|Title||Isolation and characterization of protein fractions from chickpea (Cicer arietinum L.) and oat (Avena sativa L.) seeds using proteomic techniques|
Chickpea Cicer arietinum L.) and oat Avena sativa L.) seeds are important sources of protein ingredients with potential nutritional, functional and bioactive properties. Protein fractions were prepared from chickpea and oat using sequential extractions with distilled water albumins), NaCl solution globulins) and NaOH solution glutelins), respectively. Molecular characteristics of individual protein fractions were investigated using polyacrylamide gel electrophoresis Native- and SDS-PAGE, and 2-DGE) in combination with reversed-phase high performance liquid chromatography RP-HPLC). Tryptic peptide sequences were identified using proteomic techniques including 1D trypsin in-gel digestion, liquid chromatography electrospray ionization tandem mass spectrometry LC-ESI-MS/MS) analysis and Mascot MS/MS ion search. Sequence similarity and potential bioactivity of proteins were examined using BLAST and BIOPEP analysis, respectively. Native-PAGE results showed chickpea and oat globulin fractions C-Gb and O-Gb) contained proteins corresponding to legumin 11S) and avenalin 12S), respectively. SDS-PAGE revealed that chickpea albumin and globulin fractions C-Ab and C-Gb) showed protein bands with MWs related to legumin 11S) and pea vicilin 7S) while chickpea glutelin fraction C-Gt) showed protein bands with MWs related to rice glutelin´╝Ť oat protein fractions O-Ab, O-Gb and O-Gt) showed protein bands with MWs related to oat 12S globulin avenalin). alpha- and beta-subunits of globulin and glutelin fractions from chickpea and oat were identified with estimated MWs ranging from 31 to 45 kDa and from 21 to 31 kDa, respectively. In vitro chickpea albumin, globulin and glutelin hydrolysates showed DH of 22.8%, 28.6% and 28.8%, respectively´╝Ť SDS-PAGE revealed that legumin alpha- and beta-subunits from chickpea globulin fraction C-Gb) were hydrolyzed. The identified tryptic peptides from chickpea and oat protein fractions showed sequence homology that corresponded to chickpea legumin alpha- and beta-subunit NCBI accession number: gi|6273402´╝Ť theoretical mass 56,216 Da), chickpea provicilin precursor NCBI accession number: gi|82173888´╝Ť theoretical mass 51,390 Da) and 12S seed storage globulin 1 NCBI accession number: gi|134918´╝Ť theoretical mass 58,508 Da). Chickpea and oat glutelin fractions C-Gt and O-Gt, respectively) exhibited large sequences homology with origin to chickpea legumin and oat 12S globulin. BLAST analysis of sequence alignments gave approximately 30% similarity of AAs sequences from chickpea legumin, oat 12S globulin 1 and rice glutelin precursor´╝Ť the oat 12S globulin 1 and rice glutelin precursor exhibited relatively high sequence homology of 63%. In silico BIOPEP results showed that chickpea legumin and provicilin precursor contained 177 and 133 potential ACE-inhibitory peptides within their primary sequences, respectively´╝Ť ficain and proteinase K were the suggested proteases that could theoretically release greater numbers of predicted ACE-inhibitory peptides 34 and 35, respectively) from these two proteins.
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