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Uncertainty analysis of the microtiter plate method for determining trypsin inhibitor activity

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Abstract

Protease inhibitor activity is frequently measured in legume seeds as protease inhibitors are thought to have anti-nutritional as well as anti-carcinogenic properties. Trypsin inhibitor activity (TIA) can be measured using different methods. The microtiter plate method is very convenient and routinely used; therefore, in this study, we analyzed the measurement uncertainty of the microtiter plate method to understand what affects the measurement results, as well as to compare TIA values obtained by similar and different methods. For uncertainty analysis of TIA measurement, we used the soybean variety ‘Vojvodjanka,’ which is known to have TIA greater than 80 trypsin units inhibited (TUI) per mg of seed. We followed the Guide to the Expression of Uncertainty in Measurement (GUM) for our uncertainty analysis of the microtiter plate method for TIA testing, which we present in the form of an uncertainty budget. Absorbance measurement and preparation of sample reaction mixture took the largest percent (71 %) of overall uncertainty of TIA value. The TIA of soybean variety ‘Vojvodjanka’ was (94.1 ± 8.4) TUI/mg, and this result is consistent with those obtained by other authors. The microtiter plate method is a reliable method for TIA measurement, making seed quality testing more efficient.

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References

  1. Clemente A, Domoney C (2006) Biological significance of polymorphism in legume protease inhibitors from the Bowman–Birk family. Curr Protein Pept Sci 7:201–216

    Article  CAS  Google Scholar 

  2. Casaretto JA, Zuniga GE, Corcuere LJ (2004) Abscisic acid and jasmonic acid affect proteinase inhibitor activities in barley leaves. J Plant Physiol 161:389–396

    Article  CAS  Google Scholar 

  3. Ryan CA (1990) Protease inhibitors in plants: genes for improving defenses against insects and pathogens. Annu Rev Phytopathol 28:425–449

    Article  CAS  Google Scholar 

  4. Mandal S, Kundu P, Roy B, Mandal RK (2002) Precursor of the inactive 2S seed storage protein from the Indian mustard Brassica juncea is a novel trypsin inhibitor. J Biol Chem 277:37161–37168

    Article  CAS  Google Scholar 

  5. Chye ML, Sin SF, Xu ZF, Yeung EC (2006) Serine proteinase inhibitor proteins: exogenous and endogenous functions. In Vitro Cell Dev Biol Plant 42:100–108

    Article  CAS  Google Scholar 

  6. Lam JM, Pwee KH, Sun WQ, Chua YL, Wang XJ (1999) Enzyme-stabilizing activity of seed trypsin inhibitors during desiccation. Plant Sci 142:209–218

    Article  CAS  Google Scholar 

  7. Solmon M, Belenghi B, Delledonne M, Menachem E, Levine A (1999) The involvement of cysteine proteases inhibitor genes in the regulation of programmed cell death in plants. Plant Cell 11:431–443

    Article  Google Scholar 

  8. Kennedy AR (1998) The Bowman–Birk inhibitor from soybeans as an anti-carcinogenic agent. Am J Clin Nutr 68(suppl):1406S–1412S

    CAS  Google Scholar 

  9. Kennedy AR, Kritchevsky D, Shen WC (2003) Effects of spermine-conjugated Bowman–Birk inhibitor (spermine-BBI) on carcinogenesis and cholesterol biosynthesis in mice. Pharm Res 20:1908–1910

    Article  CAS  Google Scholar 

  10. Bode W, Huber R (2000) Structural basis of the endoproteinase-protein inhibitor interaction. Biochim Biophys Acta 1477:241–252

    Article  CAS  Google Scholar 

  11. Richardson M (1991) In: Dey PM, Harborne JB (eds) Methods in plant biochemistry: amino acids, proteins and nucleic acids, vol 5. Academic Press, New York

    Google Scholar 

  12. Macedo MLR, DizFilho EBS, Freire MGM, Oliva MLV, Sumikawa JT, Toyama MH, Marangoni S (2011) A trypsin inhibitor from Sapindus saponaria L. seeds: purification, characterization, and activity towards pest insect digestive enzyme. Protein J 30:9–19

    Article  CAS  Google Scholar 

  13. Birk Y, Gertler A, Khalef S (1963) A pure trypsin inhibitor from soya bean. Biochem J 87:281–284

    Article  CAS  Google Scholar 

  14. Bowman DE (1946) Differentiation of soy bean antitryptic factor. Proc Soc Exp Biol Med 63:547–550

    Article  CAS  Google Scholar 

  15. Tanaka AS, Sampaio MU, Marangoni S, de Oliveira B, Novello JC, Oliva ML, Fink E, Sampaio CA (1997) Purification and primary structure determination of a Bowman–Birk trypsin inhibitor from Torresea cearensis seeds. Biol Chem 378:273–281

    Article  CAS  Google Scholar 

  16. Odani S, Koide T, Ono T (1986) Wheat germ trypsin inhibitor. Isolation and structural characterization of single-headed and doubled-headed inhibitors of the Bowman–Birk type. J Biochem (Tokyo) 100:975–983

    CAS  Google Scholar 

  17. Singh RR, Appu Rao AG (2002) Reductive unfolding and oxidative refolding of a Bowman–Birk inhibitor from horsegram seeds (Dolichosbi florus): evidence for hyperractive disulfide bonds and rate-limiting nature of disulfide isomerization in folding. Biochim Biophys Acta 1597:280–291

    Article  CAS  Google Scholar 

  18. Page D, Quillien L, Duc G (2000) Trypsin inhibitory activity measurement: simplifications of the standard procedure used for pea seed. Crop Sci 40:1482–1485

    Article  CAS  Google Scholar 

  19. Champ MM (2002) Non-nutrient bioactive substances of pulses. Br J Nutr 88:307–319

    Article  Google Scholar 

  20. Gaborit T, Quillien L, Gueguen J (1993) In: Poel AVD (ed) International workshop on antinutritional factors (ANFs) in legume seeds, Wageningen, the Netherlands 1–3 Dec, EAAP Wageningen, the Netherlands

  21. Hamerstrand GE, Black LT, Glover JD (1981) Trypsin inhibitors in soy products: modification of the standard analytical procedure. Cereal Chem 58(1):42–45

    CAS  Google Scholar 

  22. Liu K, Markakis P (1989) An improved colorimetric method for determining antitryptic activity in soybean products. Cereal Chem 66(5):415–422

    CAS  Google Scholar 

  23. JCGM 100:2008 Evaluation of measurement data—guide to the expression of uncertainty in measurement. BIPM, IEC, IFCC, ILAC, ISO, IUPAC, IUPAP, OIML

  24. Pesic BM, Vucelic-Radovic VB, Barac BM, Stanojevic PS, Nedovic AV (2007) Influence of different genotypes on trypsin inhibitor levels and activity in soybeans. Sensors 7:67–74

    Article  CAS  Google Scholar 

  25. Kakade ML, Rackis JJ, McGhee JE, Puski G (1974) Determination of trypsin inhibitor activity of soy products: a collaborative analysis of an improved procedure. Cereal Chem 51:376–382

    CAS  Google Scholar 

  26. Nomenclature Committee of the International Union of Biochemistry (NC-IUB) (1979) Units of enzyme activity. Eur J Biochem 97(2):319–320

    Article  Google Scholar 

  27. Erlanger BF, Kokowsky N, Cohen W (1961) The preparation and properties of two new chromogenic substrates of trypsin. Arch Biochem Biophys 95:271–278

    Article  CAS  Google Scholar 

  28. EURACHEM/CITAC Guide CG 4 (2012) Quantifying uncertainty in analytical measurement, 3rd edn. ISBN:978-0-948926-30-3. http://www.eurachem.org

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Acknowledgments

This research was financed by the Ministry of Education and Science of the Republic of Serbia, within the Project TR 31024 ‘Increasing the market of forage crops by breeding and optimizing seed production.’

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Authors and Affiliations

  1. Institute of Field and Vegetable Crops, Maksima Gorkog 30, 21000, Novi Sad, Serbia

    Vesna Župunski, Zorica Nikolić, Gordana Petrović, Gordana Tamindžić & Đura Karagić

  2. Faculty of Technical Sciences, University of Novi Sad, Trg Dositeja Obradovića 6, 21000, Novi Sad, Serbia

    Zoran Mitrović

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  1. Vesna Župunski
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  2. Zoran Mitrović
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Correspondence to Vesna Župunski.

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Župunski, V., Mitrović, Z., Nikolić, Z. et al. Uncertainty analysis of the microtiter plate method for determining trypsin inhibitor activity. Accred Qual Assur 21, 151–160 (2016). https://doi.org/10.1007/s00769-016-1192-1

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