Physicochemical properties of ß and α'α subunits isolated from soybean ß-conglycinin.

Soy protein has shown great potential for use in biobased adhesives. ß-Conglycinin is a major component of soy protein; it accounts for 30% of the total storage protein in soybean seeds. ß-Conglycinin was isolated and purified, and its subunits' (ß, α'α) physicochemical and adhesive properties were characterized. Crude ß-conglycinin was isolated from soy flour and then purified by the ammonium sulfate precipitation method. The α'α and ß subunits were isolated from the purified ß-conglycinin by anion exchange chromatography. Yields of α'α subunits and ß subunits from 140 g of soy flour were 1.86 g (1.3%) and 0.95 g (0.67%), respectively. The minimum solubility for α'α subunits, ß subunits, and ß-conglycinin occurred in pH ranges of 4.1-5.4, 3.5-7.0, and 4.8-5.3, respectively. Transmission electron microscopy showed that the ß subunits existed as spherical hydrophobic clusters, whereas α'α subunits existed as uniformly discrete particles at pH 5.0. Differential scanning calorimetry showed that ß subunits had higher thermal stability than α'α subunits. The pH had a lesser effect on adhesion strength of the ß subunits than on that of the α'α subunits. The adhesives made from ß subunits also showed greater water resistance than those from α'α subunits and ß-conglycinin. Soy protein rich in ß subunits is likely a good candidate for developing water-resistant adhesives.

Micrometer-sized fibrillar protein aggregates from soy glycinin and soy protein isolate.

Long, fibrillar semiflexible aggregates were formed from soy glycinin and soy protein isolate (SPI) when heated at 85 degrees C and pH 2. Transmission electron microscopy analysis showed that the contour length of the fibrils was approximately 1 microm, the persistence length 2.3 microm, and the thickness a few nanometers. Fibrils formed from SPI were more branched than the fibrils of soy glycinin. Binding of the fluorescent dye Thioflavin T to the fibrils showed that beta-sheets were present in the fibrils. The presence of the fibrils resulted in an increase in viscosity and shear thinning behavior. Flow-induced birefringence measurements showed that the behavior of the fibrils under flow can be described by scaling relations derived for rodlike macromolecules. The fibril formation could be influenced by the protein concentration and heating time. Most properties of soy glycinin fibrils are comparable to beta-lactoglobulin fibrils.

Novel method for the production of pure glycinin from soybeans.

A novel method for the purification of glycinin from soy meal is presented. The method is based on the isoelectric precipitation of glycinin by using carbon dioxide as a volatile precipitant. Gaseous CO(2) was pressurized into the protein solution, thus lowering the pH and initiating glycinin precipitation. Pressurization and, consequently, acidification were done in a slow and controlled manner, with the end point of pH 6.4. The acidity of the protein solution was well controlled via the pressure of gaseous CO(2). In this way simultaneous precipitation of other soybean proteins was prevented and very pure glycinin was obtained. Approximately 40% of the glycinin present in the protein solution was recovered with purity as high as 98%. The purification process was successfully performed on both small and large scales, without affecting glycinin purity.

Quantification of particle sizes with metal replication under standard freeze-etching conditions: a gold ball standard for calibrating shadow widths was used to measure freeze-etched globular proteins.

The real size of platinum-carbon (Pt-C) replicated particles is not directly equivalent to either its metal-coated diameter or its shadow width. This paper describes two indirect methods, shadow widths and coated particle diameters, for determining a particle's actual size beneath a Pt-C replication film. Both produce equivalent measurements using the same standardized conditions: 2.3 nm Pt-C films deposited at a 45 degree angle on an approximately -100 degrees C surface in a 10(-6) torr vacuum. For the first method, gold balls nucleated in a partial pressure of helium and deposited on flat indirect carbon films (root mean square roughness of 0.8 nm) on 400 mesh grids were used as test particles for calibrating shadow widths as a function of particle size. The gold ball test specimens were replicated, and a distribution of Pt-C shadow widths orthogonal to the Pt-C deposition direction was measured and averaged for gold balls 1.5 +/- 0.25 nm, 2.0 +/- 0.25 nm, etc. The diameter of each gold ball was measured within the Pt-C film along with its shadow width because the Pt-C did not obscure or adhere well to the gold. The shadow width distributions for each gold size do not differ significantly from log normal. Two proteins, the lactose repressor and the mitochondrial ATPase, F1, were also used as replication test objects. Negative staining of both proteins was conducted to measure their average diameters. In the second method, a distribution of Pt-C-coated lac repressor diameters perpendicular to the shadow direction was measured. The Pt-C film thickness measured on the quartz crystal monitor was subtracted from the average metal-coated protein diameter to obtain the lac repressor's diameter. The Pt-C-coated particle diameter distributions also did not differ significantly from log normal. While doing this work it was discovered that outgassing the Pt-C electron gun greatly affected Pt-C film granularity: 19 sec produced a high contrast, granular Pt-C film, whereas 120 sec yielded a low contrast, less granular Pt-C film. Both gold balls and protein particles were subjected in separate experiments to either 19 or 120 sec of outgassing of the Pt-C gun prior to Pt-C replication. Outgassing had a profound effect on the average size of the Pt-C shadow widths on both gold and protein particles. The Pt-C gun outgassing procedure also determined the smallest replicated particle that could be resolved. The frequency of some smaller gold ball sizes detected after replication was reduced disproportionately with 19 sec vs. 120 sec outgassing. However, Pt-C gun outgassing did not affect the average measured diameter of the Pt-C-coated protein particles. The "geometric assumption" that each metal-coated particle creates a shadow width the same size as the metal-coated particle diameter was tested using a globular protein. Pt-C replication of protein particles at a 45 degree and 20 degree angle could not confirm the geometric assumption because an average shadow width was always significantly larger than its average Pt-C-coated particle diameter. A model for how the large shadow widths are formed is presented. Gold balls were also replicated at a 45 degree angle with current high resolution conditions at a substrate temperature of -185 degrees C, and the results of these replicas were compared to the results reported here at approximately -100 degrees C.

Secondary structure of 11 S globulin in aqueous solution investigated by FT-IR derivative spectroscopy.

Secondary structure of 11 S globulin, a major storage protein of soybean seeds, has been investigated in aqueous solution by FT-IR spectroscopy. Conformational changes in the native protein upon thermal and chemical denaturation have been monitored by observing changes in the frequency position and peak intensity of the various bands. The frequency of the Amide I band of the native protein shifts by 4 cm-1 from 1,643 cm-1 to 1,647 cm-1 when denatured, while the corresponding intensity of the Amide I band compared to the native protein, decreases by 30 and 67%, respectively, for the urea and thermally denatured proteins, indicating gross conformational changes in the secondary structure. Trifluoroethanol, an alpha-helix promoter shifts the Amide I band from 1,643 cm-1 to 1,651 cm-1, typical of alpha-helix, with a corresponding increase in intensity by 14% relative to the native protein. Derivative spectroscopy, allowing resolution of overlapping bands, shows that the native protein mainly consists of beta-sheet, beta-turns and disordered structure with very little alpha-helix. On denaturation, beta-sheet disappeared almost completely with urea, while this is less so with thermal denaturation.