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

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

Diego F Coêlho, Thais Peron Saturnino, Fernanda Freitas Fernandes, Priscila Gava Mazzola, Edgar Silveira, Elias Basile Tambourgi

Biomed Res Int. 2016;2016:8409183.

PMID: 26925415

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.

Chemicals Related in the Paper:

Catalog Number	Product Name	Structure	CAS Number	Price
AP102110747	Azocasein		102110-74-7	Price

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