Goux W J, Venkatasubramanian P N
Biochemistry. 1986 Jan 14;25(1):84-94. doi: 10.1021/bi00349a013.
In this study, water proton relaxation rate (PRR) enhancements have been used to characterize the binding of metal ions to native ovalbumin, ovalbumin in which phosphate has been enzymatically cleaved from one or both of the two protein phosphoserines, and a heat-stabilized form of the protein (S-ovalbumin). With Scatchard plots constructed from water PRR enhancements, it was found that native ovalbumin and S-ovalbumin had one strong binding site for Mn2+ ion (KD approximately equal to 6.0 X 10(-4) M). Alkaline phosphatase treated ovalbumin, a protein having a single phosphoserine, had one Mn2+ binding site of slightly weaker affinity (KD approximately equal to 8.3 X 10(-4) M), while acid phosphatase treated ovalbumin, a dephosphorylated protein, had two much weaker Mn2+ ion binding sites (KD approximately equal to 1.3 X 10(-3) M). Competitive binding studies on the native protein suggested that Zn2+ ion competes with Mn2+ for the single strong-affinity site (KD approximately equal to 6.1 X 10(-3) M) while Mg2+ and Ca2+ do not. In a second set of experiments, the paramagnetic contribution to the 31P spin-lattice (T1P) and spin-spin (T2P) relaxation times at three separate magnetic field strengths was measured. Correlation times tau c characterizing Mn2+-31P dipolar relaxation were estimated from the ratios of T1P/T2P at a single field and from the ratios of spin-lattice relaxation rates at three different field strengths. The correlation times so obtained, ranging from about 0.7 to 7.7 ns at the three field strengths, were used in calculating distances from the bound Mn2+ ion to the phosphoserines of native ovalbumin, S-ovalbumin, and alkaline phosphatase treated ovalbumins. It was determined that the phosphate of phosphoserine-68 was 5.95 +/- 0.26 and 6.29 +/- 0.18 A from the Mn2+ in the native and alkaline phosphatase treated protein, respectively, and 6.99 +/- 0.30 A away from the Mn2+ in S-ovalbumin. The phosphate of phosphoserine-344 was determined to be 5.31 +/- 0.20 and 5.75 +/- 0.10 A from the Mn2+ ion in native ovalbumin and S-ovalbumin, respectively. The 13C nucleus of [1-13C]galactose enzymatically transferred to the nonreducing end of the ovalbumin oligosaccharide chain was not found to be significantly relaxed by Mn2+ bound to the protein, even at 1:1 stoichiometric ratio of metal:protein. Using this, we estimate the nonreducing terminal of the ovalbumin oligosaccharide to be at least 39 A from the metal ion binding site on the protein.
在本研究中，水质子弛豫率（PRR）增强已被用于表征金属离子与天然卵清蛋白、其中一个或两个蛋白质磷酸丝氨酸上的磷酸已被酶切的卵清蛋白以及该蛋白质的热稳定形式（S-卵清蛋白）的结合情况。通过由水质子PRR增强构建的Scatchard图，发现天然卵清蛋白和S-卵清蛋白对Mn2+离子有一个强结合位点（KD约等于6.0×10(-4)M）。碱性磷酸酶处理的卵清蛋白，一种具有单个磷酸丝氨酸的蛋白质，有一个亲和力稍弱的Mn2+结合位点（KD约等于8.3×10(-4)M），而酸性磷酸酶处理的卵清蛋白，一种去磷酸化的蛋白质，有两个弱得多的Mn2+离子结合位点（KD约等于1.3×10(-3)M）。对天然蛋白质的竞争性结合研究表明，Zn2+离子与Mn2+竞争单个强亲和力位点（KD约等于6.1×10(-3)M），而Mg2+和Ca2+则不竞争。在第二组实验中，测量了在三个不同磁场强度下顺磁对31P自旋晶格（T1P）和自旋-自旋（T2P）弛豫时间的贡献。通过在单个磁场下T1P/T2P的比值以及在三个不同磁场强度下自旋晶格弛豫率的比值估算了表征Mn2+-31P偶极弛豫的相关时间τc。如此获得的相关时间在三个磁场强度下范围约为0.7至7.7纳秒，用于计算从结合的Mn2+离子到天然卵清蛋白、S-卵清蛋白和碱性磷酸酶处理的卵清蛋白的磷酸丝氨酸的距离。已确定在天然和碱性磷酸酶处理的蛋白质中，磷酸丝氨酸-68的磷酸基团与Mn2+的距离分别为5.95±0.26埃和6.29±0.18埃，在S-卵清蛋白中与Mn2+的距离为6.99±0.30埃。已确定在天然卵清蛋白和S-卵清蛋白中，磷酸丝氨酸-344的磷酸基团与Mn2+离子的距离分别为5.31±0.20埃和5.75±0.10埃。即使在金属与蛋白质的化学计量比为1:1时，未发现与蛋白质结合Mn2+显著弛豫[1-13C]半乳糖酶促转移至卵清蛋白寡糖链非还原端的13C核。据此，我们估计卵清蛋白寡糖的非还原端距蛋白质上的金属离子结合位点至少39埃。
