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BioMed Research International
Volume 2016, Article ID 8409183, 6 pages
Research Article

Azocasein Substrate for Determination of Proteolytic Activity: Reexamining a Traditional Method Using Bromelain Samples

1Chemical Engineering School, Campinas State University (UNICAMP), Avenida Albert Einstein 500, 13083-852 Campinas, SP, Brazil
2Faculty of Pharmaceutical Sciences, Campinas State University (UNICAMP), Rua Sérgio Buarque de Holanda 250, 13083-859 Campinas, SP, Brazil
3Biochemistry and Genetics Institute, Federal University of Uberlândia (UFU), Avenida Getúlio Vargas, 230 Centro, 38700-128 Patos de Minas, MG, Brazil

Received 26 November 2015; Accepted 12 January 2016

Academic Editor: Pengjun Shi

Copyright © 2016 Diego F. Coêlho et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


Given the importance of protease’s worldwide market, the determination of optimum conditions and the development of a standard protocol are critical during selection of a reliable method to determine its bioactivity. This paper uses quality control theory to validate a modified version of a method proposed by Charney and Tomarelli in 1947. The results obtained showed that using azocasein substrate bromelain had its optimum at 45°C and pH 9 (Glycine-NaOH 100 mM). We also quantified the limit of detection (LoD) and limit of quantification (LoQ) in the above-mentioned optimum (0.072 and 0.494 mg·mL−1 of azocasein, resp.) and a calibration curve that correlates optical density with the amount of substrate digested. In all analysed samples, we observed a significant decrease in response after storage (around 17%), which suggests its use must be immediately after preparation. Thus, the protocol presented in this paper offers a significant improvement, given that subjective definitions are commonly used in the literature and this simple mathematical approach makes it clear and concise.