Rodriguez A T, Colmenarejo G, Montero F
Departamento de Bioquímica y Biologia Molecular I, Facultad de Ciencias Químicas, Universidad Complutense de Madrid, Spain.
Arch Biochem Biophys. 1991 Oct;290(1):133-42. doi: 10.1016/0003-9861(91)90599-e.
In this paper, we study the dependence of the Tm (melting temperature) of complexes formed between double-stranded deoxypolynucleotides and pure destabilizer nonspecific ligands on Kc (intrinsic association constant), nc (apparent site size), and wc (cooperativity constant). Using the Sequence Generating Function (SGF) method, we have found a simple, analytical relationship between the Tm and these interaction parameters. The validity of this relationship depends strongly on the sigma value (sigma being the nucleation parameter of the deoxypolynucleotide). Through the equation so obtained, it is possible to evaluate Kc, nc, and wc from the melting temperature of three experimental thermal denaturation profiles at different r (ligand/deoxypolynucleotide ratio) values. However, when wc greater than 100, a degeneration in the wc and Kc values appears, and the study of the free deoxypolynucleotide region in the melting profile is necessary in order to accurately evaluate these two parameters. The method has been checked using complexes formed with poly(d(A-T].poly(d(A-T] and both bovine pancreatic ribonuclease and protein GP32 of phage T4 as experimental models. The applicability of the method here developed is discussed in relation to the nature of the ligands and the sigma and wc values.
在本文中，我们研究了双链脱氧多核苷酸与纯去稳定剂非特异性配体形成的复合物的解链温度（Tm）对固有缔合常数（Kc）、表观位点大小（nc）和协同常数（wc）的依赖性。使用序列生成函数（SGF）方法，我们发现了Tm与这些相互作用参数之间的简单解析关系。这种关系的有效性强烈依赖于σ值（σ是脱氧多核苷酸的成核参数）。通过由此得到的方程，可以根据不同配体/脱氧多核苷酸比率（r）值下三个实验热变性曲线的解链温度来评估Kc、nc和wc。然而，当wc大于100时，wc和Kc值会出现退化，为了准确评估这两个参数，有必要研究解链曲线中游离脱氧多核苷酸区域。已使用聚（d（A - T）·聚（d（A - T））与牛胰核糖核酸酶以及噬菌体T4的蛋白质GP32形成的复合物作为实验模型对该方法进行了检验。本文所开发方法的适用性根据配体的性质以及σ和wc值进行了讨论。
