II. 21. Characterization of composition of hordeins in normal- and high-lysine barleys.
Alicja Kapala, Polish Academy of Sciences, Poznan, Poland.
Electrophoretic analysis of hordeins in cationic buffer system (Reisfeld et al., 1962) demonstrated distinct differences between cultivated varieties and high-lysine forms (unpublished data). The observed differences concern relative intensity of protein bands Rp 0.57 and Rp 0.71. In general, electrophoretic patterns of high-lysine barleys are distinguished by a relatively strong staining of the bands (Figure 2-H). The purpose of the present studies was to obtain additional information on hordein similarities and differences in normal- and high-lysine barleys.
For a detailed analysis of hordeins two cultivated varieties, Polish variety Gryf and Danish variety Carlsberg II, and three well-known high-lysine forms, Hiproly, Risö mutant 56 and Risö mutant 1508, were chosen. Hordeins were extracted with 50% iso-propanol containing 0.6% 2-mercaptoethanol (Køie and Nielsen, 1977) and treated with urea - 2-mercaptoethanol. The fractionation was performed on Sephadex G-100 column. Chromatographic fractions were submitted to disc electrophoresis in acidic buffer system.
Three chromatographic fractions (I-III) of hordeins were obtained with approximate mol. wt. of 100,000 (Fraction I), 55,00 (Fraction II) and 20,000 (Fraction III).
The elution profiles of the analysed barley forms differ distinctly (Figure 1). The two cultivars -- Gryf and Carlsberg II -- show similar elution profiles; Fraction I is dominant (about 65%), while the amount of Fraction III is only about 5%. In high-lysine barleys Hiproly and Risö mutant 56 the content of Fraction I is lower (by about 15%) in comparison with the varieties Gryf and Carlsberg II, but the lower content of Fraction I is compensated by a higher content of Fraction III (by about 15%). Special attention should be paid to the elution profile of hordeins of Risö mutant 1508. Hordeins of Risö mutant 1508 were characterized by the highest content of Fraction III (about 45%) and by the lowest content of Fraction III.
Fig. 1. Elution profiles of hordeins on Sehadex
G-100 with 3M urea, 0, 1M acetic acid, 0,01M Brij 35
Fig. 2. Electrophoretic patterns of total
seed hordeins (H) and chromatographic fractions (I-III) of cultivated varieties
and high-lysine forms
High-lysine forms differed from normal forms in the proportion of the three separated fractions of hordeins; high-lysine barleys appeared to have a higher proportion of the low-molecular-weight fraction. The fact is quite interesting. Køie et al., (1976) and Mesrob et al., (1970) displayed differences in the amino acid composition of hordein subfractions; the subfraction with low-molecular-weight was the richest in lysine and other essential amino acids.
Moreover, results of the present studies showed differences in composition of hordeins within the high-lysine barleys. Risö mutant 1508 differed from the other high-lysine forms not only by a higher content of the hordein components with mol. wt. of 20,000 but also by the specific, electrophoretic pattern of these proteins.
Acknowledgment:
I thank Professor DR hab. J. Przybylska for continuous help and criticism.
References:
Køie, B., J. Ingversen, A.J. Andersen, H. Doll, and B.0. Eggum.
1976. In: Evaluation of seed protein alterations by mutations breeding.
Research Coordination meeting, Hahnenklee, 1975, IAEA, Vienna, 55-61.
Køie, B. and G. Nielsen. 1977. In: Techniques for the separation of barley and maize proteins (B.J. Miflin, P.R. Shewry, Eds). Publ. by the Commission of the European Communities, Luxembourg.
Mesrob, B., M. Petrova and P. Ivanov. 1970. Biochim. Biophys. Acta, 200: 459-465.
Reisfeld, R.A., U.J. Lewis and D.E. Williams. 1962. Nature, 195:281-283.
This work was supported by the Polish Academy of Sciences under project 09.1, subproblem A part I.08-01.
