Coêlho Diego F, Saturnino Thais Peron, Fernandes Fernanda Freitas, Mazzola Priscila Gava, Silveira Edgar, Tambourgi Elias Basile
Chemical Engineering School, Campinas State University (UNICAMP), Avenida Albert Einstein 500, 13083-852 Campinas, SP, Brazil.
Faculty of Pharmaceutical Sciences, Campinas State University (UNICAMP), Rua Sérgio Buarque de Holanda 250, 13083-859 Campinas, SP, Brazil.
Biomed Res Int. 2016;2016:8409183. doi: 10.1155/2016/8409183. Epub 2016 Jan 27.
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.
鉴于蛋白酶在全球市场的重要性，在选择可靠的生物活性测定方法时，确定最佳条件并制定标准方案至关重要。本文运用质量控制理论对1947年Charney和Tomarelli提出的一种方法的改进版本进行验证。所得结果表明，使用偶氮酪蛋白底物时，菠萝蛋白酶在45°C和pH 9（100 mM甘氨酸 - 氢氧化钠）条件下具有最佳活性。我们还对上述最佳条件下的检测限（LoD）和定量限（LoQ）进行了量化（分别为0.072和0.494 mg·mL⁻¹偶氮酪蛋白），并绘制了将光密度与消化底物量相关联的校准曲线。在所有分析样品中，我们观察到储存后响应显著下降（约17%），这表明该试剂必须在制备后立即使用。因此，鉴于文献中通常使用主观定义，而本文提出的方案采用这种简单的数学方法，使其清晰简洁，有了显著改进。
