Grain hardness is the most important characteristic affecting the quality of common wheat end-products. In fact, it influences milling and properties of flours, being soft wheat flour typically used for biscuits (cookies) and cakes and hard wheat flour for bread. Wheat grain hardness appears to be determined by the degree of adhesion between the starch granules and the protein matrix, which is regulated by a protein called friabilin, isolated from prime starch granules (Greenwell and Schofield, 1986). Friabilin is present in larger amounts in soft than in hard wheats and consists of two proteins (Jolly et al 1993; Morris et al 1994), the N-terminal sequences of which were found to be identical to those of two binding lipids proteins, called puroindoline-a (Pin-a) and puroindoline-b (Pin-b) (Gautier et al 1994). Hardness is the result of the expression of one major gene, Ha, located on the short arm of chromosome 5D, controlling the occurrence of friabilin in the whole grain (Jolly et al 1993). Further evidence supporting the relationship between friabilin and Ha-locus comes from the observation that several mutations in Pin-a and -b are consistently associated with hard endosperm texture (Giroux and Morris 1997; Giroux and Morris 1998). Pins are cysteine rich proteins (10 cysteine residues forming 5 disulphide bridges) having a unique tryptophan-rich domain and a molecular mass of approximately 13,000 (Douliez et al 2000). Furthermore, they are strongly basic proteins with calculated isoelectric points of pI 10.5 for Pin-a and pI 10.7 for Pin-b (Gautier et al 1994). Methods for the isolation of Pins from flour (Blochet et al 1993) and starch granules (Jolly et al 1993, Morris et al 1994, Oda and Schofield 1997) were previously reported in the literature. In these methods purification of Pins was achieved combining Triton X-114 partitioning, gel filtration and cation exchange chromatography techiniques. Here we present an improvement of the Blochet et al (1993) method based on preparative acid electrophoresis which allows Pin-b to be isolated from wheat flour in good yield and with a high degree of purity.
Isolation of wheat Puroindoline-b from flour by preparative acid electrophoresis
Calucci L;
2003
Abstract
Grain hardness is the most important characteristic affecting the quality of common wheat end-products. In fact, it influences milling and properties of flours, being soft wheat flour typically used for biscuits (cookies) and cakes and hard wheat flour for bread. Wheat grain hardness appears to be determined by the degree of adhesion between the starch granules and the protein matrix, which is regulated by a protein called friabilin, isolated from prime starch granules (Greenwell and Schofield, 1986). Friabilin is present in larger amounts in soft than in hard wheats and consists of two proteins (Jolly et al 1993; Morris et al 1994), the N-terminal sequences of which were found to be identical to those of two binding lipids proteins, called puroindoline-a (Pin-a) and puroindoline-b (Pin-b) (Gautier et al 1994). Hardness is the result of the expression of one major gene, Ha, located on the short arm of chromosome 5D, controlling the occurrence of friabilin in the whole grain (Jolly et al 1993). Further evidence supporting the relationship between friabilin and Ha-locus comes from the observation that several mutations in Pin-a and -b are consistently associated with hard endosperm texture (Giroux and Morris 1997; Giroux and Morris 1998). Pins are cysteine rich proteins (10 cysteine residues forming 5 disulphide bridges) having a unique tryptophan-rich domain and a molecular mass of approximately 13,000 (Douliez et al 2000). Furthermore, they are strongly basic proteins with calculated isoelectric points of pI 10.5 for Pin-a and pI 10.7 for Pin-b (Gautier et al 1994). Methods for the isolation of Pins from flour (Blochet et al 1993) and starch granules (Jolly et al 1993, Morris et al 1994, Oda and Schofield 1997) were previously reported in the literature. In these methods purification of Pins was achieved combining Triton X-114 partitioning, gel filtration and cation exchange chromatography techiniques. Here we present an improvement of the Blochet et al (1993) method based on preparative acid electrophoresis which allows Pin-b to be isolated from wheat flour in good yield and with a high degree of purity.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
