvents appears to parallel the polarity and size of the solvent molecule. 2. 2. The inhibition could be removed by dialysis or by passing the antibody-urea-solvent mixture through Sephadex G-25. However, the redialyzed material was sensitive to readdition of solvent while untreated antibody was not. When dioxane was used as initial solvent, dioxane inhibited. If the original solvent was tetrahydrofuran, dioxane or tetrahydrofuran inhibited. When tetrahydropyran was used first, all three inhibited. Similar inhibition was observed with the urea-dioxane-treated anti-ribonuclease system. 3. 3. The urea-dioxane-treated material reacted with horse anti-rabbit γ-globulin serum as well as intact γ-globulin did.