NEW METHODOLOGY FOR DETERMINATION OF METHIONINE AND ISOLATION OF METHIONINE-RICH PROTEINS FROM PHASEOLUS VULGARIS L.
Abstract
An improved methodology for the gas chromatographic determination of methionine in proteins and crude plant materials was developed. This methodology relies on the ability of cyanogen bromide to cleave methionine and release methylthiocyanate. Various parameters affecting the reaction and the chromatographic analysis of methylthiocyanate were studied. The reaction time was shortened to one hour for crude samples, to twenty minutes for proteins, while five minutes were required for the chromatographic analysis. The selected column packing was stable after 1,500 injections. The oxides of methionine and derivatives of S-methylcysteine present naturally in many seeds do not release methylthiocyanate. The methionine and protein contents of 83 improved navy bean cultivars and breeding lines were determined. An average of 1.4 percent methionine (g/100g protein) and 24 percent (g/100g seed meal) protein were found. A negative correlation was found to exist between methionine and crude protein. Trypsin inhibitors present in beans can be considered good sources of cystine. A protein fraction rich in both cystine and methionine would be of great interest from the view of genetic improvement of beans. A trypsin inhibitor fraction was isolated by means of affinity chromatography. Its methionine content was determined by gas liquid chromatography and was found to contribute insignificantly to the total methionine content of the bean (0.1 percent, g/100g inhibitor protein). By studying the influence of pH and ionic strength on the extraction of navy bean proteins simple fractionation schemes were developed for obtaining protein isolates rich in methionine. The yield of each isolate and its methionine content were determined. One such fraction was obtained by acid precipitation at pH 4.1 and represents 18-20 percent of the total extracted bean protein. Two proteins representing approximately 85% of this fraction were obtained by gel filtration and ammonium sulphate precipitation. One of them was found to have relatively high methionine content (2.5 percent). It was eluted as a single peak from DEAE-cellulose and gave a single band by polyacrylamide gel electrophoresis. By SDS-polyacrylamide gel electrophoresis it was resolved depending on the variety into two or three major subunits. Its amino acid composition and carbohydrate content were determined.
Degree
Ph.D.
Subject Area
Food science
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