Protein binding heavily modulates drug activity. Therefore, the binding features need to be elucidated when chemistry researchers study new molecules (metal complexes) to be used as drugs. This paper concerns the experimental and data treatment aspects of the mechanistic analysis of the binding to a fluorescent protein (the golden standard serum albumin) by using direct fluorescence titrations. Fluorescence data are not rarely only qualitatively used, neglecting further treatments which could offer a precious detailed picture of the behavior of the drug. We aim to spread a mechanistic approach, discussing the critical aspects for correctly designing the experiments and treating the data. The researcher may confirm adduct formation and evaluate binding constants (Stern-Volmer K_SV or other types of K). Also, we discuss here, with the help of literature examples, the correct use of temperature dependence of K to extract thermodynamic parameters, comment on enthalpy-entropy compensation, together with the use of synchronous spectra and exchange experiment to gain information on the binding type and site. We think that this tutorial/critical synopsis can be of help for the increasing community dealing with these experiments, which are valuable but often much more tricky than it might appear at first sight.

蛋白质结合严重调节药物活性。因此，当化学研究人员研究用作药物的新分子（金属配合物）时，需要阐明结合特征。本文涉及通过使用直接荧光滴定对荧光蛋白（黄金标准血清白蛋白）结合进行机械分析的实验和数据处理方面。荧光数据不仅很少被定性地使用，还忽略了可以提供药物行为的宝贵详细图片的进一步治疗。我们旨在传播一种机械方法，讨论正确设计实验和处理数据的关键方面。研究人员可以确认加合物的形成并评估结合常数 (Stern-Volmer K _SV或其他类型的 K）。此外，我们在这里借助文献实例讨论正确使用 K 的温度依赖性来提取热力学参数，评论焓熵补偿，以及使用同步光谱和交换实验来获取有关结合类型的信息和网站。我们认为本教程/关键概要可以帮助越来越多的社区处理这些实验，这些实验很有价值，但通常比乍一看要复杂得多。