Skip to main content
SpringerLink
Log in

An optimized protocol to assess trypsin activity in biological samples

  • Original Paper
  • Published:
Monatshefte für Chemie - Chemical Monthly Aims and scope Submit manuscript

Abstract

Trypsin is an enzyme that facilitates digestion. It is found in the small intestine and may also be synthesized by bacteria, plants, and fungi. However, it is mainly synthesized for commercial use from cattle pancreases. Serum trypsin determination seems to be a specific diagnostic for acute pancreatitis. Therefore, the development of improved trypsin activity protocols is essential for clinical pathology and pharmaceutical research. In this study, a simple spectrophotometric procedure for trypsin, a pancreatic protease, was developed. The current protocol utilized a previously known peroxidase-like reaction to estimate the trypsin activity. An additional acidification step is employed to improve the efficiency of the protocol. Trypsin has the ability to preferentially cleave the natural compound cytochrome c into heme-peptide fragments. In our study, the resulting peroxidase-like activity was catalyzed by the oxidation of 3,3ʹ,5,5ʹ-tetramethylbenzidine in the presence of H2O2. Sulfuric acid was added to stop the enzymatic reaction before recording the absorbance at 450 nm. To optimize the formation of the end product, we used the response surface methodology to apply the Box–Behnken design to assess the assay's precision. The reliability of this new method was compared to a Bland–Altman plot analysis of trypsin activity in matched samples using the standard procedure. The protocol allowed for trypsin investigations in the 5–1500 ng/cm3 range, with a detection limit of 0.691 ng/cm3. The protocol demonstrated higher accuracy in the measurement of 500 ng/cm3 trypsin solution, with a relative standard percentage error of 1.4–2.2%. This new protocol was verified against a Bland–Altman plot analysis of trypsin activity in matched samples using the Thiamine–Trypsin test, confirming its potential for application in pharmaceutical development and disease treatment. Our study demonstrated a simple, rapid, low-cost, sensitive, and selective method for assessment of the trypsin enzyme, which can be used to study the clinical importance and pharmaceutical significance of the trypsin enzyme.

Graphical abstract

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

Data availability

The authors confirm that the data supporting the findings of this study are available within the article.

References

  1. Amrithaashri S, Kavitha S, Archana Santhanam V, Gayathri R (2021) Ann Rom Soc Cell Biol 20:750

    Google Scholar 

  2. Ramírez-Larrota JS, Eckhard U (2022) Biomolecules 12:306

    PubMed Central  PubMed  Google Scholar 

  3. Kaur J, Singh PK (2020) Crit Rev Anal Chem 52:949

    PubMed  Google Scholar 

  4. Carroll EL, Bailo M, Reihill JA, Crilly A, Lockhart JC, Litherland GJ, Lundy FT, McGarvey LP, Hollywood MA, Martin SL (2021) J Mol Sci 22:5817

    CAS  Google Scholar 

  5. Guo L, Yao JH, Zheng C, Tian HB, Liu YL, Liu SM, Cai CJ, Xu XR, Cao YC (2019) Animal 13:1899

    CAS  PubMed  Google Scholar 

  6. Jia L, Yang Y, Liu X, Chen S, Zhu J (2019) Anal Methods 11:4822

    CAS  Google Scholar 

  7. Wood LD, Canto MI, Jaffee EM, Simeone DM (2022) Gastroenterology 163:386

    PubMed  Google Scholar 

  8. Ko J, Cho J, Petrov MS (2020) Diabetes Res Clin Pract 159:107974

    CAS  PubMed  Google Scholar 

  9. Elagami H, Sinha CK, Davenport M (2022) Meconium ileus and surgical features of cystic fibrosis. Handbook of pediatric surgery. Springer, Cham, p 165

    Google Scholar 

  10. Shah D, Mital K (2018) Adv Ther 35:31

    CAS  PubMed  Google Scholar 

  11. dos Santos DM, dos Santos CW, de Souza CB, de Albuquerque FS, dos Santos Oliveira JM, Pereira HJ (2020) Biocatal Agric Biotechnol 25:101584

    Google Scholar 

  12. Cai M, Ding C, Cao X, Wang F, Zhang C, Xian Y (2019) Anal Chim Acta 1056:153

    CAS  PubMed  Google Scholar 

  13. Zhang Q, Li W, Chen J, Wang F, Wang Y, Chen Y, Yu C (2013) Chem Commun 49:3137

    CAS  Google Scholar 

  14. Duan X, Li N, Wang G, Su X (2020) Talanta 219:121171

    CAS  PubMed  Google Scholar 

  15. Zhang L, Qin H, Cui W, Zhou Y, Du J (2016) Talanta 161:535

    CAS  PubMed  Google Scholar 

  16. Miao P, Liu T, Li X, Ning L, Yin J, Han K (2013) Biosens Bioelectron 49:20

    CAS  PubMed  Google Scholar 

  17. Xue W, Zhang G, Zhang D (2011) Analyst 136:3136

    CAS  PubMed  Google Scholar 

  18. Li F, Chen Y, Lin R, Miao C, Ye J, Cai Q, Huang Z, Zheng Y, Lin X, Zheng Z, Weng S (2021) Anal Chim Acta 1148:338201

    CAS  PubMed  Google Scholar 

  19. Zhao W, Li B, Xu S, Zhu Y, Liu X (2020) Anal Chim Acta 1117:25

    CAS  PubMed  Google Scholar 

  20. Ling L, Xiao C, Wang S, Guo L, Guo X (2019) Talanta 200:236

    CAS  PubMed  Google Scholar 

  21. Stoytcheva M, Zlatev R, Cosnier S, Arredondo M, Valdez B (2013) Biosens Bioelectron 41:862

    CAS  PubMed  Google Scholar 

  22. Chen G, Shi H, Ban F, Zhang Y, Sun L (2015) Microchim Acta 182:2469

    CAS  Google Scholar 

  23. Coscueta ER, Pintado ME, Picó GA, Knobel G, Boschetti CE, Malpiedi LP, Nerli BB (2017) Food Chem 214:156

    CAS  PubMed  Google Scholar 

  24. Chen S, Fu J, Zhou S, Wu X, Tang S, Zhao P, Zhang Z (2021) Microchem J 168:106449

    CAS  Google Scholar 

  25. Gu X, Yang G, Zhang G, Zhang D, Zhu D (2011) ACS Appl Mater Interfaces 3:1175

    CAS  PubMed  Google Scholar 

  26. Xu S, Zhang F, Xu L, Liu X, Ma P, Sun Y, Wang X, Song D (2018) Sens Actuators B 273:1015

    CAS  Google Scholar 

  27. Dwivedi AK, Iyer PK (2013) J Mater Chem B 1:4005

    CAS  PubMed  Google Scholar 

  28. Zhang L, Du J (2016) Biosens Bioelectron 79:347

    CAS  PubMed  Google Scholar 

  29. Bally RA, Gribnau TC (1989) J Clin Chem Clin Biochem 27:791

    CAS  PubMed  Google Scholar 

  30. Palladino P, Torrini F, Scarano S, Minunni M (2020) Anal Bioanal Chem 412:7861

    CAS  PubMed Central  PubMed  Google Scholar 

  31. Manohar M, Joseph J, Selvaraj T, Sivakumar D (2013) Int J Sci Eng Res 4:620

    Google Scholar 

  32. Ktari N, Khaled HB, Nasri R, Jellouli K, Ghorbel S, Nasri M (2012) Food Chem 130:467

    CAS  Google Scholar 

  33. Souza AA, Amaral IP, Santo AR, Carvalho LB Jr, Bezerra RS (2007) Food Chem 100:1429

    CAS  Google Scholar 

  34. El Hadj AN, Hmidet N, Bougatef A, Nasri R, Nasri M (2009) J Agric Food Chem 57:10943

    Google Scholar 

  35. Klomklao S, Benjakul S, Visessanguan W, Kishimura H, Simpson BK (2006) J Agric Food Chem 54:5617

    CAS  PubMed  Google Scholar 

  36. Dittrich BU (1992) Polar Biol 12:269

    Google Scholar 

  37. Balti R, Barkia A, Bougatef A, Ktari N, Nasri M (2009) Food Chem 113:146

    CAS  Google Scholar 

  38. Bougatef A, Souissi N, Fakhfakh N, Ellouz-Triki Y, Nasri M (2007) Food Chem 102:343

    CAS  Google Scholar 

  39. Klomklao S, Benjakul S, Visessanguan W, Simpson BK, Kishimura H (2005) Process Biochem 40:3061

    CAS  Google Scholar 

  40. Doğan NÖ (2018) Turk J Emerg Med 18:139

    PubMed Central  PubMed  Google Scholar 

  41. Committee for Medicinal Products for Human Use (2011) Guideline on bioanalytical method validation. European Medicines Agency

    Google Scholar 

  42. Dean A, Voss D, Draguljić D (2017) Response surface methodology. Design and analysis of experiments. Springer, Cham, p 565

    Google Scholar 

  43. Nunes CA, Freitas MP, Pinheiro AC, Bastos SC (2012) J Braz Chem Soc 23:2003

    CAS  Google Scholar 

  44. Gerke O (2020) Diagnostics 10:334

    PubMed Central  PubMed  Google Scholar 

  45. Parashar M, Jain A (2021) J Adv Sci Res 12:100

    CAS  Google Scholar 

  46. Shrivastava A, Gupta VB (2011) Chron Young Sci 2:21

    Google Scholar 

Download references

Acknowledgements

We thank Dr. Alaa Tariq, College of Science, University of Babylon, for his assistance in providing chemicals and facilitating chemical experiments.

Author information

Authors and Affiliations

  1. Chemistry Department, College of Science, University of Babylon, Hilla City, Babylon Governorate, 51002, Iraq

    Mahmoud Hussein Hadwan, Saba S. M. Al-Obaidy, Lamia A. Almashhedy & Mohammed A. Kadhum

  2. Department of Medical Laboratories Techniques, Al-Mustaqbal University College, Babylon, Iraq

    Hawraa Saad Al-Kawaz & Dunia Abbas Khudhair

  3. Faculty of Medicine, Golestan University of Medical Sciences, Gorgan, Iran

    Asad M. Hadwan

  4. College of Medicine, University of Babylon, Hilla City, Babylon Governorate, 51002, Iraq

    Muntadher M. Hadwan

Authors
  1. Mahmoud Hussein Hadwan
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Saba S. M. Al-Obaidy
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Hawraa Saad Al-Kawaz
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Lamia A. Almashhedy
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Mohammed A. Kadhum
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Dunia Abbas Khudhair
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Asad M. Hadwan
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Muntadher M. Hadwan
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Mahmoud Hussein Hadwan.

Ethics declarations

Ethics approval and consent to participate

Ethics Committee (University of Babylon, College of Science, Iraq), Ref. no.: 3221 Date: 3/9/2021.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Hadwan, M.H., Al-Obaidy, S.S.M., Al-Kawaz, H.S. et al. An optimized protocol to assess trypsin activity in biological samples. Monatsh Chem 154, 267–277 (2023). https://doi.org/10.1007/s00706-022-03028-1

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00706-022-03028-1

Keywords

Search

Navigation