Gonias S L, Reynolds J A, Pizzo S V
Biochim Biophys Acta. 1982 Aug 10;705(3):306-14. doi: 10.1016/0167-4838(82)90252-7.
Circular dichroism spectroscopy, sedimentation velocity and ultraviolet difference spectroscopy were used to compare alpha 2-macroglobulin, alpha 2-macroglobulin-trypsin complex and alpha 2-macroglobulin-methylamine complex. The circular dichroic spectrum of native alpha 2-macroglobulin is significantly changed in shape and magnitude following reaction with either trypsin or methylamine. The spectra of alpha 2-macroglobulin-trypsin and alpha 2-macroglobulin-methylamine are, however, indistinguishable. The ultraviolet difference spectrum between alpha 2-macroglobulin-methylamine and native alpha 2-macroglobulin displays a tyrosine blue shift consistent with the exposure of several tyrosine residues to solvent. The conformational change which occurs in alpha 2-macroglobulin during reaction with methylamine follows pseudo-first-order kinetics. T 1/2 was 10.5 min for the reaction with 200 mM methylamine at pH 8.0 and 45 min for the reaction with 50 mM methylamine, also at pH 8.0. Reaction of methylamine with alpha 2-macroglobulin results in loss of trypsin-binding activity which appears to be a direct consequence of the conformational change induced by methylamine. A sedimentation coefficient (S0(20),W) of 20.5 was determined for alpha 2-macroglobulin-methylamine compared to a value of 18.5 for unreacted alpha 2-macroglobulin. This increase in sedimentation velocity is attributed to a 10% decrease in alpha 2-macroglobulin Stokes radius. alpha 2-Macroglobulin-trypsin complex prepared by reaction of the protease at a 2-fold molar excess with the inhibitor was a S0(20),W of 20.3. Although this sedimentation coefficient does reflect compacting of the alpha 2-macroglobulin structure compared to native alpha 2-macroglobulin, it is not large enough to rule out significant protrusion of the proteases from pockets in the alpha 2-macroglobulin structure.
利用圆二色光谱、沉降速度和紫外差光谱对α2-巨球蛋白、α2-巨球蛋白-胰蛋白酶复合物和α2-巨球蛋白-甲胺复合物进行了比较。天然α2-巨球蛋白与胰蛋白酶或甲胺反应后，其圆二色光谱在形状和幅度上发生了显著变化。然而，α2-巨球蛋白-胰蛋白酶和α2-巨球蛋白-甲胺的光谱无法区分。α2-巨球蛋白-甲胺与天然α2-巨球蛋白之间的紫外差光谱显示酪氨酸蓝移，这与几个酪氨酸残基暴露于溶剂中一致。α2-巨球蛋白与甲胺反应过程中发生的构象变化遵循准一级动力学。在pH 8.0条件下，与200 mM甲胺反应的T1/2为10.5分钟，与50 mM甲胺反应（同样在pH 8.0）的T1/2为45分钟。甲胺与α2-巨球蛋白反应导致胰蛋白酶结合活性丧失，这似乎是甲胺诱导的构象变化的直接结果。测定α2-巨球蛋白-甲胺的沉降系数（S0(20),W）为20.5，而未反应的α2-巨球蛋白的值为18.5。沉降速度的增加归因于α2-巨球蛋白斯托克斯半径减小10%。通过使蛋白酶以2倍摩尔过量与抑制剂反应制备的α2-巨球蛋白-胰蛋白酶复合物的S0(20),W为20.3。尽管该沉降系数确实反映了与天然α2-巨球蛋白相比α2-巨球蛋白结构的紧凑性，但它还不足以排除蛋白酶从α2-巨球蛋白结构中的口袋中显著突出的可能性。
