Grotjan H E, Schanbacher B D, Keel B A
Animal Science Department, University of Nebraska. Lincoln 68583-0908.
J Chromatogr. 1991 Jul 19;549(1-2):141-52. doi: 10.1016/s0021-9673(00)91425-0.
In a previous study [Keel et al., Biol, Reprod., 36 (1987) 1102] the ovine luteinizing hormone (oLH) in pituitary extracts was chromatofocused on pH 10.5-7 gradients after equilibration in 25 mM triethylamine-HCl, pH 11.0, by gel permeation. Under these conditions, some immunoreactive oLH flowed through the columns unrestricted and this was interpreted to represent extremely basic isoforms. However, when selected flow-through peaks were re-chromatofocused, each was contaminated with other isoforms of oLH. In order to clarify this dilemma, various methods of sample preparation and application were systematically compared. Consistent with previous observations, variable amounts of the immunoreactive oLH in pituitary extracts equilibrated in triethylamine by gel permeation, dialysis, flow dialysis or ion-retardation chromatography eluted as flow-through peaks when chromatofocused. In contrast, when the ionic components in the pituitary homogenization buffer were removed by these methods as well as ultrafiltration and the proteins were applied to the resin in the elution buffer (1:45 Pharmalyte 8-10.5-HCl, pH 7.0), none of the immunoreactive oLH in pituitary extracts eluted as a flow-through peak. Thus, it appears that oLH eluting as a flow-through peak results from incomplete binding of the hormone to the chromatofocusing resin when applied in triethylamine.
在之前的一项研究中[基尔等人，《生物繁殖》，36 (1987) 1102]，垂体提取物中的绵羊促黄体生成素（oLH）在通过凝胶渗透在25 mM三乙胺 - HCl（pH 11.0）中平衡后，在pH 10.5 - 7梯度上进行色谱聚焦。在这些条件下，一些免疫反应性oLH不受限制地流过柱子，这被解释为代表极碱性同工型。然而，当选择的流过峰重新进行色谱聚焦时，每个峰都被oLH的其他同工型污染。为了澄清这一困境，系统地比较了各种样品制备和上样方法。与之前的观察结果一致，通过凝胶渗透、透析、流动透析或离子阻滞色谱在三乙胺中平衡的垂体提取物中，不同量的免疫反应性oLH在进行色谱聚焦时以流过峰的形式洗脱。相比之下，当通过这些方法以及超滤去除垂体匀浆缓冲液中的离子成分，并且将蛋白质以洗脱缓冲液（1:45 Pharmalyte 8 - 10.5 - HCl，pH 7.0）上样到树脂上时，垂体提取物中的免疫反应性oLH均没有以流过峰的形式洗脱。因此，似乎以流过峰形式洗脱的oLH是由于在三乙胺中应用时激素与色谱聚焦树脂的不完全结合所致。
