Ikeuchi Y, Nakagawa K, Endo T, Suzuki A, Hayashi T, Ito T
Laboratory of Chemistry and Technology of Animal Products, Department of Bioscience and Biotechnology, Graduate School of Agriculture, Kyushu University, Fukuoka 812-8581, Japan.
J Agric Food Chem. 2001 Aug;49(8):4052-9. doi: 10.1021/jf001364d.
This study was conducted to assess the effect of high hydrostatic pressure on monomer beta-lactoglobulin (BLg) at acid pH by fluorescence spectroscopy under pressure and by circular dichroism (CD) and (1)H NMR spectroscopies after release of pressure. The intrinsic (tryptophan) fluorescence measurement and the study of 8-anilinonaphthalene-1-sulfonate (ANS) binding to BLg indicated that at pH 2.0 the recovery of center of spectral mass or ANS fluorescence was almost complete upon pressure release. No difference in (1)H NMR spectra was observed between pressurized and unpressurized BLg. In addition, NMR detection of the H/D exchange of aromatic protein indicated that the conformation of the vicinity of tryptophan residues could be refolded almost completely after release of pressure. These results seemingly confirm that the pressure-induced denaturation of BLg at pH 2.0 is reversible. However, cis-parinaric acid binding ability of pressurized BLg was largely lost, although its retinol binding ability was the same as its unpressurized one. Furthermore, CD spectra of the far-UV region and 2D NMR spectra evidently revealed the difference in the conformation of the molecule between unpressurized and pressurized BLg. These results are interpreted as an existence of partially fragile structure in the BLg molecule by high pressure.
本研究旨在通过压力下的荧光光谱法以及压力释放后的圆二色性（CD）和核磁共振（¹H NMR）光谱法，评估高静水压对酸性pH条件下单体β-乳球蛋白（BLg）的影响。色氨酸的固有荧光测量以及8-苯胺基萘-1-磺酸盐（ANS）与BLg结合的研究表明，在pH 2.0时，压力释放后光谱质量中心或ANS荧光几乎完全恢复。加压和未加压的BLg之间未观察到¹H NMR光谱的差异。此外，对芳香族蛋白质的H/D交换进行的NMR检测表明，压力释放后色氨酸残基附近的构象几乎可以完全重新折叠。这些结果似乎证实了pH 2.0时压力诱导的BLg变性是可逆的。然而，加压后的BLg与顺式-十八碳四烯酸的结合能力大大丧失，尽管其与视黄醇的结合能力与未加压时相同。此外，远紫外区域的CD光谱和二维NMR光谱明显揭示了未加压和加压的BLg分子构象的差异。这些结果被解释为高压作用下BLg分子中存在部分易损结构。
