Purification and properties of three albumins from Triticum aestivum seeds

Abstract

Three albumins were isolated from bread wheat seeds by gelfiltration on Sephadex G-100 and differential preparative disc-electrophoresis on polyacrylamide gel. The proteins were obtained in suficient yield and purity to carry out characterisation studies. The electrophoretic mobilities of the three albumins in polyacrylamide discs in glycine- Tris medium, p H 9.5, were 0.28, 0.34 and 0.39 (referred to that of bromophenol blue taken as I ) . The isoelectric points were 6'40, 6.40 and 5.35 and the molecular weights 17,700,18,200 and 18,900, respectively. The amino acid compositions o f the purified albumins were very similar. The correlation of the purified albumins with those isolated from bread wheats by other authors is discussed. Introduction'SOLUBLE' proteins from bread and Triticum durum wheat are usually separated using electrophoretic m e t h o d~. l -~ However, the heterogeneity of protein extracts, even after a preliminary fractionation, seriously limits the evaluation of these results. Therefore, attempts were made to extract and purify species-specific proteins from, for instance, bread wheat in order to prepare immune sera against the proteins. Several varieties of both species could then be tested by immunodiffusion and the specificity of such fractions could be statistically analysed. In this way it was possible to characterise an albumin specific for bread wheat.10-12The albumin fraction from the seeds of Triticum aestivum or Triticum durum, extracted with 0. S M -N~C~ and precipitated with between 0 . 9 3~ and 1.33 M ammonium sulphate, was gelfiltered on Sephadex G-100. Four major fractions of different molecular weights were obtained (A, B, C, D) (Cantagalli, P., unpublished results). In disc-electrophoresis the lowest molecular weight fraction (Db) from bread wheat had a significantly different composition from the same fraction obtained from T. durum wheat (Dd) (Cantagalli, P., unpublished results).Therefore, an attempt was made to isolate and characterise the albumins present in fraction D b . Experimental Extraction and purification of albuminsAlbumins were extracted according to the method of Cantagalli et a1.13 1000 g of bread wheat whole flour (a mixture of varieties Autonomia and Generoso) were suspended in 2000 ml of phosphate buffer, pH 6.6 ( O -O~~M M -N~~H P O~, O .~~I M -K & P O~, 0-48~-NaCi), at 4"c and left overnight. The suspension was then centrifuged at 2700 Y g and the albumins were separated from the supernatant by salting out with between 0 . 9 3~ and 1 ' 33111 ammonium sulphate. The precipitate obtained was collected by centrifuging at 2700 x g and washed three times with 1 *33M ammonium sulphate. The yield of albumins was approximately 1 g. Gel-filtration on SephadexAbout 1 g of albumins was dissolved in 25 ml of buffer solution, pH 7.4 (0.008~-NazHP04, 0.001rvr-KH2P04, 0.137~-NaC1, 0*003~-KC1), applied on a Sephadex G-100 column (90 x 6 cm) and eluted with the same buffer at a rate of 1 .O ml/min, according to the method of Cantagalli et al.13 * Laboratorio Chimico Pro...

Purification and Characterization of ?-Amylase Inhibitors in Wheat (Triticum Aestivum Var. Anza)

“…Since then several workers have studied the a-amylase inhibitors in wheat (Shainkin and Birk 1970;Silano et al 1973; Saunders and Lang 1973;Petrucci et al 1974Petrucci et al ,1975Buonocore et al 1976;De Ponte et al 1976). Earlier, others had worked on the same wheat albumins without knowing that they had the ability t o inhibit a-amylase (Nimmo et al 1963; Fish and Abbott 1969;Ewert 1969; Feillet and Nimmo 1970;Sodini et al 1970; Cantagalli et al 1971). Shainkin and Birk (1970) purified and characterized two different a-amylase inhibitors.…”

Gel electrophoretic analysis of peanut proteins and enzymes. I. Characterization of DEAE-cellulose separated fractions

“…Gel electrophoresis has been used extensively to characterize and identify proteins and enzymes in biological systems (Cantagalli et al, 1971;Cherry et al, 1970Cherry et al, , 1971aCherry et al, ,b, 1972Dawson, 1971 Tombs, 1963) and to detect experimentally induced modifications in these molecules (Jensen, 1959;Neucere, 1972;Neucere et al, 1972). Cherry et al (1971b) and Cherry and Ory (1973a,b,c) have shown that the electrophoretic protein and enzyme profiles of individual peanuts are very complex, with much variation within the cultivars examined.…”

A new monomeric α-amylase inhibitor from the tetraploid emmer wheat is mostly active against stored product pests

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