MAGNETIC RESONANCE STUDIES OF INTERACTION OF MANGANOUS ION WITH BOVINE SERUM ALBUMIN
MAGNETIC RESONANCE STUDIES OF INTERACTION OF MANGANOUS ION WITH BOVINE SERUM ALBUMIN
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DOI：
10.1021/bi00905a003
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发表时间：
1963-01-01
期刊：
影响因子：
2.9
通讯作者：
COHN, M
中科院分区：
文献类型：
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作者：
MILDVAN, AS;COHN, M
用脉冲核磁共振测量了结合态锰对水质子纵向核磁弛豫速率（1/T1）的影响。该参数的值用增强系数[epsilon]b表示，即相同浓度下束缚态Mn与自由态Mn的质子弛豫率之比。观察到的增强值用于确定Mn2+与牛血清白蛋白的结合位点数量和结合常数，并将[epsilon]b的变化与蛋白结构变化联系起来。AtpH7.5, [mu] = 0.2, T = 24[度]，牛血清白蛋白与一个Mn2+紧密结合，Ka = 2.7[正负]0.6 × 10m4 -1, [epsilon] b = 11.5[正负]0.8，大约5个锰离子弱，Ka = 3.3[正负]0.6 × 10m3 -1, [epsilon] b = 6.5[正负]0.3。电子自旋共振法测定的游离Mn2+的结合常数和结合位点数与增强法测定的结合Mn2+的结合常数和结合位点数一致。对于第一个结合位点，Ka随pH单调增加，表明配体的pK值为6.1、6.8和8.5，但[epsilon] b的变化范围从pH 6以下的2到pH 7.5以上的11，表明配体的pK = 7.0。用对汞苯甲酸盐滴定牛血清白蛋白的一个巯基对Ka没有影响，但对[epsilon]b有轻微的增加。尿素（[大于或等于]2M）、氯化胍（[大于或等于]2M）和癸基硫酸盐（0.1 M）均能降低Ka和[epsilon]b。当变性剂的浓度在0.3 M到6 M之间变化时，胍和尿素在不同阶段对[epsilon] b的改变是不同的
A new physical parameter of bound manganese, its effect on the longitudinal nuclear magnetic relaxation rate of water protons (1/T1), has been measured by pulsed nuclear magnetic resonance. The values of this parameter are expressed in terms of enhancement [epsilon]b, the ratio of the proton relaxation rate of bound Mn to that of free Mn at the same concentration. The observed enhancement values were used to determine the number of binding sites and the binding constants of Mn2+ to bovine serum albumin and to correlate changes in [epsilon]b with structural changes induced in the protein. AtpH7.5, [mu] = 0.2, T = 24[degree], bovine serum albumin binds one Mn2+ tightly, Ka = 2.7 [plus or minus] 0.6 X 104M-1, with [epsilon] b = 11.5 [plus or minus] 0.8, and approximately five manganous ions weakly, Ka = 3.3 [plus or minus] 0.6 X 103 M-1, with [epsilon] b = 6.5 [plus or minus] 0.3. Binding constants and numbers of binding sites determined from measurements of free Mn2+ from electron spin resonance agreed well with those of bound Mn2+ determined from enhancement. For the first binding site, Ka increased monotonically with pH, suggesting ligands with pK values of 6.1, 6.8, and 8.5, but [epsilon] b varied from 2 below pH 6 to 11 above pH 7.5, implicating a group with pK = 7.0. Titration of the one sulfhydryl group of bovine serum albumin with p-mercuribenzoate had no effect on Ka but slightly increased [epsilon]b. Urea ([greater than or equ]2M), guanidinium chloride ([greater than or equ]2M), and decyl sulfate (0.1 M) decreased both Ka and [epsilon]b. Guanidinium and urea each alter [epsilon] b differently and in several stages as the concentration of denaturant is varied between 0.3 M and 6 M. Guanidinium