Identification, distribution and partial characterization of wheat starch-associated proteins.

Abstract

Wheat starch is composed of two populations of starch granules: large 'A' granules and small 'B' granules. A procedure for the separation of wheat starch into its component populations was developed. Digital image analysis showed that the resulting preparations contained 1% or less of the other granule population and were representative of the corresponding starch fraction in flour. The relative surface areas and volumes of these two types of starch granules were calculated and the results indicated that for an equal volume of starch granules, the small granules have 3.5--4.5 times the surface area of the large granules. The protein content of each population was determined from the nitrogen content. The small granules were found to contain 0.35% protein as compared to 0.15% protein in the large granules. Microspectrofluorometry was used as a means of indirectly measuring protein content and establishing the distribution of protein within the granules, before and after treatments to extract the protein. Extracted proteins from the soft wheat variety Fredrick were analyzed by SDS-PAGE to determine their distribution between the large and small starch granules. The large starch granules were found to contain one major protein and the small granules eight major proteins. These nine proteins were then isolated by electroelution and a partial characterization was carried out by determining amino acid compositions, N-terminal sequences, protease susceptibilities, peptide maps, and carbohydrate content. The results indicated that the nine proteins have several properties in common and they may be composed of similar structural units. In particular, the amino acid compositions, protease susceptibilities and peptide maps varied only slightly. However, there were a few differences among the proteins. Some of the them tested positive for carbohydrate, while others did not. N-terminal sequence analysis indicated that most of the N-termini were blocked, and the two distinct sequences obtained showed no homology to the primary structure of any known protein. One of the major differences between hard and soft wheats is the degree to which the starch granules are damaged during milling. The extent of damage can be visualized by certain stains, the most common being Congo Red. However, another related dye, Hessian Bordeaux, was found to stain damaged granules much more intensely. The maximum absorbance of starch granules stained with Hessian Bordeaux was more than five times that of starch granules stained with an equivalent concentration of Congo Red. Hessian Bordeaux is therefore a more useful stain than Congo Red for the microscopic detection of damaged starch granules.