III. 1. New techniques for evaluating lysine content in hordeins.

T. K. Blake, Program in Genetics and Cell Biology, Washington State University, Pullman, Washington 99164 (U.S.A.)

Hordeins, the storage proteins of barley, comprise approximately 50% of the total seed protein in common barley varieties (Shewry et al., 1980). The biological value of the hordeins as a protein source for monogastric animals is primarily limited by low lysine content (Eggum, 1980), typically O to 1.5 mole%.

Hordeins are among the most polymorphic proteins yet identified in barley (Doll and Brown, 1979). The high levels of genetic variation at the Hor-l and Hor-2 loci suggests the possibility that biologically significant variation in lysine content within the hordeins may be available if appropriate selection techniques can be developed. Requirements for such techniques include 1) small-scale purification and quantification of hordeins from single seeds, 2) rapid separation of the hordeins into genetically meaningful groups, and 3) a lysine-specific protein stain compatible with the separation system. This paper describes preliminary techniques developed to study the nutritional significance of polymorphism within the hordeins.

Techniques:
Half seeds are crushed and extracted with 55% propan-l-ol, 2% 2-mercaptoethanol at 60°C according to the method of Shewry et al. (1978). Hordeins are precipitated from the supernate by the addition of 2 volumes of 100% 2-propanol and overnight-storage at -20°C.

Protein content is estimated in the propanol/mercaptoethanol extract by the method of Bradford (1976), using purified hordeins as standards. Standard curves from Advance, Hiproly, Karl, and Risø 56 are shown in Figure 1. Duplicate Kjeldahl analyses were performed on all standard solutions. All r² values are greater than O.98.

Figure 1. Bradford assay standard curves for hordeins purified from four barley varieties and redissolved in 55% propan-l-ol, 2% 2-Mercaptoethanol. Absorbance at 595nm correlated with protein content of standard solutions as determined by micro-Kjeldahl analysis. Coefficients of determination (r² values) for each standard curve are greater than 0.98, and have a linear response in the Bradford assay over a range of 60 micrograms to over 200 micrograms hordein.

Fluorescamine [4-phenylspiro(furan-2(3H), l'phthalan)3,3'-dione] has been characterized as an excellent lysine specific stain for proteins (Seitz, 1980; Ragland et al., 1976). To test the specificity of fluorescamine for lysyl residues the stain was reacted with proteins of known amino acid content. These included four calf thymus histone fractions and lysozyme. Ten microgram samples stained with fluorescamine at pH 9.0 in .04 M Borate buffer were electrophoresed with SDS-PAGE gels as described below. Protein fluorescence in the gel was measured using a Beckman Model 220 fluorescence densitometer. The absorbance of 50 microgram samples was measured at 390 nm. Absorbance and fluorescence were correlated with lysine mole% (Fig. 2). The highly significant r² values confirm that fluorescamine preferentially reacts with lysyl residues in proteins.

Figure 2. Correlations between absorbance of 390nm and lysine content in solutions containing 50 micrograms calf thymus histones and lysozyme (top line), and in gel fluorescence as determined by fluorescence densitometry and lysine content using ten microgram aliquots of the same samples.

The separation system used to evaluate fluorescamine stained and nonstained hordeins is a modification of the Laemmli (1970) discontinuous SDS-PAGE method. Fluorescamine stained samples are diluted with 3 volumes sample buffer before final sample preparation. Twenty micrograms of nonstained hordein or 40 micrograms of fluorescamine stained protein are electrophoresed into 8.4 to 16.8%T, 0.8%C polyacrylamide gels in the presence of 0.1% SDS (Fig. 3).

Figure 3. Gel 1: Coomassie Blue Stained Hordeins
Samples from left: Risø 1508, Advance, Advance/Risø 7, Risø 7/Advance, TR441, TR441/Risø 7, Risø 7/TR441, Risø 7/Advance, TR441, PON 47-79, Mex 188, WA 10086-79, CI 3947, Bomi, Carlsberg II.

Figure 3. Gel 2: Fluorescamine Stained Hordeins
Samples from left: Advance, Advance, Carlsberg II, Carlsberg II, Risø 56, PI 382605, TR441, TR441/Risø 7, Risø 7/TR441, Risø 7, CI 3947, Risø 1508, Bomi/Risø 1508, Bomi, Carlsberg II, Risø 56, Risø 56, Good Delta.

Fluorescamine treated and non-treated samples yield qualitatively similar results, although some intensity differences may be noted between hordeins from different cultivars. This differential fluorescence may be related to either differences in lysine content between the electrophoretically different proteins, or varying protein content in the different bands. By comparing fluorescence intensity with intensity of staining with a less lysine-specific protein stain, e.g., Coomassie Blue, an estimate of the relative lysine content of each band may be obtained. We hope to use this technique to identify introductions from the world barley collection with nutritionally desirable hordeins and incorporate the best lines into our feed barley breeding program.

References:

Bradford, M. M., 1976. Anal. Biochem. 72:248.

Eggum, B. O., 1973. In: Nuclear Techniques for Seed Protein Improvement. IAEA, Vienna.

Laemmli, U. K., 1970. Nature 227:680-685.

Ragland, W. L., J. L. Pace, and D. L. Kemper, 1974. Anal. Biochem. 59:24-29.

Seitz, W. R., 1980. CRC Critical Reviews in Anal. Chem. 8:367-405.

Shewry, P. R., J. R. Ellis, H. M. Pratt, ald B. J. Miflin, 1978. J. Sci. Food Agric. 29:587-596.

Shewry, P. R., J. M. Field, M. A. Kirkman, A. J. Faulks, and B. J. Miflin, 1980. J. Exp. Bot. 31:393-407.

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