Wicar S, Mulkerrin M G, Bathory G, Khundkar L H, Karger B L
Barnett Institute, Northeastern University, Boston, Massachusetts 02115.
Anal Chem. 1994 Nov 15;66(22):3908-15. doi: 10.1021/ac00094a011.
As a continuation of a previous paper on the retention behavior of recombinant human growth hormone (rhGH) in reversed phase chromatography at pH 6.5 (Oroszlan, P., et al. Anal. Chem. 1992, 64, 1623-1631) the effect of 1-propanol (1-PrOH) and acetonitrile on the conformation of rhGH at this pH has been investigated by circular dichroism (CD), second-derivative UV spectroscopy, fluorescence anisotropy, fluorescence quenching, and fluorescence lifetime measurements. Addition of 1-PrOH up to a concentration of 10% (v/v) does not cause any significant changes in protein structure. However, above this concentration, a transition from the native to a new state is observed; the transition is completed above 30% (v/v) of 1-PrOH, the composition for completion being dependent on temperature. This change in structure correlates with retention changes observed in reversed phase chromatography. The new rhGH conformation retains much of the alpha-helicity and possesses a slightly expanded hydrodynamic radius relative to native rhGH. Second-derivative UV spectroscopy suggests that the hydrogen bond between Trp 86 and Asp 169, spanning two alpha-helices, remains intact. On the other hand, the near-UV CD intensity changes from positive to negative in the Trp region of the spectrum, signaling an alteration in the Trp environment. In addition, fluorescence quenching measurements with trichloroethanol reveal greater accessibility to solvent of the Trp residue after the conformational transition has occurred. From the results, it is concluded that a molten globule state (compact state retaining much of the secondary structure of the native state but with a disrupted tertiary structure) is produced with the addition of > 30% (v/v) 1-PrOH.(ABSTRACT TRUNCATED AT 250 WORDS)
作为之前一篇关于重组人生长激素（rhGH）在pH 6.5的反相色谱中保留行为的论文（奥罗斯兰，P.等人，《分析化学》，1992年，64卷，1623 - 1631页）的延续，通过圆二色性（CD）、二阶导数紫外光谱、荧光各向异性、荧光猝灭和荧光寿命测量，研究了1 - 丙醇（1 - PrOH）和乙腈在此pH下对rhGH构象产生的影响。添加浓度高达10%（v/v） 的1 - PrOH不会引起蛋白质结构的任何显著变化。然而，高于此浓度时，会观察到从天然状态到新状态的转变；在1 - PrOH浓度高于30%（v/v）时转变完成，完成转变所需的组成取决于温度。这种结构变化与反相色谱中观察到的保留变化相关。新的rhGH构象保留了大部分α - 螺旋结构，并且相对于天然rhGH，其流体动力学半径略有增大。二阶导数紫外光谱表明，跨越两个α - 螺旋的色氨酸86和天冬氨酸169之间的氢键保持完整。另一方面，在光谱的色氨酸区域，近紫外CD强度从正变为负，这表明色氨酸环境发生了改变。此外，用三氯乙醇进行的荧光猝灭测量表明，构象转变发生后，色氨酸残基对溶剂的可及性更高。从结果可以得出结论，添加> 30%（v/v）的1 - PrOH会产生一种熔球态（一种紧密状态，保留了天然状态的大部分二级结构，但三级结构被破坏）。 （摘要截取自250字）
