R.J. Dimler

Starch gel electrophoresis of wheat gluten proteins with concentrated urea.

Abstract Starch gel electrophoresis using aluminum lactate buffer (pH 3.1) containing 3 M urea demonstrated nine components in wheat gluten; four were not previously detected by the Tiselius method. One of the components appears to be high molecular weight and unable to migrate in starch gels. Gliadin was shown to be composed of eight components, whereas glutenin appeared to be identical with the high molecular weight α1 component of whole gluten. Electrophoresis of wheat gluten in Veronal (pH 7.9) and sodium glycinate (pH 9.7) buffers containing 3 M urea failed to resolve gluten into distinct components. When examined in aluminum lactate buffer containing 3 M urea, the water-soluble protein fraction isolated from a gluten preparation was composed of at least nine electrophoretic components.

Characterization and properties of the phosphomannan from Hansenula hostii NRRL Y-2448

Abstract The phosphorylated mannan produced in good yield from glucose by the bisexual diploid yeast, Hansenula holstii NRRL Y-2448, appears to be the first significantly phosphorylated polysaccharide to be obtained from a yeast or a nonpathogen. Isolated as the potassium salt, this water-soluble polysaccharide derivative has constituents in the proportion, d -mannose:phosphorus:potassium::5:1:1. The product has [ α ] D 25 + 103 ° (in 0.1 M potassium chloride), M N (reducing power) 103,000 ± 10,000, M W (light scattering) of the order of 16 × 10 6 , S 20, w = 44, and an unusually homogeneous molecular distribution for an unfractionated, native polymer. The single titration equivalence-point of the polyacid at pH 7.2 indicates a phosphodiester structure. Weak acid liberates secondary hydrogen ions and causes molecular degradation; dilute alkali appears to cause no structural change. Aqueous solutions show exceptional resistance to microbial attack. The brilliantly clear aqueous solutions have properties characteristic of a plastic, thixotropic, polyelectrolyte. The viscosity-concentration curve shows a viscosity maximum of 2500 cp. at 1.5% polysaccharide concentration and a minimum of 1700 cp. at 3%. At suitable concentrations of phosphomannan and borax, complexing and cross-linking occur; the presence of potassium chloride augments these effects.

AMIDE GROUPS AS INTERACTION SITES IN WHEAT GLUTEN PROTEINS: EFFECTS OF AMIDE-ESTER CONVERSION.

Abstract The side-chain amide groups of gluten and its fractions, glutenin and gliadin, were converted in part to ester groups by reaction with methanol containing hydrogen chloride. The transformation occurred with little cleavage of peptide linkages. Properties that were changed significantly included solubility in various systems, intrinsic viscosity, and cohesion of the hydrated mass. The changes indicate that the large number of side-chain amide groups in wheat gluten and its component proteins participate in association between the protein molecules and also between the proteins and various solvents. The concept of hydrogen bonding provides a reasonable explanation for the association through amide groups and for the effects of amideester conversion. The amide groups are also partially responsible for the cohesive and elastic characteristics of hydrated vital wheat gluten.

Reduction and starch-gel electrophoresis of wheat gliadin and glutenin

Abstract Reduction of the disulfide bonds of wheat gliadin and glutenin followed by electrophoresis in starch gel revealed the presence of some components which are perhaps common to both proteins. There were marked quantitative differences in the distribution of components. The release of 20 or more electrophoretic components from the previously unresolved glutenin fraction is further evidence that extensive intermolecular disulfide bonding is responsible for its high molecular weight. Intermolecular disulfide bonding is present only to a limited extent in the gliadin fraction.

HYDROGEN ION EQUILIBRIA OF WHEAT GLUTENIN AND GLIADIN.

Abstract Hydrogen ion titration curves of twice-precipitated wheat glutenin and gliadin have been obtained in 3 M urea plus 0.15 M KCl at 25 °C. The data have been analyzed by equations treating the electrostatic effect as an empirical factor. The ionizing groups per 10 5 g. glutenin, and their intrinsic p K s at 25 °C. are: 38 carboxyl (4.76), 13 imidazole (6.6), 2 α-amino (8.4), 1 sulfhydryl (9.4), 23 tyrosyl (10.42), and 12 lysyl (10.6); those for gliadin are: 22 carboxyl (4.85), 14 imidazole (6.45), 2 α-amino (8.0), 1 sulfhydryl (10.0), 16 tyrosyl (10.31), and 5 lysyl (10.7). The guanidyl groups with p K greater than 13 do not affect the titration curves except through the net charge Z of glutenin and gliadin. The ionizing groups all appear to be normal. Empirical values of the electrostatic factor, w , are significantly larger for carboxyl groups than for tyrosyl groups. This change in w suggests that the conformations of both molecules depend on pH.

Hydrogen-ion equilibria of wheat gluten

Abstract Hydrogen-ion titration curves of wheat gluten have been studied in 3 M urea plus 0.15 M KCl at 25 °C. Ionizing groups per 105 g. gluten and their intrinsic pKs at 25 δC. are: 29 carboxyl (4.77), 15 imidazole (6.43), 2 α-amino (8.4), 1 sulfhydryl (10.1), 20 tyrosyl (10.26), and 9 lysyl (10.78). The guanidyl groups (pK > 13) affect the titration curve only through the net charge, Z, of gluten. The ionizing groups all appear to be normal although the intrinsic pK of the imidazole groups is on the low side. The empirical electrostatic factor w at acid pH was considerably larger than it was at alkaline pH, and this difference suggests a conformation change has occurred. The titration results agree with the amino acid and other analyses within experimental error.

Chromatographic fractionation of wheat gluten on carboxymethylcellulose columns

Abstract A Chromatographic procedure has been developed to isolate purified gluten components in preparative amounts. These isolated proteins correspond to electrophoretic components demonstrated in wheat gluten protein. The chromatography of wheat gluten was accomplished by stepwise elution from Carboxymethylcellulose (CMC) using buffers with increasing hydrogen-ion concentrations. All except the α1 component were obtained by this procedure. Although the strong selective binding of the α1 component to CMC required elution with 8 M urea, the intractable nature of the α1 component isolated in this manner prevented its use in other investigations. The behavior of isolated gluten proteins upon rechromatography and their electrophoretic characteristics suggest that the peaks in gluten patterns represent individual proteins and that these peaks are not due to association effects.

Conformational studies of wheat gluten, glutenin, and gliadin in urea solutions at various pH's

Abstract Viscosity, sedimentation velocity, ultraviolet difference spectra, and optical rotatory dispersion measurements were carried out on wheat gluten, glutenin, and gliadin in 3 M urea plus 0.11 M KCl plus 0.02 M buffer at pH 3–10 at 25 °C or room temperature. An increase in intrinsic viscosity and a decrease in sedimentation coefficient for glutenin at pH 10 compared with that at pH 4 are consistent with an increase in asymmetry of the protein molecule. Parameters from optical rotatory dispersion studies on glutenin also indicate a conformational change at pH 10. Some increases in intrinsic viscosity were also observed for gluten and gliadin at pH 10, but the increase for gliadin might not be significant. The absence of tyrosine and tryptophan peaks in the ultraviolet difference spectra of gluten, glutenin, and gliadin suggests that these two amino acids are not involved in any interaction with other groups.

HYDROGEN-ION EQUILIBRIA OF WHEAT GLUTEN IN GUANIDINE-HYDROCHLORIDE SOLUTIONS.

Abstract Hydrogen-ion titration curves of wheat gluten have been studied in 2 and 4 M guanidine-hydrochloride (G-HCl) at 25 °C. The data were analyzed by equations treating the electrostatic effect as an empirical factor. The ionizing groups per 10 5 gm gluten and their intrinsic p K s at 25 °C in 2 and 4 M G-HCl are: 29 carboxyl (4.54, 4.48), 15 imidazole (6.49, 6.56), 2 α-amino (8.5, 7.7), 1 sulfhydryl (10.0, 9.5), 20 tyrosyl (9.97, 9.97), and 9 lysyl (10.4, 10.4). The number and intrinsic p K of guanidyl groups cannot be determined in G-HCl solutions. The ionizing groups all appear normal. The empirical electrostatic factor w at acid pH was considerably larger than it was at alkaline pH, and a larger decrease in w was observed in alkaline than in acid solutions when the solvent was changed from 2 to 4 M G-HCl. These changes in w suggest that the conformation of gluten depends on pH and that the conformation in acid solution is more stable. Different methods for purifying G-HCl are discussed.