Kakalis L T, Kumosinski T F, Farrell H M
U.S. Department of Agriculture, Eastern Regional Research Center, Philadelphia, PA 19118.
Biophys Chem. 1990 Oct;38(1-2):87-98. doi: 10.1016/0301-4622(90)80043-7.
High-resolution, natural abundance 13C[1H] (100.5 MHz), 31P[1H] (161.8 MHz) and 1H (400.0 MHz) NMR spectroscopy was used to identify the calcium-binding sites of bovine casein and to ascertain the dynamic state of amino acid residues within the casein submicelles (in 125 mM KCl, pD = 7.4) and micelles (in 15 mM CaCl2/80 mM KCl, pD = 7.2). The presence of numerous, well-resolved peaks in the tentatively assigned 13C-NMR spectra of submicelles (90 A radius) and micelles (500 A radius) suggests considerable segmental motion of both side chain and backbone carbons. The partly resolved 31P-NMR spectra concur with this. Upon Ca2+ addition, the phosphoserine beta CH2 resonance (65.8 ppm vs DSS) shifts upfield by 0.2 ppm and is broadened almost beyond detection; a general upfield shift (up to 0.3 ppm) is also observed for the 31P-NMR peaks. The T1 values of the alpha CH envelope for submicelles and micelles are essentially identical corresponding to a correlation time of 8 ns for isotropic rotation of the caseins. Significant changes in the 31P T1 values accompany micelle formation. Data are consistent with a loose and mobile casein structure, with phosphoserines being the predominant calcium-binding sites.
利用高分辨率、天然丰度的13C[1H]（100.5兆赫兹）、31P[1H]（161.8兆赫兹）和1H（400.0兆赫兹）核磁共振光谱来鉴定牛乳酪蛋白的钙结合位点，并确定酪蛋白亚胶粒（在125毫摩尔氯化钾中，pD = 7.4）和胶粒（在15毫摩尔氯化钙/80毫摩尔氯化钾中，pD = 7.2）内氨基酸残基的动态状态。在初步指定的亚胶粒（半径90埃）和胶粒（半径500埃）的13C - 核磁共振光谱中存在大量分辨率良好的峰，这表明侧链和主链碳都有相当大的片段运动。部分解析的31P - 核磁共振光谱也证实了这一点。加入Ca2 + 后，磷酸丝氨酸βCH2共振（相对于DSS为65.8 ppm）向上场移动0.2 ppm，并且几乎变宽到无法检测；在31P - 核磁共振峰中也观察到一般的向上场移动（高达0.3 ppm）。亚胶粒和胶粒的αCH包络的T1值基本相同，对应于酪蛋白各向同性旋转的相关时间为8纳秒。胶粒形成时31P T1值有显著变化。数据与松散且可移动的酪蛋白结构一致，磷酸丝氨酸是主要的钙结合位点。
