Facultad de Ciencias Químicas, Universidad de Colima, Carr. Colima-Coquimatlán km. 9, C.P. 28400 Coquimatlán, Colima, Mexico; Facultad de Ciencias Biológicas y Agropecuarias, Universidad de Colima, Carr. Colima-Manzanillo km. 40, C.P. 28100 Tecomán, Colima, Mexico; Consejo Nacional de Ciencia y Tecnología, Laboratorio de Agrobiotecnología, Tecnoparque CLQ, Universidad de Colima, Carretera Los Limones-Loma de Juárez, C.P. 28629 Colima, Colima, Mexico.
Facultad de Ciencias Biológicas y Agropecuarias, Universidad de Colima, Carr. Colima-Manzanillo km. 40, C.P. 28100 Tecomán, Colima, Mexico; Consejo Nacional de Ciencia y Tecnología, Laboratorio de Agrobiotecnología, Tecnoparque CLQ, Universidad de Colima, Carretera Los Limones-Loma de Juárez, C.P. 28629 Colima, Colima, Mexico; Centro Nacional de Referencia de Control Biológico, Carr. Tecomán-Estación FFCC km 1.5, Col. Tepeyac, C.P. 28110 Tecomán, Colima, Mexico.
Food Chem. 2022 Dec 1;396:133681. doi: 10.1016/j.foodchem.2022.133681. Epub 2022 Jul 15.
A microvolumetric method for surface hydrophobicity (H) determination of proteins using a Nanodrop fluorospectrometer was developed. This method reduces the protein and fluorophore quantities that are necessary for sample preparations and readings by two and three orders of magnitude, respectively, compared to conventional methods. In addition, readings can be obtained in just 2-6 s. Bovine serum albumin (BSA) and 1-anilino 8-naphthalene sulfonic acid (ANS) were used for the first optimization of appropriate fluorophore-protein conditions for H determination (20 μM ANS, 0.5-4 μM BSA, pH 5). Based on validation guidelines, the novel method shows linear behavior, good intraday precision, accuracy, and sensitivity. This method was robust against several factors, as determined by a Youden-Steiner test. Additional surface hydrophobicity determinations using several proteins demonstrate suitable method applicability. The present microvolumetric method provides a reliable technique to determine the H of proteins for pharmaceutical, biotechnological, and food applications.
一种使用 Nanodrop 荧光分光光度计测定蛋白质表面疏水性 (H) 的微量体积法已经被开发出来。与传统方法相比，该方法分别将样品制备和读取所需的蛋白质和荧光染料的量减少了两个和三个数量级。此外，读取时间仅需 2-6 秒。牛血清白蛋白 (BSA) 和 1-苯胺基-8-萘磺酸 (ANS) 被用于首次优化适合 H 测定的合适荧光染料-蛋白质条件（20 μM ANS、0.5-4 μM BSA、pH 5）。根据验证指南，新方法显示出线性行为、良好的日内精密度、准确性和灵敏度。通过 Youden-Steiner 检验，该方法对多种因素具有稳健性。使用几种蛋白质进行的其他表面疏水性测定表明该方法具有良好的适用性。本微量体积法为药物、生物技术和食品应用中蛋白质的 H 测定提供了一种可靠的技术。
