Ivanov Iu D, Bukharina N S, Pleshakova T O, Frantsuzov P A, Krokhin N V, Ziborov V S, Archakov A I
Biofizika. 2011 Sep-Oct;56(5):939-44.
An approach to measure the activity of single oligomers of the heme-containing enzyme the cytochrome P450 CYP102A1 (CYP102A1) by atomic force microscopy (AFM) has been developed. It was found that the amplitude of fluctuations of the height of single CYP102A1 molecules performing the catalytic cycle is twice as great as the amplitude of fluctuations of the height of the same enzymes in the inactive state. It was shown that the amplitude of height fluctuations of a CYP102A1 protein globule depends on temperature, the maximum of this dependence being observed at 22 degrees C. The activity of a single CYP102A1 molecule in the unit amplitude of height fluctuations of a protein globule reduced to the unit time was 5+/-2 Ac. The elasticity of a single protein molecule was measured from the deformation of this molecule by the action of an AFM probe. The use of AFM probes of different geometry made i t possible t o determine t he integral andlocal Young's modulus for the monomers of the protein putidaredoxin reductase from the cytochrome P450 CYP101 (P450cam)-containing monooxigenase system, which were 37+/-117 and 1+/-3 MPa, respectively.
已开发出一种通过原子力显微镜（AFM）测量含血红素酶细胞色素P450 CYP102A1（CYP102A1）单个寡聚体活性的方法。研究发现，进行催化循环的单个CYP102A1分子高度波动的幅度是处于非活性状态的相同酶高度波动幅度的两倍。结果表明，CYP102A1蛋白质球状体高度波动的幅度取决于温度，在22摄氏度时观察到这种依赖性的最大值。在蛋白质球状体高度波动单位幅度下，单个CYP102A1分子在单位时间内的活性为5±2 Ac。通过AFM探针作用使单个蛋白质分子变形来测量其弹性。使用不同几何形状的AFM探针能够确定来自含细胞色素P450 CYP101（P450cam）的单加氧酶系统的蛋白质铁氧还蛋白还原酶单体的积分杨氏模量和局部杨氏模量，分别为37±117和1±3 MPa。
