Neil W. Taylor

Electrophoresis and fractionation of wheat gluten.

Abstract Electrophoretic patterns of wheat gluten previously reported in the literature are asymmetric with respect to the ascending and descending boundaries. Primary objective of the present work was to find electrophoresis conditions that would give symmetrical diagrams. Such patterns would aid in establishing the number of components in gluten and their relative concentrations. An analytical tool also would be provided for following the progress of fractionation experiments. Lack of such a tool has been a handicap in previous attempts to fractionate gluten. Several suitable buffer systems were found. They include sodium chloroacetate-chloroacetic acid, sodium acetate-hydrochloric acid, sodium phosphate-phosphoric acid, all at pH 3.1 and nominal ionic strengths of about 0.03, and aluminum lactate-lactic acid at pH 3.1 and nominal ionic strengths of 0.03–0.12. Electrophoretic patterns obtained in these buffers demonstrate that gluten of bread wheats is composed of at least four major and one minor components representing, in order of decreasing mobility, relative concentrations among themselves of approximately 44, 22, 15, 16, and 3 %. There is evidence that the peak containing 15 % of the total gluten is composed of two components. In addition, the patterns show the presence of a small amount of relatively fast-moving material believed to be either globulin or albumin rather than true gluten protein. While aluminum lactate-lactic acid buffer does not resolve the two faster moving components, the excellent symmetry of the patterns obtained in this medium at relatively high gluten concentration recommends its use for following the progress of fractionation experiments and for investigating the effect on gluten composition of such variables as gluten quality and method of preparation. A partial separation of the electrophoretic components was obtained by precipitation of gluten from acid solution by increasing pH or ionic strength. The relationships between electrophoretic pattern and gluten quality and method of preparation were also studied.

Molecular weights of wheat gluten fractions

Abstract Molecular weights of glutenin and gliadin fractions differing in electrophoretic mobility have been determined in an ultracentrifuge using approach to sedimentation equilibrium. γ-Gliadin is homogeneous and has a molecular weight of 47,000. β-Gliadin has a molecular weight of 42,000. Although it contains four electrophoretic components, each apparently has approximately the same molecular weight. Glutenin is heterogeneous, has a weight-average molecular weight of 2–3 million, and contains small molecules (very approximately 50,000) as well as very large ones. The observed molecular weight is believed to be real; that is, it results from chemical bonds rather than physical aggregation.

Sexual agglutination in yeast: V. Small particles of 5-agglutinin☆

Abstract An improved preparation of the sex-specific agglutinin from Hansenula wingei mating type 5 has been made by digesting the cell walls with subtilisin. It is similar to the previously reported agglutinin derived by digestion with snail enzymes in its specificity of agglutinating H. wingei mating type 21 only, and in sensitivity of its agglutinating activity to mercaptoethanol. It has less protein, 4%, a small sedimentation rate, 13.1 Svedbergs at 25 °, and a much smaller molecular weight, 570,000, and is more homogeneous in size than the previous preparation. After purification on phosphocellulose, it was shown to be pure in the sense that more than 85% was absorbed by H. wingei type 21 cells.

Wheat gluten and its glutenin component: viscosity, diffusion and sedimentation studies.

Abstract Gluten from Ponca wheat was fractionated by precipitation with aqueous buffers into glutenin and the soluble gliadin fraction. The glutenin in pH 3.1 buffers had a high intrinsic viscosity, which increased markedly with lowering of ionic strength. It had a broad distribution of sedimenting components of high molecular weight and was highly heterogeneous in diffusion. Glutenin thus approximates a randomly coiled polyelectrolyte with a broad distribution of molecular size.

Purification of sexual agglutination factor from the yeast Hansenula wingei by chromatography and gradient sedimentation

Abstract The agglutination factor from mating type 5 of Hansenula wingei was fractionated by various techniques. The factor was heterogeneous in chromatography on phospho-cellulose and notably broad in its sedimentation distribution diagram. Highly active fractions could be obtained either from chromatography on phospho-cellulose or from fractions sedimenting at rates greater than 100 Svedbergs. The most active fractions had particle weights greater than 108, and only a few of these particles per cell were required to agglutinate the opposite mating type. The agglutinating factor appears to be a protein-mannan complex.

Sexual agglutination in yeast: VII. Significance of the 1.7S component from reduced 5-agglutinin☆

Abstract The sex-specific agglutinin from mating type 5 of Hansenula wingei was purified by absorption on and elution from cells of type 21. When purified 5-agglutinin was reduced with thiols, its ability to agglutinate type 21 was destroyed and approximately six small fragments ( s 25 = 1.7S, M = 12,000) were released from the major reduced component. Approximately five disulfide bonds were present in the average 5-agglutinin particle, as estimated from 35 S label. Allowing for variation among different preparations, it appears that each particle of 1.7S component is linked to the major reduced component by one disulfide bond. After fractionation, neither the 1.7S reduced component nor the major one were specifically absorbed by type 21 cells, but mixtures of both components were partially absorbed by type 21 cells after the disulfide bonds had been reformed by oxidation. It is concluded that the 1.7S component is not the complete combining site of 5-agglutinin, but that both reduced components carry parts of the combining site, which is stabilized by the disulfide bond joining them.

Sexual agglutination in yeast: IV. Minimum particle count per cell for fast-sedimenting 5-agglutinin☆☆☆

Abstract Fast-sedimenting fractions of sexual agglutination activity digested from Hansenula wingei mating type 5 (5-agglutinin) were obtained by centrifuging in guanidine-HCl or in NaCl gradients under conditions designed to produce the highest specific activity. The particles of 5-agglutinin in these fractions ranged from 120 to 300 million molecular weight as determined by light scattering. An average of 1.7 particles of 5-agglutinin per cell was found at the agglutination endpoint. This number is reasonably close to the theoretical limit of 0.5 particle per cell, and is comparable to the value of approximately unity for other precipitating and agglutinating systems.

Sexual agglutination in yeast. VI. Role of disulfide bonds in 5-agglutinin.

Abstract The sex-specific agglutinin from mating type 5 of Hansenula wingei contained six disulfide bonds per particle. It could he inactivated by disulfide-cleaving agents and converted to a mixture of two components having sedimentation rates of approximately 2 and 13 S, compared with the original of s 25, w = 13.1. The 2S component made up 9% and the 13S, 81% of the total material. On dialysis in pH 7.6 buffer, the reduced products regained 25–77% of the original agglutinative activity. After separation of the two reduced components by gel filtration, tests disclosed that activity was regained gained only when both components were dialyzed together. Evidently, 5-agglutinin contains a sensitive, interchain disulfide bond system which unites several agglutinating elements in each particle with a large inert matrix.