Considine Thérèse, Singh Harjinder, Patel Hasmukh A, Creamer Lawrence K
Riddet Centre, Massey University, Private Bag 11 222, Palmerston North, New Zealand.
J Agric Food Chem. 2005 Oct 5;53(20):8010-8. doi: 10.1021/jf050841v.
Bovine beta-lactoglobulin B (beta-LG) is susceptible to pressure treatment, which unfolds it, allowing thiol-catalyzed disulfide bond interchange to occur, facilitating intermolecular bonding (both noncovalent and disulfide). In the present study, beta-LG was mixed with sodium dodecyl sulfate (SDS), all-trans-retinol (retinol), or 8-anilino-1-naphthalenesulfonate (ANS) on a 1:1.1 molar basis, and aliquots were held at pressures between 50 and 800 MPa for 30 min at pH 7.2 and 20 degrees C. Polyacrylamide gel electrophoresis (PAGE) showed that beta-LG alone (control) was converted into a non-native monomer and a series of dimers, trimers, etc., at pressures beyond 100 MPa; SDS inhibited the formation of non-native species up to 200 MPa, and neither retinol nor ANS inhibited the formation of the non-native species as effectively as SDS. At pressures beyond 350 MPa, SDS ceased to have any inhibitory effect, but both ANS and retinol showed significant inhibition. The near- and far-UV CD patterns and the ANS fluorescent data were consistent with the PAGE data, but the retinol fluorescent data did not show sufficient change to interpret. The results suggested that there were three discernible structural stages. In Stage I (0.1-150 MPa), the native structure is stable; in Stage II (200-450 MPa), the native monomer is reversibly interchanging with non-native monomers and disulfide-bonded dimers; and in Stage III (>500 MPa), the free CysH in non-native monomer and dimer interacts with -S-S- bonds to produce high molecular weight aggregates of beta-LG. SDS inhibited the Stage I to Stage II transition at 200 MPa, and ANS and retinol inhibited the Stage II to Stage III transition at 600 MPa.
牛β-乳球蛋白B（β-LG）对压力处理敏感，压力会使其展开，从而使硫醇催化的二硫键交换发生，促进分子间键合（包括非共价键和二硫键）。在本研究中，β-LG与十二烷基硫酸钠（SDS）、全反式视黄醇（视黄醇）或8-苯胺基-1-萘磺酸盐（ANS）按1:1.1的摩尔比混合，在pH 7.2和20℃下，将等分试样在50至800MPa的压力下保持30分钟。聚丙烯酰胺凝胶电泳（PAGE）显示，单独的β-LG（对照）在压力超过100MPa时会转化为非天然单体以及一系列二聚体、三聚体等；SDS在高达200MPa时抑制非天然物种的形成，视黄醇和ANS都不如SDS有效地抑制非天然物种的形成。在压力超过350MPa时，SDS不再有任何抑制作用，但ANS和视黄醇都显示出显著的抑制作用。近紫外和远紫外圆二色光谱图以及ANS荧光数据与PAGE数据一致，但视黄醇荧光数据没有显示出足以进行解释的变化。结果表明存在三个可区分的结构阶段。在阶段I（0.1 - 150MPa），天然结构稳定；在阶段II（200 - 450MPa），天然单体与非天然单体和二硫键连接的二聚体可逆地交换；在阶段III（>500MPa），非天然单体和二聚体中的游离半胱氨酸巯基与-S-S-键相互作用，产生β-LG的高分子量聚集体。SDS在200MPa时抑制阶段I到阶段II的转变，ANS和视黄醇在600MPa时抑制阶段II到阶段III的转变。
