Jerold A. Bietz

Separation of cereal proteins by reversed-phase high-performance liquid chromatography

Cereal proteins have been extremely difficult to purify and characterize owing to their heterogeneity, poor solubility and tendency to polymerize. High-performance liquid chromatography (HPLC) on a 300 A reversed-phase (RP)(C18) support (Syn Chropak RP-P), using acetonitrile as organic modifier in the presence of trifluoroacetic acid, has been found to be capable of high-resolution separations of these proteins; the resolution is often better than that obtained by any other chromatographic or electrophoretic method. Examples are presented showing separations of low-molecular-weight gliadins, omega-gliadins and ethanol-soluble reduced glutenin subunits from wheat and of zein from corn. In addition, proteins may be directly extracted from ground single kernels and subsequently analyzed by RP-HPLC; applications in genetic studies, in breeding programs and in varietal identification are proposed. In addition to its high resolution, RP-HPLC is superior to most other methods in speed, sensitivity, reproducibility and suitability for quantitation. Polypeptide chains of molecular weight up to 133,000 are recovered in high yields and the column capacity is high, demonstrating that RP-HPLC is suitable for both preparative and analytical separations of proteins. RP-HPLC resolves proteins primarily on the basis of differences in surface hydrophobicity, so it therefore complements, rather than duplicates, other techniques that separate proteins on the basis of size or charge. RP-HPLC promises to become an invaluable technique for the fractionation and characterization of proteins from cereals and other sources.

Detection of quality differences among wheats by high-performance liquid chromatography

Abstract Proteins extracted with various solvents from small wheat flour samples were analyzed by reversed-phase high-performance liquid chromatography (RP-HPLC) and size-exclusion HPLC (SE-HPLC). RP-HPLC analysis of gliadin proteins can identify wheat varieties and, for durum wheats (used for pasta products), can rapidly determine quality. After lower-molecular-weight proteins are extracted, high-molecular-weight glutenin proteins can be reduced, alkylated, isolated and analyzed by RP- or SE-HPLC. The ratio of high- to low-molecular-weight glutenin subunits can be used to determine the quality of wheats used to make bread.

Reversed-phase high-performance liquid chromatography of reduced glutenin, a disulfide-bonded protein of wheat endosperm

Abstract The subunits of glutenin, the disulfide-bonded, high-molecular weight protein fraction of wheat, have been separated by reversed-phase high-performance liquid chromatography (RP-HPLC) on large-pore silica-based columns. Glutenin was first isolated from bulk samples, and effects of dissociating, reducing and alkylating agents on subsequent RP-HPLC separations of glutenin subunits were compared. Eight RP-HPLC columns (C 18 , C 8 , diphenyl or cyanopropyl) were compared to achieve optimal resolution of glutenin subunits. Proteins were eluted by a gradient of increasing acetonitrile concentration in the presence of 0.1% trifluoroacetic acid and were detected at 210 nm. Excellent separations occurred when glutenin was dissociated in the presence of 8 M urea or 6 M guanidine hydrochloride, reduced with 5% 2-mercaptoethanol or 0.1 % dithiothreitol, alkylated with 4-vinylpyridine and chromatographed on C 18 or C 8 bonded-phase columns. Using these conditions, fifteen to twenty major glutenin subunits were resolved. Glutenin subunits were then extracted from single kernels of aneuploid lines of the variety Chinese Spring and fractionated by RP-HPLC. Four early eluting peaks were shown to contain major glutenin subunits coded by the long arms of chromosomes 1D and 1B, and thus were assumed to correspond to the high-molecular-weight subunits. Their elution volume indicated that these subunits have lower surface hydrophobicities than do most lower-molecular-weight glutenin subunits which eluted later. Significant differences in glutenin subunit composition among bread wheat varieties suggest possible relationships between specific polypeptides and breadmaking quality, and show that RP-HPLC of glutenin subunits has the potential of identifying most wheat varieties. These results demonstrate that RP-HPLC is a valuable complement to other chromatographic and electrophoretic methods for analysis of glutenin subunits.

Assessment of the potential breadmaking quality of hard wheats by reversed-phase high-performance liquid chromatography of gliadins

The development of reversed-phase high-performance liquid chromatography has provided a method for analysis of large numbers of very small samples of wheat proteins much more quickly and accurately than was previously possible. This paper describes the comparison of the amount of a specific gliadin fraction in a given variety with the general baking (breadmaking) score previously determined for that variety. Correlation coefficients between this gliadin fraction and breadmaking quality of hard red spring wheats grown in three different areas of the country during one year are significant at the 5% or 1% level. This method could, if found to be suitable for other varieties over a number of years, provide a quick method to analyze for breadmaking potential of new wheat varieties on as little as half-kernels or 5 mg of wheat flour.

Reversed-phase high-performance liquid chromatography of durum wheat gliadins: Relationships to durum wheat quality

Durum wheat gliadins have been analyzed by RP-HPLC (reversed-phase high-performance liquid chromatography) for the first time. Gliadins extracted from each particular durum variety were resolved to give a complex chromatographic pattern that was unique to that variety. However, four chromatographic fractions, which were eluted under hydrophobic conditions (45·2, 45·6, 47·4 and 49·1% acetonitrile, respectively), could be used to classify durum varieties into two groups. Proteins in these fractions were purified by preparative RP-HPLC and analyzed by aluminium lactate-polyacrylamide gel electrophoresis (AL—PAGE), which indicated that two of these fractions contained gliadin bands 45 or 42 that have been shown previously to be correlated closely with gluten strength and weakness, respectively. Therefore, RP—HPLC can be used also to screen for gluten quality characteristics in durum varieties in a way analogous to that in which electrophoresis may be used. A rapid, highly-automated RP-HPLC analytical procedure was developed to screen for durum varieties with unacceptably weak gluten characteristics. Each determination takes less than 10 min, permitting approximately 200 analyses per 24 h. Because of its convenience and rapidity, RP-HPLC of durum wheats has considerable potential as an alternative to electrophoresis as an analytical tool for screening for gluten traits when large numbers of samples must be screened, as in early generation of wheat breeding.

Separation of alcohol-soluble maize proteins by reversed-phase high performance liquid chromatography*

Three alcohol-soluble protein fractions from maize endosperm [zein; water-soluble, alcohol-soluble reduced glutelin (wsASG); and water-insoluble, alcohol-soluble reduced glutelin (wiASG)] were extracted simultaneously with a mixture of ethanol and water (70:30 v/v) containing 5% (v/v) 2-mercaptoethanol and 0·5% (w/v) Na acetate (70% EtOH-0·5% Na acetate-5% ME) from near-isogenic sugary-1 (su1), floury-2 (fl2), opaque-2 (o2) and normal (+) maize. Subsequent fractionation by reversed-phase high performance liquid chromatography (RP-HPLC) on a C18 column using an aqueous acetonitrile (CH3CN)-trifluoroacetic acid (CF3COOH) solvent gradient gave good resolution, and accurate quantification was achieved by monitoring the absorbance of column eluates at 210 nm. The relative amounts of these three alcohol-soluble protein fractions varied significantly in both normal and mutant lines, and RP-HPLC patterns were specific for each inbred and genotype. A correlation coefficient of -0·90 was found between combined peak absorbance per gram protein and the lysine content of the protein. Two su1 maize inbreds, having more methionine than normal counterparts due to increased ASG, also had more 15,000 mol.wt high methionine ASG subunits as revealed by RP-HPLC, indicating that the amounts of high methionine (wi) and high histidine, high proline (ws) ASG vary regularly in mutant genotypes. This rapid and simple extraction method, in conjunction with RP-HPLC, may replace sequential extraction techniques. In conjunction with RP-HPLC, this procedure provides another new method for determining protein quality and genotype of maize.

Isolation and characterization of a methionine-rich protein from Maize Endosperm

A 17,000 mol. wt methionine-rich (met-rich) polypeptide from alcohol-soluble reduced glutelin (ASG) of maize endosperm from the hybrid WF9 × Br 38 was purified by a combination of differential solubility and ion exchange chromatography. Antiserum specific for this polypeptide was prepared against a similar fraction purified by Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE) from the inbred W64A. This polypeptide (17,000 mol. wt met-rich ASG) is distinct from zein and other ASG fractions in that it contains the highest levels of methionine (10·8 mol%), glycine, alanine, tyrosine and arginine, and the lowest levels of proline, valine, isoleucine, histidine and phenylalanine among these proteins. Its N-terminal sequence, determined for 36 residues, shows numerous dipeptide (Ala-Gly and Gly-Leu) repeats and it shares no homology with either zein or proline-rich ASG fractions; this lack of homology was also indicated by results from immunological studies.