An Evaluation of Techniques for the Extraction of Hordein and Glutelin from Barley Seed and a Comparison of the Protein Composition of Bomi and RisØ 1508

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Abstract
Whole seed of barley (cv. Julia) was ground, and the meal extracted to remove lipids, non-protein nitrogen compounds, albumins, and globulins. Four procedures for extracting hordein and glutelin from this meal were then compared. The composition of the isolated fractions was monitored by amino acid analysis and SDS polyacrylamide gel electrophoresis. More hordein was extracted by 55% (v/v) propan-2-ol containing 2% (v/v) 2-mercaptoethanol at 60 °C than at 20 °C or by sequential extraction with 55% propan-2-ol alone followed by 55% propan-2-ol plus 0.6% 2-mercaptoethanol. After hordein extraction glutelins were successfully extracted from the residual meal by reduction and alkylation in buffer containing 8 M urea, and were precipitated by dialysis against water. Small amounts of hordein were recovered from the alkylated glutelin by washing with hot 70% (v/v) ethanol plus 0.7% (v/v) acetic acid. The acid alcohol-insoluble glutelin was free from hordein polypeptides. Glutelins were also extracted sequentially using borate buffer at pH 10 with 0.6% mercaptoethanol followed by the same buffer with 1% SDS. Two procedures were used to compare the hordein and glutelin composition of endosperms of high lysine (RisØ 1508) and normal (Bomi, Julia) barley varieties. The hordein extracted at 60°C by 55% propan-2-ol plus 2% 2-mercaptoethanol represented almost 50% of the total N of the endosperm of Bomi and Julia, and 16% of RisØ 1508. The high lysine mutant (RisØ 1508) had more glutelin and salt-soluble nitrogen than Bomi. Electrophoretic analysis of the component polypeptides of the hordein of Bomi and RisØ 1508 showed several differences in the bands present, and in their relative proportions. In contrast the hordein-free glutelins of all three varieties appeared to have similar polypeptide compositions. Investigation of the salt-soluble fraction confirmed that the high lysine gene in RisØ 1508 results in increases in both protein and non-protein nitrogen components. The results obtained on the distribution of nitrogen between the various fractions in the seeds of Bomi and RisØ 1508 and on the amino acid analysis and polypeptide composition differ considerably from those published by other workers, in which a classical Osborne type extraction was used, and we conclude that such methods should not be used for barley.