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PubMed1977

Purification of several proteolytic enzymes by tosyl- and carbobenzoxy-triethylene-tetramine-sepharoses.

Tosyl-triethylenetetramine-Sepharose (Tos-T-Sepharose) and carbenzoxytriethylenetetramine-Sepharose (Z-T-Sepharose) were found to be adsorbents utilizable in the purification of several microbial and animal proteases. The former Sepharose derivative adsorbed alpha-chymotrypsin, trypsin, subtilisin, thermolysin and neutral subtilopeptidase at neutral pH range, and acid proteases such as pepsin and Rhizopus niveus protease at pH 3.5-6.5. alpha-Chymotrypsin and trypsin were eluted with 0.1 N acetic acid and Rhizopus protease with 0.5 N acetic acid, thermolysin with 1 M guanidine-HCl or 33% ethyleneglycol, whilst pepsin was recovered by elution with 2 M guanidine-HCl at pH 3.5. The binding of neutral subtilopeptidase and subtilisin to this adsorbent was comparatively weak and both the enzymes were recovered by elution with 0.5 M NaCl at neutral pH. On the other hand, Z-T-Sepharose was found to bind tightly to these proteolytic enzymes except neutral subtilopeptidase. Trypsin and alpha-chymotrypsin were released from the adsorbent column with 1 M p-toluenesulfonate, and subtilisin with 1 M guanidine-HCl or 33% ethyleneglycol at neutral pH region. By these chromatographic procedures, the specific activities of these proteolytic enzymes increased effectively. Comparison of the binding abilities of acetyl-, benzoyl-, tosyl- and carbobenzoxy-T-Sepharoses to these enzymes suggests that hydrophobicity of tosyl and carbobenzoxy groups plays an important role in the enzyme-adsorbent interaction.

Acetylation↗
PubMed1981

Activation of human T and non-T lymphocytes by sepharose-bound concanavalin A and the differential effect of macrophages.

The mitogenic effect of insoluble concanavalin A (Con A) bound to Sepharose beads on human peripheral blood mononuclear cells has been investigated. Sepharose-Con A, with a maximum activity at 100-400 micrograms/ml and an optimal incubation time of 3 or 4 days, stimulated the mononuclear cells with a high mitogenic response. Sepharose-Con A also stimulated the monocyte-depleted lymphocytes and their T- and non T-enriched fractions with a definite mitogenic response, although the levels of response were much lower than those of unpurified mononuclear cells. Addition of the adherent monocytes strongly augmented the Sepharose-Con A responses of monocyte-depleted lymphocytes and their T-enriched fractions, whereas it gave no effect on those of non-T-enriched fractions. Culture fluids of adherent monocytes also augmented the Sepharose-Con A responses of monocyte-depleted lymphocytes. These results indicate that, although insoluble Sepharose-Con A activates both human T and B cells, the response of T cells but not B cells may be dependent in part on the presence of monocytes or a monocyte-derived soluble factor(s). Thus, it is concluded that Sepharose-Con A may be a useful mitogen to elucidate the role of macrophages or their soluble factors in activation of human T and B cells.

Cell Separation↗
PubMed2004

Fractionation of soluble proteins in Escherichia coli using DEAE-, SP-, and phenyl sepharose chromatographies.

In an effort to simplify a complex mixture of soluble proteins from Escherichia coli, methods to fractionate the samples prior to two-dimensional (2D) gel electrophoresis were developed. These methods involve the use of DEAE-Sepharose, SP-Sepharose, and phenyl Sepharose chromatographic columns and the fractionation of the protein mixtures based on differential anionic, cationic, and hydrophobic properties of the proteins, respectively. Fractionation of the soluble proteins from an E. coli extract with DEAE-Sepharose resulted in a threefold increase in the number of detectable 2D gel spots. These gel spots were amenable to protein identification by using in-gel trypsin digestions, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and peptide mass fingerprinting. Significantly, the DEAE-Sepharose column fractionation effectively partitioned the soluble proteins from the cell extracts. Similarly, an SP-Sepharose column was used to fractionate the soluble proteins from E. coli and resulted in over a twofold increase in the number of detectable gel spots. Lastly, fractionation of the cell extract with the phenyl Sepharose column resulted in a threefold increase in the number of detectable 2D gel spots. This work describes an easy, inexpensive way to fractionate the soluble proteins in E. coli and a way to better profile the E. coli proteome.

Bacterial Proteins↗
PubMed1989

A simplified method for the preparation of WGA-Sepharose 4B and its use in the purification of MN blood group antigens.

A simplified method for the preparation of wheat germ agglutinin(WGA)-Sepharose 4B by coupling highly purified WGA, prepared by improved affinity chromatography, with BrCN activated Sepharose 4B in a solution of high carbonate buffer is described. The amount of WGA linked to Sepharose 4B was 82.40% (3.07 mg WGA per ml Sepharose 4B). MN blood group antigens of human erythrocyte membranes purified with WGA-Sepharose 4B affinity chromatography showed a single band on sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS/PAGE). The yield of the antigens from 400 mL fresh blood was 32-40 mg. The WGA-Sepharose 4B column could be used several times without loss of activity.

Electrophoresis, Polyacrylamide Gel↗
PubMed1988

Affinity purification of cytosolic epoxide hydrolase using derivatized epoxy-activated Sepharose gels.

Improved affinity chromatography procedures for the purification of cytosolic epoxide hydrolase are described. An earlier affinity purification method using immobilized 7-methoxycitronellyl thiol (MCT) sporadically produced final enzyme preparations containing major impurities. To eliminate these impurities, we tested alternate ligands, spacer arms, and ligand concentrations. A series of alkyl and aryl thiols coupled to epoxy-activated Sepharose were found to exhibit markedly different binding characteristics as compared with commercially available alkyl- and aryl-Sepharose gels. Using one of these new matrices, benzylthio-Sepharose, cytosolic epoxide hydrolase from mouse liver was purified over 100-fold, appeared homogeneous by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and was obtained with 60-90% recovery of enzyme activity. The impurities previously observed with the MCT-Sepharose procedure were reduced or eliminated by using an MCT ligand concentration of 5 microequivalents per gram or less. MCT-Sepharose and benzylthio-Sepharose provide rapid and convenient one-step procedures for obtaining purified cytosolic epoxide hydrolase from numerous species and tissues.

Adult↗
PubMed1988

Affinity chromatography of fibrinogen on Lens culinaris agglutinin immobilized on CNBr-activated sepharose: study of the active groups involved in nonspecific adsorption.

Fibrinogen binds specifically to Lens culinaris agglutinin coupled to CNBr-activated Sepharose. However, a fraction of the retained fibrinogen remains tightly bound to the gel and is eluted only by electrophoretic desorption. The irreversible binding of fibrinogen results from the interaction of fibrinogen specifically bound to the immobilized lectin with some reactive groups still present on the Sepharose matrix. Therefore, the active groups present on lectin-Sepharose after different treatments and their influence on the irreversible binding of fibrinogen have been studied. After the coupling step some cyanate esters remain on the gel, but they are neutralized under all the conditions studied. In addition, imidocarbonates formed under the basic conditions used to activate Sepharose, and carbonates resulting from acid treatment of the gel, are also present. Carbonates seem to be the main active groups involved in the irreversible binding of fibrinogen to lectin-Sepharose. Imidocarbonates also contribute to the nonspecific binding, although to a lesser extent than carbonates. Treatment of CNBr-activated Sepharose with 0.1 M HCl prior to the coupling step and neutralization, after coupling, with 0.1 M ethanolamine, pH 9.5, for 24 h at room temperature reduce the nonspecific binding to less than 9% of the fibrinogen fraction retained by the column. This percentage is appreciably smaller than that obtained by neutralization for 2 h at room temperature with either 0.1 M Tris-HCl, pH 8.0 (congruent to 66%), or 1 M ethanolamine, pH 9.0 (congruent to 23%).

Adsorption↗
PubMed1985

Preparation of steroid radioligands for ultrafiltration assays by a solid-phase transport globulin method using concanavalin A-Sepharose.

Concanavalin A-Sepharose (Con A-Sepharose) was applied to separate non-protein-bound and albumin-bound radioactive impurities from steroid radioligands. Con A-Sepharose gel, plasma, and steroid radioligand were mixed, incubated, and then washed with buffer. This method was compared with an affinity diafiltration method which separates non-protein-bound radioactive impurities with a filter membrane. 3H-Water and 3H-estrone sulfate, chosen to serve as molecules representative of non-protein-bound and albumin-bound impurities, were removed quite effectively by the Con A-Sepharose method, while 85% of 3H-estrone sulfate could not be removed by the diafiltration method. Plasma unbound cortisol (F) and testosterone (T) values determined by ultrafiltration using 3H-F and 3H-T prepared by the Con A-Sepharose method were significantly lower than those determined using the radioligands unprocessed or prepared by the diafiltration method. The whole procedure of the Con A-Sepharose method takes only 3-4 hours. This method is a simple, rapid, and effective technique for preparation of steroid radioligands for plasma protein binding studies.

Female↗
PubMed1984

Separation of heparin on Sepharose CL-4B in the presence of high concentrations of ammonium sulphate.

Heparin was fractionated by chromatography on Sepharose CL-4B/3.8-2.0 M ammonium sulphate in 0.01 M hydrochloric acid at 4 degrees C based on a principle different from that of gel filtration--possibly due to multiple interaction mechanisms including those based on hydrophobic bonds. The results of the fractionation were very similar to those of chromatography on Phenyl-Sepharose CL-4B/3.8-2.0 M ammonium sulphate in 0.01 M hydrochloric acid at room temperature. That is, the separation was related to the molecular size distribution, N-acetyl content and anticoagulant activities. As the result of studies on a set of Sepharose 4B gels with different degrees of cross-linking, it has been shown that the introduction of the cross-linked structure, -O-CH2-CH(OH)-CH2-O-, into the gel matrix enhances the interaction between the Sepharose CL-4B gel and heparin, indicating that heparin retention by the highly cross-linked Sepharose 4B gel surpasses that by Phenyl-Sepharose CL-4B.

Ammonium Sulfate↗
PubMed1985

Quantitative characterization of the binding of plasminogen to intact fibrin clots, lysine-sepharose, and fibrin cleaved by plasmin.

The binding of human Glu- and Lys-plasminogens to intact fibrin clots, to lysine-Sepharose, and to fibrin cleaved by plasmin was quantitatively characterized. On intact fibrin clots, there was one strong binding site for Glu-plasminogen with a dissociation constant, Kd, of 25 microM and one strong binding site for Lys-plasminogen with a Kd of 7.9 microM. In both cases, the number of plasminogen binding sites per fibrin monomer was 1. Also, a much weaker binding site for Glu-plasminogen was observed with a Kd of about 350 microM. Limited digestion of fibrin by plasmin created additional binding sites for plasminogen with Kd values similar to the binding of plasminogen to lysine-Sepharose. This was predictable given the observations that plasminogen binds to lysine-Sepharose and can be eluted with epsilon-aminocaproic acid [Deutsch, D.G., & Mertz, E.T. (1970) Science (Washington, D.C.) 170, 1095-1096] and that plasmin preferentially cleaves fibrin at the carboxy side of lysyl residues [Weinstein, M.J., & Doolittle, R.F. (1972) Biochim. Biophys. Acta 258, 577-590], because the structures of the lysyl moiety in lysine-Sepharose and of epsilon-aminocaproic acid are identical with the structure of a COOH-terminal lysyl residue created by plasmin cleavage of fibrin. The Kd for the binding of Glu-plasminogen to lysine-Sepharose was 43 microM and for fibrin partially cleaved by plasmin 48 microM. The Kd for the binding of Lys-plasminogen to lysine-Sepharose was 30 microM. With fibrin partially cleaved by plasmin, there were two types of binding sites for Lys-plasminogen, one with a Kd of 7.6 microM and the other with a Kd of 44 microM.(ABSTRACT TRUNCATED AT 250 WORDS)

Affinity Labels↗
PubMed1984

Lack of binding of serum glycoprotein hormone alpha subunit to concanavalin A-Sepharose reflects increased branching of the oligosaccharide chains.

The lectin-binding properties of serum alpha subunit were studied by lectin affinity chromatography. Normal individuals and most patients with pituitary tumours produced alpha subunit which bound specifically to Concanavalin A-Sepharose (Con A). Some patients with pituitary tumours produced both Con A-reactive alpha subunit and alpha subunit which did not bind to Con A. Concanavalin A-Sepharose-binding alpha subunit from all sources bound strongly to Ricinus communis agglutinin-Sepharose after treatment with neuraminidase. Serum alpha subunit from those patients with pituitary tumours, which did not bind to Con A, bound to wheat germ agglutinin-Sepharose, exhibiting both weakly binding and strongly binding forms. Serum alpha subunit from both patients and controls, which did bind to Con A, showed only weak affinity for wheat germ agglutinin-Sepharose. Neither the low affinity nor the high affinity of serum alpha subunit from any source for wheat germ agglutinin-Sepharose was affected by neuraminidase. These findings show that (a) the predominant pattern of glycosylation of serum alpha subunit from normal controls is a Con A-reactive, biantennate complex oligosaccharide and (b) that the structural alteration which results in serum alpha subunit which does not bind to Con A in some patients with pituitary tumours is not an absence of carbohydrate, rather the alpha subunit contains highly branched, either complex or hybrid oligosaccharides.

Adult↗
PubMed1984

Multiple classes of heparan sulfate proteoglycans from fibroblast substratum adhesion sites. Affinity fractionation on columns of platelet factor 4, plasma fibronectin, and octyl-sepharose.

Both newly formed and long-term culture-generated substratum adhesion sites, generated by EGTA-mediated detachment of Balb/c SVT2 cells, were extracted with an eta-octyl-beta-D-glucopyranoside buffer containing salt and several protease inhibitors under conditions which result in maximal solubilization of the sulfate-radiolabeled proteoglycans. Because of the functional importance of heparan sulfate proteoglycans in the fibronectin-dependent cell-substratum adhesion processes of these cells, these proteoglycans were fractionated on affinity columns of octyl-Sepharose or of the heparan sulfate-binding proteins platelet factor 4 or plasma fibronectin. These affinity matrices resolved a number of both binding and nonbinding classes of heparan sulfate proteoglycan from both types of adhesion sites. In particular, the platelet factor 4 column could resolve several proteoglycans with differing binding affinities. Approximately twice as much heparan sulfate proteoglycan from newly formed sites bound to all three matrices as proteoglycan from longterm sites. The proteoglycan which bound to one matrix was then tested for binding to a second matrix; this approach resolved a number of biochemically distinct species. For example, one-half of the fibronectin-Sepharose-binding fraction from the long-term sites could also bind to platelet factor 4-Sepharose; however, over 90% of the fibronectin-binding fraction from newly formed sites could bind to platelet factor 4. A major portion of the octyl-Sepharose-binding fractions of the original extracts could bind to fibronectin-Sepharose. These studies indicate that some of these proteoglycans have overlapping affinities for fibronectin, platelet factor 4, and octyl-Sepharose and that a portion of the heparan sulfate proteoglycan from these adhesion sites cannot bind to any of these affinity matrices. These results are discussed with regard to the functional significance of these various heparan sulfate proteoglycans in mediating adhesion to extracellular matrices containing fibronectin or platelet factor 4.

Animals↗
PubMed1993

Heparin-sepharose binding growth factors in peritoneal fluid of women with endometriosis.

OBJECTIVE: To characterize and to purify the growth substances for mouse embryo fibroblasts (NIH/3T3) and endometrial-like cells of the peritoneal fluid of women with endometriosis. DESIGN: A pool of 102 mL of peritoneal fluid (PF) collected from 24 women with laparoscopic evidence of endometriosis was dialyzed and thereafter chromatographed onto carboxymethyl (CM)-sepharose, heparin-sepharose, cartridges of C18 silica, and reverse-phase high-performance liquid chromatography (reverse-phase HPLC). Mitogenic activity throughout various steps of chromatography was monitored by the increase of the (3H)-thymidine incorporation into NIH/3T3 fibroblasts and enzymatically isolated epithelial cells of rabbit endometrium. RESULTS: Peritoneal fluid of women with endometriosis contained mitogen(s) for NIH/3T3 fibroblasts that were retained on and eluted from CM-sepharose, heparin-sepharose, cartridges of C18 silica, and reverse-phase HPLC columns performed sequentially. Mitogenic material purified 2,500-fold exerted bioactivity on NIH/3T3 and the enzymatically isolated epithelial cells from rabbit endometrium. CONCLUSION: Peritoneal fluid of women with endometriosis contains heparin-binding growth factors that can be purified by sequential chromatography on CM-sepharose, heparin-sepharose, and reverse-phase HPLC columns. The presence in the PF of potent mitogens for fibroblasts and endometrium-derived epithelial cells could play an important role in the pathogenesis of endometriosis.

3T3 Cells↗
PubMed1973

The properties of subunits of avidin coupled to sepharose.

Avidin that had been coupled to Sepharose 4B activated with CNBr retained over 90% of its biotin-binding capacity. When low concentrations of CNBr were used about 75% of the protein could be removed from the Sepharose by washing with guanidinium chloride (6 m). The remaining 25%, the covalently bound subunits, had an almost undiminished capacity for biotin but a decreased affinity. Addition of avidin subunits in guanidinium chloride to the coupled subunits followed by dilution or dialysis restored the original biotin-binding capacity and affinity. Three classes of binding sites were present in preparations of the subunits. About 25% were weak (K=5x10(-8)m), about one third exchanged their biotin in a few minutes (K approximately 10(-10)m) and the remainder were indistinguishable from the native tetramer. The last-named exchanged their bound biotin at a similar rate at pH5 and at pH2, they did not lose their biotin in 6 m-guanidinium chloride and they were resistant to tryptic digestion in the absence of biotin. The proportion of these stable sites could be increased to 65% when the subunits coupled to Sepharose were incubated at 37 degrees C. This increase was reversed by guanidinium chloride, which suggested that it was caused by a temperature-dependent association of covalently linked subunits. This in turn implies a temperature-dependent mobility of the agarose matrix of the Sepharose. Analysis of the spatial distribution of subunits within the Sepharose beads led to the conclusion that the association of subunits implied that they could move through distances greater than 20nm (several hundred A). This mobility and consequent formation of tetramer was greatly decreased when avidin subunits were coupled to Sepharose that had been cross-linked with divinyl sulphone.

Avidin↗
PubMed1986

Affinity chromatography of peptidylarginine deiminase from rabbit skeletal muscle on a column of soybean trypsin inhibitor (Kunitz)-Sepharose.

We designed a simple procedure for the purification of peptidylarginine deiminase, which catalyzes the deimination of arginyl residues in protein, from rabbit skeletal muscle using substrate affinity chromatography. Of the immobilized substrate ligands tested, i.e. protamine and soybean trypsin inhibitor (Kunitz) (STI), STI-Sepharose was found to be an effective affinity adsorbent for purification of the enzyme. The specific binding of peptidylarginine deiminase to STI-Sepharose was observed in the presence of calcium ion, and the enzyme could be selectively eluted from the affinity adsorbent by washing with chelator. A 1,800-fold purification with a 50% yield was achieved in the three-step procedure, which involved DEAE-Sephacel ion-exchange and STI-Sepharose affinity chromatography. The purified enzyme was homogeneous on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The specific activity and the recovery were considerably higher than have been obtained by any procedures previously reported. The specific interaction of peptidylarginine deiminase with STI immobilized on Sepharose was also investigated quantitatively by frontal affinity chromatography. In this method, a peptidylarginine deiminase solution was applied continuously to an STI-Sepharose column and the retardation of the elution front was measured as a parameter of the strength of the interaction. The dissociation constant for the enzyme with STI was found to be 2.3 X 10(-7)M. This value was in good agreement with that obtained by kinetic analysis in our previous studies. Peptidylarginine deiminase required millimolar Ca2+ for the binding to STI-Sepharose. The Ca2+ dependence of the enzyme binding was quite similar to that of the enzymatic activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1987

Analysis of the bond between encephalomyocarditis virus and its human erythrocyte receptor by affinity chromatography on virus-sepharose columns.

Affinity chromatography was used to analyse the bond between encephalomyocarditis (EMC) virus and glycophorin, the receptor for EMC virus on human erythrocytes. Between 60 and 80% of glycophorin added to virus-Sepharose columns was retained compared with 10 to 20% retention on glycine-Sepharose columns. Elution with 0.2 M-NaCl released about 80 to 90% of the retained glycophorin from virus-Sepharose columns but little from glycine-Sepharose. Glycophorin remaining on either the virus or glycine columns after 0.2 M-NaCl treatment was released with Triton X-100, suggesting that this material was aggregated and trapped non-specifically. The sensitivity of the EMC virus-glycophorin bond to 0.2 M-NaCl was confirmed by showing that the radiolabelled EMC virus that bound to human erythrocyte membranes in 0.02 M-phosphate buffer was released when washed with this buffer containing 0.2 M-NaCl. It was concluded that weak ionic interactions were involved in this virus-receptor bond. Treatment of glycophorin with neuraminidase prevented it binding to EMC virus-Sepharose indicating the requirement for sialic acid for receptor activity. However, other sialylated molecules did not bind. Only one chymotryptic peptide of glycophorin (CH0) bound to EMC virus-Sepharose, confirming that this peptide contains the virus-binding site as had been previously suggested using other techniques. The effects of two non-ionic detergents and two anionic detergents on the virus-glycophorin bond were examined. In each case a very low concentration of detergent disrupted the bond and the concentration required was in parallel with that which dissolved erythrocyte membranes. It appears that multivalent glycophorin aggregates are required for stable bond formation.

Chromatography, Affinity↗
PubMed1998

Purification of elongation factors EF-Tu and EF-G from Escherichia coli by covalent chromatography on thiol-sepharose.

The elongation factors EF-Tu and EF-G of Escherichia coli are involved in the transport of aminoacyl-tRNA to ribosomes and the translocation of ribosomes on mRNA, respectively. Both possess cysteine residues that are important for activity. We took advantage of this property to design a purification protocol based on thiol-Sepharose chromatography, a method involving thiol-disulfide interchange between protein thiol groups and the glutathione-2-pyridyl-disulfide conjugate of the affinity resin. Bacterial cells were lysed by a lysozyme-EDTA method, and the lysate supernatant was purified by chromatography on, first, DEAE-Sephacel and, then thiol-Sepharose. Both elongation factors were purified in a single procedure, since DEAE-Sephacel fractions containing both factors were loaded on the thiol-Sepharose column. Thiol-Sepharose chromatography efficiently separates each elongation factor from all contaminating proteins. The purified elongation factors were characterized by SDS-PAGE, protein sequencing, and biological activity. The specific reactivities of the elongation factors with thiol-Sepharose allow their efficient purification and suggest that they possess hitherto undiscovered properties connected with their reactive thiols.

Amino Acid Sequence↗
PubMed1990

Thyroxine binding to human serum albumin immobilized on sepharose and effects of nonprotein albumin-binding plasma constituents.

125I-thyroxine (125I-T4) binding to human serum albumin (HSA) covalently attached onto CNBr-activated Sepharose (HSA-Sepharose) was studied. 125I-T4 binding to HSA-Sepharose was rapid and saturable. Nonlinear curve-fitting analysis of binding isotherms revealed two classes of binding sites. The values of dissociation constants of high and low affinity sites were 2.19 +/- 0.53 x 10(-6) M and 2.69 +/- 0.78 x 10(-5) M, respectively. The number of binding sites of the high and the low affinity sites were 1.28 +/- 0.46 mol/mol and 23.5 +/- 9.7 mol/mol of HSA, respectively. Fatty acids and bilirubin competitively inhibited the high-affinity binding of 125I-T4 to HSA-Sepharose without affecting the low-affinity binding. 8-anilino-1-naphthalene sulfonic acid (ANS) inhibited the high affinity T4 binding via reduction of the binding capacity. Unlabeled T4 showed little inhibition of ANS binding to HSA, as measured by fluorescence intensity. These results suggest that ANS allosterically inhibits the high-affinity T4 binding to HSA-Sepharose.

Anilino Naphthalenesulfonates↗
PubMed1991

Purification and characterization of three toxins and two agglutinins from Abrus precatorius seed by using lactamyl-Sepharose affinity chromatography.

Three toxins, abrin-I, -II, and -III, and two agglutinins, APA-I and -II, were purified from the seeds of Abrus precatorius by lactamyl-Sepharose affinity chromatography followed by gel filtration and DEAE-Sephacel column chromatography. Abrin-I did not bind on DEAE-Sephacel column chromatography and the bound abrin-II, abrin-III, APA-I, and APA-II were eluted with a sodium acetate gradient. The identity of each protein was established by sodium dodecylsulfate-polyacrylamide gel electrophoresis and isoelectric focusing. The relative molecular weights are abrin-I, 64,000; abrin-II and abrin-III, 63,000 each: APA-I, 130,000; and APA-II, 128,000. Isoelectric focusing revealed microheterogeneity due to the presence of isoforms in each protein. Toxicity and binding studies further confirmed the differences among the lectins. The time course of inhibition of protein synthesis in thymocytes by the toxins showed lag times of 78, 61, and 72 min with Ki's of 0.55, 0.99, and 0.74 ms-1 at a 0.63 nM concentration of each of abrin-I, -II, and -III, respectively. A Scatchard plot obtained from the equilibrium measurement for the lectins binding to lactamyl-Sepharose beads showed nonlinearity, indicating a cooperative mode of binding which was not observed for APA-I binding to Sepharose 4B beads. Further, by the criterion of the isoelectric focusing profile, it was shown that the least toxic abrin-I and the highly toxic abrin-II isolated by lactamyl-Sepharose chromatography were not retained on a low-affinity Sepharose 4B matrix, which signifies the necessity of using a high-affinity matrix for the purification of the lectins.

Abrin↗