**2. Material and methods**

*In vitro* protein digestibility was studied in 10 lines and seven F1 hybrids of the grain sorghum (*Sorghum bicolor* (L.) Moench) (Table 1).

To study *in vitro* protein digestibility the modified method of whole-grain flour pepsin treatment was used (Oria et al. 1995). For each variety 25 mg of flour was treated with 5 ml of 0.15% pepsin solution (P7000 Sigma-Aldrich) in the 0.1 M potassium-phosphate buffer (pH 2.0) for 120 min at 37 0С with repeated shaking. Analysis of seed storage protein (kafirin) spectra was performed before and after pepsin treatment by SDS-PAG electrophoresis (SDS-PAGE) in reducing conditions. SDS-PAGE was carried out in the 12.5% (w/v) acrylamide separating gel (0.375 M TRISHCl, pH 8.8) and 4% stacking gel (0.125 M TRIS, pH 6.8) according to modified Laemmli method (Laemmli, 1970). SDS-reducing buffer: 62.5 mM TRISHCl, pH 6.8, 20% glycerol, 2% SDS, 5% β-mercaptoethanol; running buffer: 25.0 mM TRISHCl, 192 mM glycine, 0.1% SDS, pH 8.3; spacer thikness 1.00 mm. Gels were electrophoresed at 20-23 ma for about 5 hr. Gels were stained with Coomassie Brilliant Blue G-250 or R-250 (Diezel et al, 1972).

together by disulphide (S-S) bonds, which are formed by sulphur-containing amino acids (Nunes et al., 2005). In the native state, both mono- and oligomers are present, while in 'reduced' extracts (i.e. with addition of 5% 2-mercaptoethanol that destroys S-S bonds) only

The causes of the poor kafirin digestibility appear to be multi-factorial (Duodu et al., 2003). Among these factors are chemical structure of kafirin molecules, some of which (γ- and βkafirins) are abundant with sulfur-containing amino acids that are capable to form S-S bonds, resistant to protease digestion; interactions of kafirins with non-protein components such as polyphenols and polysaccharides; and spatial organization of different kafirins in

Among the methods that were developed for investigation of sorghum protein digestibility (Pedersen & Eggum, 1983; Mertz et al., 1984; Aboubacar et al., 2003), pepsin digestion of the flour proteins with subsequent gel electrophoresis is the most informative. This method, originally applied by B. Hamaker and co-workers (Weaver et al., 1998; Aboubacar et al., 2001) has been used in a number of studies (Nunes et al., 2004; Wong et al., 2010). Application of this method allowed to isolate sorghum lines with high protein digestibility (Weaver et al., 1998) and to find out that γ-kafirin plays an important role in resistance of sorghum seed storage proteins to protease digestion, namely, γ-kafirin forms a disulfidebound enzyme-resistant layer at the periphery of protein bodies that restricts access of proteases to the inferior-located and more easily digested α-kafirins (Oria et al., 2000).

In our investigations (Italianskaya et al., 2009), we studied the protein digestibility in different sorghum lines and hybrids using this method and revealed significant polymorphism for *in vitro* kafirin digestibility as well as the strong genetic bases of this trait. In this paper, we summarize the results of these studies, which allowed isolating sorghum lines and F1 hybrids with increased nutritive value. In addition, we demonstrate that kafirin polymorphism may be used in genetic experiments, namely, in determination of genetic

*In vitro* protein digestibility was studied in 10 lines and seven F1 hybrids of the grain

To study *in vitro* protein digestibility the modified method of whole-grain flour pepsin treatment was used (Oria et al. 1995). For each variety 25 mg of flour was treated with 5 ml of 0.15% pepsin solution (P7000 Sigma-Aldrich) in the 0.1 M potassium-phosphate buffer (pH 2.0) for 120 min at 37 0С with repeated shaking. Analysis of seed storage protein (kafirin) spectra was performed before and after pepsin treatment by SDS-PAG electrophoresis (SDS-PAGE) in reducing conditions. SDS-PAGE was carried out in the 12.5% (w/v) acrylamide separating gel (0.375 M TRISHCl, pH 8.8) and 4% stacking gel (0.125 M TRIS, pH 6.8) according to modified Laemmli method (Laemmli, 1970). SDS-reducing buffer: 62.5 mM TRISHCl, pH 6.8, 20% glycerol, 2% SDS, 5% β-mercaptoethanol; running buffer: 25.0 mM TRISHCl, 192 mM glycine, 0.1% SDS, pH 8.3; spacer thikness 1.00 mm. Gels were electrophoresed at 20-23 ma for about 5 hr. Gels were stained with Coomassie Brilliant

monomers were detected (El Nour et al., 1998).

the protein bodies of endosperm cells.

structure of endosperm in sorghum.

Blue G-250 or R-250 (Diezel et al, 1972).

sorghum (*Sorghum bicolor* (L.) Moench) (Table 1).

**2. Material and methods** 


In parenthesis: brief designation used in the paper. F1 hybrids were obtained using male-sterile counterparts of fertile lines; they are designated as A2 or М35-1А depending on the type of male sterility-inducing cytoplasm.

Table 1. The grain sorghum entries used in this investigation

For quantitative estimation of kafirin digestibility the SDS-PAGE banding patterns were scanned by laser densitometer ULTROSCAN XL (LKB-Pharmacia) with wavelength 633 nm. The protein quantity in each fraction was expressed as the area (mm2) of the appropriate peak on densitogram, which was calculated by Software LKB 2222 (Version 3.00). In some experiments, the SDS-PAGE banding patterns were analyzed by Scangel program (developed by Dr. A.F. Ravich). The protein quantity in each fraction and in each lane of electrophoregramm was expressed as the amount of dots in the appropriate protein band. Experiments were performed in two replications. The data on digestibility of kafirins (the ratio of protein peak area before and after pepsin digestion) were subjected to variance analysis using the program Agros (Version 2.09; Dr. S. Martynov, Wheat Genetic Resources Department, N.I. Vavilov Institute of Plant Production, St. Petersburg, Russia).

In some lines and hybrids, the dependence of *in vitro* protein digestibility from *in vitro* starch digestibility was studied. In this experiment, the flour, firstly, was subjected to amylolitic enzyme treatment according to the method of B.V. McCleary (McCleary et al., 2002) using Megazyme Resistant Starch Kit (Megazyme Co, Ireland). The pellet remained after removal of solubilised starch was used for pepsin treatment according to the method described above, and, after that, the protein spectrum of the sample was studied by SDS-PAGE.

In order to use kafirins as markers of genetic structure of endosperm the modified technique of SDS-PAGE was applied. In these experiments, AS-1a line of the grain sorghum, which is characterized by a low frequency of parthenogenic embryo formation (Elkonin et al., 2012)

Gel Electrophoresis as a Tool to Study Polymorphism and

proteins peak area sum. Mean data of two replications.

pepsin digestion (54.5% digestibility level) (Table 3).

(Italianskaya et al., 2009)

experiments.

value.

et al., 1993).

Nutritive Value of the Seed Storage Proteins in the Grain Sorghum 467

(4-10%) that is in concordance with the literature data (Shull et al., 1991; Waterson

KVV-45 13.2 37.3 2.0 3.9 Milo-10 12.8 30.7 5.3 7.2 А2 KVV-97 13.1 24.3 3.2 5.7 А2 KVV-97/P-614 13.3 31.4 4.4 5.3 P-614 10.7 26.9 5.5 5.5 A2 KVV-114 10.8 26.0 4.1 6.9 A2 KVV-114/V-4w 9.5 24.9 4.6 8.9 V-4w 10.3 33.1 3.4 10.2 1 Relative content of each fraction is expressed as percentage of its peak area from the total endosperm

Table 2. Relative content of different kafirin fractions in some sorghum lines and F1 hybrids

After pepsin digestion the amount of protein in kafirin fractions substantially reduced (Figs. 1; 2). Different sorghum lines and cultivars differed significantly by this trait. For example, among the entries presented in Figure 2 the highest digestibility level had VIR-120 – 90.8% (lanes 1 and 2), while the kafirins of line KVV-3 (lanes 9 and 10) were the most resistant to

In our previous study (Italianskaya et al., 2009), we observed significantly higher variation among the lines. For example, in the cultivar Volzhskoe-4 (V-4, registered standard), the amount of undigested γ- and α-kafirins after pepsin digestion was 80% and 73% from their initial contents, respectively. The total amount of undigested kafirins in cv. V-4 was 70% (digestibility level was 30%). At the same time, in the line KVV-45, the total amount of undigested proteins was 37% (digestibility level was 63%). Percentage of undigested α1 and γ-kafirins in the line KVV-45 was only 25% and 30%, respectively. The differences in kafirin spectra between this line and cv. V-4 before and after pepsin treatment are clearly seen in the Figure 3. Further investigation confirmed a high level of protein digestibility in this line (78.4%) (Table 3). Perhaps, the line KVV-45 contains mutation(s) in the genes encoding structure or deposition of kafirin molecules and, therefore, is of a great interest for future

Remarkably, in subsequent investigation it was found that in the line Topaz the digestibility level was even higher than in the KVV-45 and reached 89% (see chapter 4). This value is sufficiently high; it corresponds to digestibility level of whole grain flour protein of the best condenced-tannin-free sorghum entries (Axtell et al., 1981, and other reports, as cited in Duodu et al., 2003). One should expect that this line would have high nutritive

One should note high digestibility of the β-kafirin fractions in majority of lines. This fact contradicts to hypothesis that explains poor kafirin digestibility by formation of S-S bonds because β-kafirins as well as γ-kafirins contain a high amount of cystein, a sulfur-containing amino acid (Belton et al, 2006). In addition, in all lines, the polypeptides with molecular

γ α1 α2 β

Line, F1 hybrid Protein fraction, % 1

was used. Emasculated panicles of this line were pollinated with the pollen of the line Volzhskoe-4w homozygous for dominant gene *Rs*, conditioning purple color of coleoptiles, seedling leaves and stem. To study the origin of the kernels (apomictic or sexual) with the aid of the kafirin polymorphism, the kernels were split into two parts. The part with an embryo was put in a tray on a moisture filter paper to study the phenotypic traits of a seedling (expression of the *Rs* gene). Another part was used in SDS-PAGE to study its kafirin spectrum. In these experiments, gels were electrophoresed at constant voltage (70 V) for about 15 hr. Gels were stained with AgNO3 solution.
