William B. Watt

Glycoprotein II The Isolation and Characterization of a Major Antigenic and Non-Haemagglutinating Glycoprotein from Phaseolus Vulgaris

Summary 1. The isolation of Glycoprotein II from the seeds of kidney bean is described. The purification was achieved by extraction of the pH 5 insoluble proteins of the seeds at pH 8.3 followed by high-voltage electrophoresis and chromatography on Sephadex G-200 and DEAE-cellulose columns of the soluble proteins. 2. Glycoprotein II was found to be in the form of a monomer and essentially homogeneous at neutral and slightly alkaline pH values by disc gel electrophoresis, immuno-diffusion and -electrophoresis, velocity and equilibrium ultracentrifugation. Its monomer molecular weight was 140 000 ± 2000. Its hydrodynamic properties indicated a voluminous and asymmetric (β = 2.56 · 10−6; ve = 5.72 · 10−18 ml) particle with large amounts of entrained solvent. Between pH 3.4 and 6.6 it was mainly in the form of a tetramer (molecular weight of about 560 000) with smaller amounts of monomer and higher oligomers also present. These oligomers were found not to be in a rapid chemical equilibrium and their conversion rates were infinitely slow at these pH values. Between pH 2.2 and 3.4 the glycoprotein was almost exclusively in the form of the monomer. 3. Glycoprotein II was also shown to be homogeneous by chromatography and velocity and equilibrium ultracentrifugation in dissociating media. It was however dissociated to about one quarter (35 000–43 000) of the monomer molecular weight in these solvents. 4. The carbohydrate part was mainly composed of D-mannose and D-glucosamine. No uronic acids were present. The amino acid analyses revealed a definite deficiency in the sulphur-containing amino acids, especially in cyst(e)ine. Phosphorus-containing compounds were shown to be absent. 5. Glycoprotein II was found to be a strongly antigenic protein. It had however no haemagglutinating activity for rabbit erythrocytes.

Isolectins of Phaseolus vulgaris. A comprehensive study of fractionation

Abstract The results of fractionation studies and chemical, physical and immunochemical investigations established the existence of a range of closely related glycoproteins in the albumin fraction of the seeds of “haricot” kidney bean ( Phaseolus vulgaris ). The isoelectric point of these glycoproteins was found by isoelectric focusing to vary between about pH 4.5 and 6.9. Their subunit composition, however, was very similar; they were all made up of two types of subunits, 30 000 ± 1000 and 35 000 ± 1000, respectively, in a ratio of about 3:1. The chemical composition of these glycoproteins bore a close resemblance to each other. However, small differences were also found and these might have accounted for the charge heterogeneity. An immunochemically related glycoprotein was also found in the globulin fraction. This, however, was made up by subunits of the size of 30 000 ± 1000 and was different in amino acid composition. These glycoproteins were shown to be agglutinins, or isolectins, of red or white blood cells. They were also found to interact with other cells, such as far cells, and to be able to bind carbohydrate-type materials. However, these isolectins had negligible effects on lymphocyte transformation. The extent of all activities was strongly dependent on the isoelectric point of the isolectins. The specificity of the interaction, however, was apparently the same; i.e. they were all inhibited by the same compounds including N- acetyl- d -galactosamine and glycopeptides containing N- acetyl- d -glucosamine , mannose and galactose and obtained from fetuin. The possible involvement of these isolectins in bean toxicity is discussed.

Isolation and properties of protein fractions from navy beans (Phaseolus vulgaris) which inhibit growth of rats

Abstract A protein fraction which inhibits growth of weanling rats was obtained from Phaseolus vulgaris seeds of the Sanilac variety by extraction with sodium chloride solution and removal of extraneous materials by precipitation at pH 4.0 and absorption on 1:1 bentonite-Celite. The protein was precipitated by 0.75 saturation with ammonium sulfate. The growth inhibiting protein fraction contained at least four different proteins separable by electrophoresis, ultracentrifugation, immunodiffusion, and gel chromatography. Only one of the soluble proteins inhibited rat growth, and it comprised about 35% of the total growth inhibiting fraction and had hemagglutinating activity, stimulated leucocytes, contained very little trypsin inhibitor activity, and had an isoelectric point of pH 5.2 and a mol. wt of approx. 110 000. A protein which was insoluble at pH 4 and collected as a precipitate also inhibited growth of rats but because of the small amount isolated and its insolubility, it was not further studied.

Isolation of soybean trypsin inhibitors by affinity chromatography on anhydrotrypsin-Sepharose 4B

By repeated treatments of trypsin with phenylmethylsulfonyl fluoride (PMSF), followed by base elimination of PMS from the PMS-trypsin, a catalytically inactive anhydrotrypsin preparation of low (less than 1%) active trypsin content was obtained. Inactive material was removed by affinity chromatography on trypsin inhibitor-Sepharose 4B and the purified anhydrotrypsin with full binding capacity for trypsin inhibitors was coupled to cyanogen bromide-activated Sepharose 4B. When used below its maximum capacity for trypsin inhibitors the anhydrotrypsin-Sepharose-4B affinity column absorbed both classes of inhibitors present in soybean. When overloaded, the Kunitz type was bound preferentially. Based on this observation, conditions for the partial separation of the two types of inhibitors were worked out.

A novel method for the preparation of protein bodies by filtration in high (over 70% w/v) sucrose-containing media

Abstract A novel method for the preparation of protein bodies from the cotyledons of Phaseolus vulgaris is described in high (over 70% w/v) sucrose-containing media. Homogenates of cotyledons were filtered through no. 15 Ballotini beads and the filtrates were further purified by stepwise gradient centrifugation. The organelles harvested from the layer of density of 1.32 g/ml compared favourably in composition, size and yield with those prepared by other techniques and in iso-osmotic aqueous media.

The determination of the molecular size of peptides and proteins by chromatography on bio gel P-100 columns in phenol-acetic acid-water (1:1:1,W-V-V) solvent mixture.

Abstract 1. 1.|The results of our studies of molecular sieve chromatography on a Bio Gel P-100 column in phenol-acetic acid-water (1:1:1; w/v/v) of a number of well-characterized proteins, polypeptides and other compounds of known molecular weight indicated the existence of the following empirical relationship between the elution volume and the log of the molecular weight: log 10 mol. wt. = [5.13 −(0.53 ± 0.013) x (V e /V o )] ± 0.059 Of the nearly thirty individual compounds studied cytochrome c and insulin A chain were found to deviate from the above equation. 2. 2.|Application of this and similar chromatographic techniques in other dissociating solvent systems for the investigation of insoluble and structural proteins was discussed.

Free-flow electrophoresis of proteins in phenol-containing solvents at various pH values

1. Several proteins were found to migrate when subjected to free-flow electrophoresis in buffered phenol-ethanediol-water (3:2:3, w/v/v) solvent mixtures. Mobility of these proteins changed with changing pH (apparent) values of this medium. A pH value of zero mobility for each individual protein could be estimated. 2. Founded on these observations, a high-voltage electrophoresis method in free-flowing buffer films was worked out. The method as presented here was particularly suitable for the separation of proteins on a preparative scale. Application of this and other protein fractionation techniques in dissociating media for the investigation of structural and other insoluble proteins was discussed.

Erythro- and lymphoagglutinins of Phaseolus acutifolius

Abstract A potent lymphoagglutinin which had low affinity for red cells or fetuin and another lectin which reacted strongly with red cells and fetuin but was a poor agglutinin for lymphocytes were isolated from seeds of Phaseolus acutifolius . A number of other lectin components with intermediate activity towards these cells was also isolated. All the lectins had very similar amino acid and carbohydrate composition, sedimentation patterns, partial specific volume and molecular weight values of about 116 600 and were thus smaller than the related Phaseolus vulgaris lectins (M r = 119 000). The lectins contained four subunits with only minor size and charge differences between the lympho- and erythroagglutinating subunits and their electrophoretic mobility in SDS gel electrophoresis was anomalously high. The existence of lympho- and erythroagglutinating subunits in two members of the genus Phaseolus supports their close morphological similarity.