Jennissen H P
Biochemistry. 1976 Dec 28;15(26):5683-92. doi: 10.1021/bi00671a001.
The concept of cooperativity appears to be the key to the understanding of the complex mechanisms underlying the adsorption of proteins to agaroses substituted with hydrophobic alpha-aminoalkanes. The adsorption of phosphorylase b occurs through the positive cooperative interaction of a critical number of approximately 3-5 butyl and a higher number of methyl residues with ocrresponding sites on the enzyme. The amount of adsorbed phosphorylase b per millileter of packed gel (methyl-, butyl-Sepharose) in the absence and presence of 1.1 M ammonium sulfate at temperatures between 0 and 34 degrees C is a power function of the free solute equilibrium concentration (Freundlich isotherm). In contrast, the adsorption of cyanmyoglobin to phosphocellulose is described by the Langmuir equation. The surface coverage dependent isosteric heats of adsorption for phosphorylase b indicate an endothermic reaction only on the butyl-Sepharose in the presence of high salt concentrations. Scatchard plots of the Freundlich isotherms of phosphorylase b are concave upwards, typical of negative cooperativity. Hill plots of these isotherms (5-70% saturation) yield coefficients between nH = 0.39 and 0.71. At high surface coverages, the Hill coefficients approach unity. Apparent association constants (K0.5) of 4-39 X 10(4) M-1 are calculated for the adsorption of phosphorylase b, as compared to 2-9 X 10(4) M-1 for the adsorption of cyanmyoblobin. In general, negative cooperativity of binding may be explained by changes in the affinity of the ligand for the matrix, due to the sequential multivalent adsorption, and competition of phosphorylase b molecules for the critical number of alkyl residues (nonindependence of binding) on one side and to variations in the configuration of binding and entropy on the other.
协同性的概念似乎是理解蛋白质吸附到用疏水α-氨基烷烃取代的琼脂糖上的复杂机制的关键。磷酸化酶b的吸附是通过大约3 - 5个关键数量的丁基和更多数量的甲基残基与酶上相应位点的正协同相互作用发生的。在0至34摄氏度之间，在不存在和存在1.1 M硫酸铵的情况下，每毫升填充凝胶（甲基-、丁基-琼脂糖）吸附的磷酸化酶b的量是游离溶质平衡浓度的幂函数（弗伦德利希等温线）。相比之下，氰化肌红蛋白在磷酸纤维素上的吸附可用朗缪尔方程描述。磷酸化酶b的表面覆盖依赖性等量吸附热表明，仅在高盐浓度存在下，丁基-琼脂糖上的反应是吸热的。磷酸化酶b的弗伦德利希等温线的斯卡查德图向上凹，这是负协同性的典型特征。这些等温线（饱和度为5 - 70%）的希尔图得出的系数nH在0.39至0.71之间。在高表面覆盖率下，希尔系数接近1。计算出磷酸化酶b吸附的表观缔合常数（K0.5）为4 - 39×10⁴ M⁻¹，相比之下，氰化肌红蛋白吸附的表观缔合常数为2 - 9×10⁴ M⁻¹。一般来说，结合的负协同性可以通过以下原因来解释：一方面，由于顺序多价吸附，配体与基质的亲和力发生变化，以及磷酸化酶b分子对关键数量的烷基残基的竞争（结合的非独立性）；另一方面，结合构型和熵的变化。
