A comparison of methods for the extraction and separation of hordein fractions from 29 barley varieties

Abstract

Methods have been developed for extracting and comparing the hordein fractions of barley seed as a means of identifying different varieties. A rapid method involving a single extraction with 55 % (v/v) propan-2-01 + 2% (v/v) 2-mercaptoethanol at 60°C removed slightly less nitrogen from milled grain than a more detailed procedure in which three sequential extractions were made with the same solvent. The hordein fractions extracted by these two procedures were alkylated and their component polypeptides separated by four one-dimensional and two two-dimensional systems. The results showed that the hordein extracted by both techniques had the same polypeptide composition and thus the rapid method was satisfactory for use in comparing varieties. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis and isoelectric focusing were used to compare the polypeptide composition of the hordein fractions from 29 varieties. Using the former system a total of 1 1 different patterns were recognised while further small differences between several varieties were revealed by isoelectric focusing or two-dimensional analysis. If the polypeptide patterns obtained by these techniques are considered in conjunction with other grain characters it should be possible to identify almost all of the 29 varieties.

Genetic analysis of hordein polypeptides from single seeds of barley

“…After centrifugation the supernatant containing the extracted hordein is reduced to dryness at 40°C, dissolved in 200 cl buffer containing 8-M urea (Cavins and Friedman, 1968;Shewry, Ellis, Pratt and Miflin, 1978) and 1 per cent (v/v) 2-mercaptoethanol and alkylated with 4-vinylpyridine (3.0 pd). The alkylated solution is dialysed against a solution of 1 per cent (w/v) sodium dodecylsuiphate (SDS) and electrophoresced on 175 per cent acrylamide gels containing 0l per cent SDS atpH 89 (Shewry, Ellis, Pratt and Miflin, 1978).…”

“…WE have recently developed a simple rapid procedure (Shewry, Pratt and Miflin, 1978) for the varietal identification of large numbers of single or half-seeds of barley by the analysis of hordein polypeptides.…”

“…These groups differ in amino acid composition as well as in molecular weight and electrophoretic mobility (Miflin and Shewry, 1977). Whereas a number of different "B" and "C" hordein patterns occur (Shewry, Ellis, Pratt and Miflin, 1978), the low molecular weight "A" hordein (which only accounts for 1-2 per cent of the total fractions) has a constant pattern in all varieties so far investigated and may not be a true storage protein (Miflin and Shewry, 1977). A previous study by Oram, Doll and Køie (1975) suggests that the "B" hordein pattern is controlled by co-dominant alleles at a locus close to Mi-a on chromosome 5.…”

Hordein polypeptide patterns in relation to malting quality in Brazilian barley varieties

“…Hordein was first described by Osborne (1895) as the barley seed protein soluble in aqueous alcohol. Improved methods for hordein extraction have been developed including the use of reducing agents (Shewry et al, 1978a), the modification of solvent constituents (Shewry et al, 1980b) and the addition of a buffer in the extraction medium (Doll & Andersen, 1981).…”

Cloning of double stranded DNA coding for hordein polypeptides

“…Considerable varietal heterogeneity of hordein polypeptide sizes has been reported (25,26) and genetic analysis have revealed that these polymorphisms in the polypeptide group of C-hordein are determined by one and those of the B-hordein by another locus on barley chromosome 5 (12, 22. 24, 26).…”