Soybean proteins electrophoresis

Hymowitz, T. N. Kaizuma. Dissemination of soybeans (Glycine max) Seed protein electrophoresis profiles among Japanese cultivars. Econ. Bot. 1979, 33, 311—319. 

Garcia-Ruiz C, Garcia MA, Garcia MC, Marina ML. Development of a capillary electrophoresis method for the determination of soybean proteins in soybean-rice gluten-free dietary products. 

Most of the applications of HPLC for protein analysis deal with the storage proteins in cereals (wheat, corn, rice, oat, barley) and beans (pea, soybeans). HPLC has proved useful for cultivar identihcation, protein separation, and characterization to detect adulterations (illegal addition of common wheat flour to durum wheat flour) [107]. Recently Losso et al. [146] have reported a rapid method for rice prolamin separation by perfusion chromatography on a RP POROS RH/2 column (UV detection at 230nm), sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE), and molecular size determination by MALDl-MS. DuPont et al. [147] used a combination of RP-HPLC and SDS-PAGE to determine the composition of wheat flour proteins previously fractionated by sequential extraction. 

To test this idea, a Bio-Rad Protean II electrophoresis cell and Bio-Rad Model 3000xi computer-controlled power supply were used to carry out the electrophoretic separation and recovery of pre-stained and unstained protein calibration standards (lysozyme, soybean trypsin inhibitor, carbonic anhydrase, ovalbumin, bovine serum albumin and phosphorylase B) obtained from Bio-Rad Laboratories. Standard SDS-gel electrophoresis techniques were used [167]. 

Power supply and electrophoresis apparatus Protein molecular weight standards (suggested standards phosphorylase [97,400 Da], bovine serum albumin [66,200 Da], ovalbumin [45,000 Da], carbonic an-hydrase [31,000 Da], soybean trypsin inhibitor [21,500 Da], and lysozyme [14,400 Da] at 1 mg/ml each in a single mixture)... 

Although, on the basis of end-group analysis (1 mole ofN-terminal alanine per 30,000 g of protein in 8 M urea) and electrophoretic studies,544 the soybean agglutinin appeared to be a tetrameric protein composed of identical subunits, Lotan and coworkers546 later reported resolution of two types of subunit in the ratio of 1 1, either by electrophoresis at alkaline pH in the presence of urea or detergents, or by chromatography on DEAE-cellulose in Tris buffer, pH 7.3, with 8 M urea. 

Figure 2. SDS gel electrophoresis of the products of partial cystine cleavage for several test proteins. A. molecular weight standards, B. yeast alcohol dehydrogenase. C. P-lactoglobulin, D. hen egg lysozyme, E. ovalbumin, F. calf fetal serum fetuin. Molecular weight standards are indicated by arrows on the left side of the gel and are bovine serum albumin (66,300), bovine liver glutamate dehydrogenase (55,400), porcine muscle lactate ddiydiogenase (36,500), bovine erythrocyte carbonic anhydrase (31,000), soybean trypsin inhibitor (21,500), hen egg lysozyme (14,400), bovine lung aprotinin (6,000), unresolved bovine pancreatic insulin A and B chains.

In order to establish a molecular data base for BR and begin to address questions (a) and (b) above, we have examined whether BR affects the transcription of auxin-induced genes in elongating soybean epicotyl sections using available auxin-induced soybean sequences as probes. More generally, we have shown changes in protein synthesis caused by BR in soybean epicotyls and hypocotyls by in vitro translation of mRNA (+ or - BR) followed by 2-D gel electrophoresis. 

Fig. 3. (left) SDS-PAGE of p PJNAD treated SR vesicles in the absence and presence of poly L-lysine. 0.88 mg/ml of SR was incubated at 25 C for 60 min with 20 [xM [32p]NAD (2.8 ci/mmol) and 0, 50 or 100 M,g/ml of poly L-lysine. Radiolabeling of the acid insoluble fraction (25 p.g protein) from each sample was analyzed by SDS-polyacrylamide gel electrophoresis (8). Tlie Coomassie brilliant blue-staining pattern (A) and an autoradiogram of the same gel (B) are shown. Molecular weight markers phosphorylase b (94 K), bovine serum albumin (67 K), ovalbumin (43 K), carbonic anhydrase (30 K) and soybean trypsin inhibitor (20.1 K). 

Fig. 1. Ultrogel AcA 54 chromatography of sARF I effect of dimyristoyl phosphatidylcholine (DMPC) on ADP-ribosylation of Gsa and choleragen A protein. Purified sARF I was chromatographed on a column (1.2 x 104 cm) of Ultrogel AcA 54. Fractions (1 ml) were collected and samples were (A) analyzed by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and assayed for ARF activity in a reaction mixture containing Gg (0.4 jig), choleragen (25 pg), and 100 pM GTP without (B) or with (C) 1 mM DMPC. (A) SDS-PAGE of 250 pi of fraction plus bovine serum albumin, 10 pg. Lanes 1-4, fractions 68, 72, 76 and 80 Lane 5, standard proteins, phosphorylase b, bovine serum albumin, ovalbumin, carbonic anhydrase, soybean trypsin inhibitor, a-lactalbumin. (B) and (C) Autoradiograms of ADP-ribosylated proteins. Lanes 1-4, fractions 68,72,76 and 80 Lane 5, column buffer.

Blazek V, CaldweU RA 2009. Comparison of SDS gel capillary electrophoresis with microfluidic lab-on-a-chip technology to quantify relative amounts of 7S and IIS proteins from 20 soybean cultivars. Int J Food Sci Technol 44 2127-2134. 

Although most NRs are regulated by nitrate, constitutive NRs have been reported in several species. Soybean contains two constitutive NRs, an NAD(P)H NR and an NADH NR, which are distinct from the typical inducible NADH NR present in soybean and most plant species (Nelson et ai, 1983 Streit and Harper, 1986). Tobacco plants (Muller, 1983) and cells (Marion-Poll et ai, 1984 Muller and Grafe, 1978) have moderate levels of constitutive NR activity. In tobacco the constitutive NR activity is presumed to be the NADH NR since mutants lack both the constitutive and inducible NR activities. In barley a constitutive NR was detected after electrophoresis of wild-type and mutant narla crude extracts. This NR was distinct from the major inducible NR (Heath-Pagliuso et ai, 1984). The relationship of this NR to the NAD(P)H NR in nar a is not known, although our data indicate that the NAD(P)H NR is induced by nitrate (Warner and Huffaker, 1989). Low levels of a constitutive NR have also been observed in maize (Remmler and Campbell, 1986). The significance of low levels of apparent constitutive NR should be considered with caution since NR mRNA in barley (Melzer et ai, 1989) and NR protein in maize (Oaks et al., 1988) appear to be induced by very low levels of nitrate. 

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