title: Azocasein Substrate for Determination of Proteolytic Activity: Reexamining a Traditional Method Using Bromelain Samples.
abstract: 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.
authors.affiliation: Chemical Engineering School, Campinas State University (UNICAMP), Avenida Albert Einstein 500, 13083-852 Campinas, SP, Brazil.
authors.forename: Diego F
authors.identifier: 0000-0002-2123-5891
authors.initials: DF
authors.lastname: Coêlho
authors.affiliation: Chemical Engineering School, Campinas State University (UNICAMP), Avenida Albert Einstein 500, 13083-852 Campinas, SP, Brazil.
authors.forename: Thais Peron
authors.identifier: 0000-0002-8994-1776
authors.initials: TP
authors.lastname: Saturnino
authors.affiliation: Chemical Engineering School, Campinas State University (UNICAMP), Avenida Albert Einstein 500, 13083-852 Campinas, SP, Brazil.
authors.forename: Fernanda Freitas
authors.identifier: 0000-0003-0463-787X
authors.initials: FF
authors.lastname: Fernandes
authors.affiliation: Faculty of Pharmaceutical Sciences, Campinas State University (UNICAMP), Rua Sérgio Buarque de Holanda 250, 13083-859 Campinas, SP, Brazil.
authors.forename: Priscila Gava
authors.initials: PG
authors.lastname: Mazzola
authors.affiliation: Biochemistry and Genetics Institute, Federal University of Uberlândia (UFU), Avenida Getúlio Vargas, 230 Centro, 38700-128 Patos de Minas, MG, Brazil.
authors.forename: Edgar
authors.identifier: 0000-0001-7249-639X
authors.initials: E
authors.lastname: Silveira
authors.affiliation: Chemical Engineering School, Campinas State University (UNICAMP), Avenida Albert Einstein 500, 13083-852 Campinas, SP, Brazil.
authors.forename: Elias Basile
authors.identifier: 0000-0003-3275-0810
authors.initials: EB
authors.lastname: Tambourgi
mesh_terms: D001963:Bromelains / Q000737:chemistry* / Q000494:pharmacology; D002364:Caseins / Q000737:chemistry* / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D057230:Limit of Detection; D059748:Proteolysis / Q000187:drug effects*; D013379:Substrate Specificity
chemical_list: D002364:Caseins; C026018:azocasein; D001963:Bromelains