Characterization of Zeins Fractionated by Reversed-Phase High-Performance Liquid Chromatographyl

Cereal Chern. 65(3):215-222 Zein, the major alcohol-soluble protein of corn endosperm, was extracted with 55% 2-propanol. It was then fractionated by preparative reversed-phase (CIs) high-performance liquid chromatography (RPH PLC) with an acetonitrile-triOuoroacetic acid gradient. From 15 to 17 peaks resulted for native or reduced and alkylated (R-A) zeins. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAG E) showed early eluting HPLC fractions of native zein to be primarily monomers; later-eluting fractions increasingly tend to be or to form oligomers. SDSZein, the major storage protein in corn endosperm, is usually extracted with aqueous ethanol or isopropanol. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) without ,B-mercaptoethanol (,B-ME), as well as ultracentrifugation, have shown zein to consist of almost equal amounts of dimers and monomers, with lesser amounts of higher molecular weight (mol wt) oligomers. The monomeric polypeptides have molecular weights of 19,000-25,000 (referred to here as 22,000 and 24,000 mol wt) (Turner et al 1965, Ganchev et al 1976, Tsai 1980, Paulis 1981, Landry and Sallantin 1983, Abe et aI1985). Upon SDS-PAGE in the presence of ,B-ME, zein appears as only 22,000 and 24,000 mol wt subunits. Although virtually insoluble in water, zein's conformation is characteristic of more conventional globular proteins (Danzer et al 1975). It contains 45% a-helix in 70% ethanol (Wu et al 1983). Osmotic studies in dimethylformamide and formamide (Danzer and Rees 1976) show zein to have a number average mol wt of 21,000-24,000. Some zein (B-zein) exists as an aggregate, which dissociates to monomers in formamide, but reaggregates at higher protein concentrations. In contrast, sulfhydryl analysis (Abe et al 1985) showed that almost all zein cysteine residues form intermolecular disulfide bonds in dimers, polymers, and in protein bodies (Abe et al 1981). Recently, reversed-phase high-performance liquid chromatography (RP-HPLC) was used to identify corn varieties (Bietz 1985, Smith 1986, Paulis and Bietz 1986, Paulis et al 1986, Smith and Smith 1986). To further understand the aggregated structure of zein, we used preparative RP-H PLC to fractionate native and reduced and alkylated (R-A) zeins, both solubilized in 8M urea. Resulting fractions were characterized by isoelectric focusing (IEF), acidic urea (AU) PAGE, SDS-PAGE, and amino acid analysis to determine whether oligomer formation is random or ordered, or results from disulfide interchange or association during isolation. Differences in zein surface hydrophobicities revealed by RP-HPLC show that subunit structure and conformation direct oligomer formation. "Three-dimensional" characterization of zein in terms of surface hydrophobicity, molecular weight, and charge heterogeneity, using RP-HPLC, SDS-PAGE, and IEF + AUPAG E, respectively, may be superior to two-dimensional separations based on SDS-PAGE and IEF or on AU-PAGE IEF I Presented at the 189th ACS j ational Meeting, Div. Agricultural and Food Chemistry, Miami, Florida, May 1985. 2 orthern Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture, Peoria, IL 61604. The mention offirm names or trade products does not imply that they are endorsed or recommended by the U.S. Department of Agriculture over other firms or similar products not mentioned. This article is in the public domain and not copyrightable. It may be freely reprinted with customary crediting of the source. American Association of Cereal Chemists, Inc., 1988. PAG E showed that for R-A zeins, all RP-H PLC fractions are monomers. Acidic urea PAGE and isoelectric focusing showed native zeins in each RP-HPLC fraction to contain 3-10 components, compared to 1-5 for R-A zein. Proteins differing in hydrophobicity frequently had similar electrophoretic mobilities, emphasizing zein's heterogeneity. Amino acid compositions of all RP-HPLC fractions were similar, suggesting marked homology of zein polypeptides. Preparative RP-HPLC was also useful for isolating zeins for further studies. (Hagen and Rubenstein 1980, Wilson et al 1981, Wall et al 1984, Wilson 1986). MATERIALS A D METHODS Protein Preparation Zein was isolated from maize inbred W64A (Landry et al 1983). Samples of native zein for RP-HPLC, IEF, and AU-PAGE were dissolved in 8M urea. Zeins were also reduced in 8M urea with 1% ,B-ME (16 hr at room temperature), alkylated for 1 hr with acrylonitrile, and acidified with aluminum lactate buffer to form R-A zein (Paulis and Wall 1977). RP-HPLC fractions of native zein were examined both unreduced and after reduction and alkylation. RP-HPLC A 250 X 10 mm SynChropak RP-P column (CIS) having 300 A pores was used for preparative RP-HPLC. The column was eluted at room temperature first for 10 min with 42.3% aqueous acetonitrile (CH3 C ) (+ 0.1 % trifluoroacetic acid) and then with an 80-min linear gradient from 42.3 to 57.3% CH3 C (Paulis and Bietz 1986). The column effluent was monitored at 280 nm (absorbance range = 0.4). Protein was injected as two 1.5-ml aliquots (10 mg/ml 8M urea). Collected peak fractions (Fig. I) were dried in 1.2 X 15 cm test tubes at room temperature in a Buchler Vortex-Evaporator, and stored at 4° C until analyzed. Dried fractions were dissolved in 0.5-2.0 ml of 70% (v/v) aqueous ethanol at 600-1,500 J..Lg/ml. Protein concentrations of fractions were estimated by integration after injection of 30 mg R-A or native zein. Aliquots for SDSPAGE, AU-PAGE, and IEF were dried in 4 cm disposable culture tubes by a stream of nitrogen. Corresponding fractions from replicate zein RP-HPLC fractionations were collected and lyophilized for amino acid analyses. Analytical Methods Lyophilized RP-HPLC fractions (equivalent to 1-10 mg of protein) were hydrolyzed by refluxing with 6M HCl (210 mIl mg of protein) for 24 hr. Liberated amino acids were determined on a Dionex D-300 amino acid analyzer using a physiological fluids analysis column with lithium buffers. Reported levels of methionine and cysteine include methionine sulfone and cysteic acid. Tryptophan and carboxyethylcysteine (for R-A fractions) were not determined. Lyophilized native or R-A zein and their RP-HPLC fractions (40-50 J..Lg/5-10 J..Ll sample) were analyzed by SDS-PAGE (Laemmli 1970) using a 10I5% gradient gel (Landry et al 1983). To analyze native zein, no ,B-ME was used in samples or gel. ,B-ME was added to reduce samples of native zein and to R-A zein fractions. ative and R-A zein RP-HPLC fractions were also analyzed by Vol. 65, No.3, 1988 215 IEF (pH 6-8)(Wall et a11984) and A -PAGE (Landry et aI1983). Tative fractions were reduced and alkylated for IEF as described by Paulis and Wall (1977). Gels were fixed overnight in 30% methanol containing 10% trichloroacetic acid before silver staining (Bio-Rad Laboratories 1982). RESULIS AND DISCUSSION Isolation and RP-HPLC Zein was extracted with 55% isopropanol from defatted W64A corn endosperm following salt extraction, and purified after extraction with petroleum ether, ethanol, and 55% tert-butanol by LH-20 chromatography (Landry 1979). RP-H PLC separated native zein (Fig. 1A) into about 17 overlapping peaks; 27 fractions were collected for subsequent electrophoresis and amino acid analysis. Gel filtration chromatography separates similar unreduced zein preparations into peaks corresponding to about 40% monomers, 40% dimers, and 20% higher molecular weight oligomers (Ivanov et al 1976, Rewa et al 1978, Rewa and Bruckner 1979, Paulis 1981, Landry and Sallantin 1983, Landry and Guyon 1984, Landry et al 1986). R-A zein also yields 15-17 peaks upon preparative RP-HPLC (Fig. 1B). The chromatogram contains more early-eluting protein than does that of native zein. Eighteen R-A zein RP-H PLC fractions were collected for subsequent analysis. SDS-PAGE Figure 2A shows SOS-PAGE patterns, under nonreducing conditions, of RP-HPLC fractions of native zein. Initial fractions contained mainly 22,000 mol wt monomers plus some 44,000 mol wt dimers. Later fractions become more enriched in 24,000 mol wt monomers, and dimers of 46,000 and 48,000 mol wt become evident. In late fractions (19-27), monomers diminish, dimers predominate, and trimers, tetramers, and higher molecular weight oligomers appear. SOS-PAG E under reducing conditions of native zein RPHPLC fractions (Fig. 2B) shows, as does nonreducing SOS-PAGE (Fig. 2A), that initial fractions contain primarily 22,000 mol wt monomers. Bands corresponding to 24,000 mol wt appear at about fraction 5. These polypeptides are subunits of 46,000 and 48,000 dimers, and of later-eluting higher molecular weight oligomers that contain both 22,000 and 24,000 mol wt subunits. SOS-PAGE under reducing conditions of RP-HPLC fractions of R-A zein (Fig. 2C) shows that initial fractions (1-4) contain primarily 22,000 mol wt polypeptides, corresponding in elution volume to native 22,000 mol wt zein monomers (Fig. 2B). Latereluting RP-HPLC fractions (13-18) of R-A zein are also enriched in 22,000 mol wt polypeptides. Polypeptides of 24,000 mol wt occur predominately in fractions 7-12, which correspond to those RP-HPLC fractions of native zein enriched in dimers (Fig. 2A). These results show that 22,000 and 24,000 mol wt zein I I I I I I I I I I I I I 1 I , I , I , I I I I I I I I I I , I I 1 6 ' 8 I I I I 2 4 5 I I 7 9 I 10 11 I 12 13 1141 15 16 I I I I , , I 0.2 A ~---------57.3 ---------------z -I -Co.) 0.1 -I I M -50.8 -I I ::J: ---, I Co.) I I ?i! I I I I I I , I I I , I I I I I 10 12 15 1 '8. 20 I I , I I I I I I I I I I I I I I I I I 1617181 9 I 1111 113' 14 I 1161 17 I 1,9' '21 122' 23 124125 126 127 1 0 1 I' I I I I' I I I I 'I I I I I , I , I I co I I I I I I I I I I I I I I I I I I I I I I 44.3 N c:r: