110 lines
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110 lines
14 KiB
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<document id="1E14416E4EADBAC5C957586FA9117943" ID-DOI="10.1016/j.phytochem.2018.12.018" ID-ISSN="1873-3700" ID-Zenodo-Dep="10481851" IM.bibliography_approvedBy="karina" IM.illustrations_approvedBy="karina" IM.materialsCitations_approvedBy="felipe" IM.metadata_approvedBy="felipe" IM.tables_approvedBy="karina" IM.taxonomicNames_approvedBy="karina" IM.treatments_approvedBy="karina" checkinTime="1704928208845" checkinUser="felipe" docAuthor="Mohanraj, Soundappan S., Gujjarlapudi, Mariyamma, Lokya, Vadthya, Mallikarjuna, Nalini, Dutta-Gupta, Aparna & Padmasree, Kollipara" docDate="2019" docId="0396878CFFF1FFE0FFD4FA2DFCCCFEE9" docLanguage="en" docName="Phytochemistry.159.159-171.pdf" docOrigin="Phytochemistry 159" docSource="http://dx.doi.org/10.1016/j.phytochem.2018.12.018" docStyle="DocumentStyle:9E596C34F4E94307D29315B03ACE1007.6:Phytochemistry.2014-2019.journal_article" docStyleId="9E596C34F4E94307D29315B03ACE1007" docStyleName="Phytochemistry.2014-2019.journal_article" docStyleVersion="6" docTitle="Rhynchosia sublobata subsp. protease Meikle" docType="treatment" docVersion="4" lastPageNumber="160" masterDocId="FFAFFFF4FFF0FFE1FFB0FFDBFFA0FF9C" masterDocTitle="Purification and characterization of Bowman-Birk and Kunitz isoinhibitors from the seeds of Rhynchosia sublobata (Schumach.) Meikle, a wild relative of pigeonpea" masterLastPageNumber="171" masterPageNumber="159" pageNumber="160" updateTime="1706742242453" updateUser="karina">
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<mods:title id="DF5A1B909F1D09D7F615A61688CB39B7">Purification and characterization of Bowman-Birk and Kunitz isoinhibitors from the seeds of Rhynchosia sublobata (Schumach.) Meikle, a wild relative of pigeonpea</mods:title>
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<mods:namePart id="126757355320E4A35CD9D8207062B3C4">Mohanraj, Soundappan S.</mods:namePart>
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<mods:affiliation id="703FA1793C82A665663D4B681A1065CF">Department of Plant Sciences, School of Life Sciences, University of Hyderabad, Hyderabad, 500 046, Telangana, India</mods:affiliation>
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<mods:namePart id="3719A196E0731B448E9EE4BB6FB6E099">Gujjarlapudi, Mariyamma</mods:namePart>
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<mods:namePart id="0EB63BDE0B59D3AA5E4A5EFE6CADBCE9">Lokya, Vadthya</mods:namePart>
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<mods:namePart id="F1F351759F6E1645BE792A9D191ED4B4">Mallikarjuna, Nalini</mods:namePart>
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<mods:namePart id="2CF68F9A3A6E762825CD7DA9FACFEF4A">Dutta-Gupta, Aparna</mods:namePart>
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<mods:namePart id="7E7F4B92143D29C43CBDC45CFBE04DF6">Padmasree, Kollipara</mods:namePart>
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<mods:title id="4A3E9B91D68466E5A30E1EACDA8F8D94">Phytochemistry</mods:title>
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<mods:date id="7DFAC4B93343A63C051E3772362E0C8E">2019</mods:date>
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<treatment id="0396878CFFF1FFE0FFD4FA2DFCCCFEE9" ID-DOI="http://doi.org/10.5281/zenodo.10593972" ID-Zenodo-Dep="10593972" LSID="urn:lsid:plazi:treatment:0396878CFFF1FFE0FFD4FA2DFCCCFEE9" httpUri="http://treatment.plazi.org/id/0396878CFFF1FFE0FFD4FA2DFCCCFEE9" lastPageNumber="160" pageId="1" pageNumber="160">
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<subSubSection id="C3256511FFF1FFE0FFD4FA2DFD68F995" box="[100,712,1526,1545]" pageId="1" pageNumber="160" type="nomenclature">
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<emphasis id="B94BEA88FFF1FFE0FFD4FA2DFD68F995" bold="true" box="[100,712,1526,1545]" italics="true" pageId="1" pageNumber="160">
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2.1. Purification of
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<taxonomicName id="4C3F4D19FFF1FFE0FEAAFA2DFE2AF995" authority="(Schumach.) Meikle" authorityName="Meikle" baseAuthorityName="Schumach." box="[282,394,1526,1545]" class="Magnoliopsida" family="Fabaceae" genus="Rhynchosia" kingdom="Plantae" order="Fabales" pageId="1" pageNumber="160" phylum="Tracheophyta" rank="subSpecies" species="sublobata" subSpecies="protease">R. sublobata</taxonomicName>
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protease inhibitors “RsPI” by FPLC
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The 20–60% (NH
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)
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SO
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fractionated CPI was resolved into two peaks in trypsin-affinity column when eluted with
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HCl (
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<figureCitation id="13042A1FFFF1FFE0FD01F991FD58F9C1" box="[689,760,1610,1629]" captionStart="Fig" captionStartId="2.[100,130,585,602]" captionTargetBox="[193,1399,153,560]" captionTargetId="figure-146@2.[187,1400,152,563]" captionTargetPageId="2" captionText="Fig. 1. FPLC purification of ‘RsPI’ from R. sublobata seeds. Elution profiles of (A) trypsin-affinity and (B) gel filtration columns. The protein peaks active against trypsin were marked with an asterisk (*); (C) Tricine SDS-PAGE (15%) showing different fractions of purification protocol under non-reducing conditions. Lane 1 is loaded with Protein molecular weight marker (PMWM) and lanes 2–6 are loaded with 5 or 10 μg of corresponding protein sample. The gel was stained with silver nitrate. CPI – crude protease inhibitor, TAC – Trypsin affinity chromatography, GFC – Gel filtration chromatography and SBBI – Soybean BBI." figureDoi="http://doi.org/10.5281/zenodo.10481853" httpUri="https://zenodo.org/record/10481853/files/figure.png" pageId="1" pageNumber="160">Fig. 1A</figureCitation>
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). The peak 2 with TI activity was further loaded on a Sephadex G-50 fine gel filtration chromatography column to remove any high MW contaminants retained in the trypsin affinity fractions. The pool of peak 2 fractions with significant TI activity was referred to as
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<taxonomicName id="4C3F4D19FFF1FFE0FD3FF962FCA2F950" box="[655,770,1721,1740]" class="Magnoliopsida" family="Fabaceae" genus="Rhynchosia" kingdom="Plantae" order="Fabales" pageId="1" pageNumber="160" phylum="Tracheophyta" rank="species" species="sublobata">
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<emphasis id="B94BEA88FFF1FFE0FD3FF962FCA2F950" bold="true" box="[655,770,1721,1740]" italics="true" pageId="1" pageNumber="160">R. sublobata</emphasis>
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trypsin specific protease inhibitors “RsPI” (
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<figureCitation id="13042A1FFFF1FFE0FE4FF90EFDE3F974" box="[511,579,1749,1768]" captionStart="Fig" captionStartId="2.[100,130,585,602]" captionTargetBox="[193,1399,153,560]" captionTargetId="figure-146@2.[187,1400,152,563]" captionTargetPageId="2" captionText="Fig. 1. FPLC purification of ‘RsPI’ from R. sublobata seeds. Elution profiles of (A) trypsin-affinity and (B) gel filtration columns. The protein peaks active against trypsin were marked with an asterisk (*); (C) Tricine SDS-PAGE (15%) showing different fractions of purification protocol under non-reducing conditions. Lane 1 is loaded with Protein molecular weight marker (PMWM) and lanes 2–6 are loaded with 5 or 10 μg of corresponding protein sample. The gel was stained with silver nitrate. CPI – crude protease inhibitor, TAC – Trypsin affinity chromatography, GFC – Gel filtration chromatography and SBBI – Soybean BBI." figureDoi="http://doi.org/10.5281/zenodo.10481853" httpUri="https://zenodo.org/record/10481853/files/figure.png" pageId="1" pageNumber="160">Fig. 1B</figureCitation>
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) and it showed two major bands when separated in Tricine SDS-PAGE (
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<figureCitation id="13042A1FFFF1FFE0FDFBF92AFD32F898" box="[587,658,1777,1796]" captionStart="Fig" captionStartId="2.[100,130,585,602]" captionTargetBox="[193,1399,153,560]" captionTargetId="figure-146@2.[187,1400,152,563]" captionTargetPageId="2" captionText="Fig. 1. FPLC purification of ‘RsPI’ from R. sublobata seeds. Elution profiles of (A) trypsin-affinity and (B) gel filtration columns. The protein peaks active against trypsin were marked with an asterisk (*); (C) Tricine SDS-PAGE (15%) showing different fractions of purification protocol under non-reducing conditions. Lane 1 is loaded with Protein molecular weight marker (PMWM) and lanes 2–6 are loaded with 5 or 10 μg of corresponding protein sample. The gel was stained with silver nitrate. CPI – crude protease inhibitor, TAC – Trypsin affinity chromatography, GFC – Gel filtration chromatography and SBBI – Soybean BBI." figureDoi="http://doi.org/10.5281/zenodo.10481853" httpUri="https://zenodo.org/record/10481853/files/figure.png" pageId="1" pageNumber="160">Fig. 1C</figureCitation>
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). The lower major band apparently possessed molecular mass of ca. 13 kDa or 8 kDa when compared with commercially available protein molecular weight marker (PMWM) and soybean Bowman-Birk Inhibitor (SBBI), respectively. This discrepancy is very common in TIs, particularly among those which are known to self-associate depending on the method of molecular weight determination such as ultra-centrifugation, size exclusion chromatography, SDS-PAGE, 2-D gel electrophoresis or MALDI-TOF mass spectrometry (
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<bibRefCitation id="EFAE4B6BFFF1FFE0FB98FF7BFA99FF2E" author="Bergeron, D. & Nielsen, S. S." box="[1064,1337,159,179]" pageId="1" pageNumber="160" pagination="1544 - 1549" refId="ref11740" refString="Bergeron, D., Nielsen, S. S., 1993. Partial characterization of trypsin inhibitors and Nterminal sequences of five trypsin isoinhibitors of great northern beans (Phaseolus vulgaris). J. Agric. Food Chem. 41, 1544 - 1549." type="journal article" year="1993">Bergeron and Nielsen, 1993</bibRefCitation>
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and references therein;
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<bibRefCitation id="EFAE4B6BFFF1FFE0FC31FF60FB95FF52" author="Swathi, M. & Lokya, V. & Swaroop, V. & Mallikarjuna, N. & Kannan, M. & Dutta-Gupta, A. & Padmasree, K." box="[897,1077,187,206]" pageId="1" pageNumber="160" pagination="77 - 87" refId="ref15180" refString="Swathi, M., Lokya, V., Swaroop, V., Mallikarjuna, N., Kannan, M., Dutta-Gupta, A., Padmasree, K., 2014. Structural and functional characterization of proteinase inhibitors from seeds of Cajanus cajan (cv. ICP 7118). Plant Physiol. Biochem. 83, 77 - 87." type="journal article" year="2014">Swathi et al., 2014</bibRefCitation>
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,
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<bibRefCitation id="EFAE4B6BFFF1FFE0FBF2FF60FBD2FF52" author="Swathi, M. & Mishra, P. K. & Lokya, V. & Swaroop, V. & Mallikarjuna, N. & Dutta-Gupta, A. & Padmasree, K." box="[1090,1138,187,206]" pageId="1" pageNumber="160" refId="ref15247" refString="Swathi, M., Mishra, P. K., Lokya, V., Swaroop, V., Mallikarjuna, N., Dutta-Gupta, A., Padmasree, K., 2016. Purification and partial characterization of trypsin-specific proteinase inhibitors from pigeonpea wild relative Cajanus platycarpus L. (Fabaceae) active against gut proteases of Lepidopteran pest Helicoverpa armigera. Front. Physiol. 7." type="book" year="2016">2016</bibRefCitation>
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). However, molecular mass of lower band is assumed to be closer to 8 kDa considering its similar migration pattern with SBBI as compared to PMWM marker. In contrast, the upper band which showed molecular mass of ca. 20 kDa is well in range with both PMWM and SBBI. Further, this discrepancy in molecular mass was monitored by loading both SBBI and RsPI in all gels performed in this study.
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</paragraph>
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</subSubSection>
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</treatment>
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