Hatton M W, Regoeczi E
Biochim Biophys Acta. 1975 Feb 27;379(2):504-11. doi: 10.1016/0005-2795(75)90157-9.
The features of the structure of lysine, linked to Sepharose by the alpha-amino group, which are important for affinity chromatography of rabbit plasminogem were studied. Nine lysine and lysine-like conjugates, including epsilon-aminohexanoic acid DL-norleucine, DL-alpha-aminoadipic acid, DL-alpha-epsilon-diaminopimelic acid, cadaverine L-ornithine, L-arginine and D-lysine, were prepared; Using labelled rabbit plasminogen added to plasma, the ability of each conjugate to absorb plasminogen and separate the allomeric forms, type I and type II, during epsilon-aminohexanoic acid gradient elution was compared to Sepharose-L-lysine. Plasminogen had no affinity for Sepharose-epsilon-aminohexanoic acid, and was only weakly attracted by Sepharose-norleucine, Sepharose-cadaverine and others. Sepharose-ornithine held a greater attraction to the protein but the strongest binding was obtained with Sepharose-arginine. The affinity of plasminogen type I was always less than type II for the Sepharose-lysine analogues and the recovery of type II greater than type I from Sepharose-ornithine and Sepharose-arginine. Plasminogen affinity was in the order of Sepharose-arginine greater than Sepharose-lysine greater than Sepharose-ornithine. However, because of the present difficulty in recovering plasminogen from Sepharose-arginine the use of Sepharose-lysine in the affinity chromatography of rabbit plasminogen remains unchallenged. It is concluded that binding of rabbit plasminogen to conjugates of lysine and its analogues is determined by the presence of both a free carboxyl and a free amino group and that the distance between these groups is critical.
研究了通过α-氨基与琼脂糖相连的赖氨酸的结构特征，这些特征对兔纤溶酶原的亲和色谱法很重要。制备了九种赖氨酸和类赖氨酸结合物，包括ε-氨基己酸、DL-正亮氨酸、DL-α-氨基己二酸、DL-α,ε-二氨基庚二酸、尸胺、L-鸟氨酸、L-精氨酸和D-赖氨酸；将标记的兔纤溶酶原添加到血浆中，比较了每种结合物在ε-氨基己酸梯度洗脱过程中吸附纤溶酶原并分离I型和II型同种异型的能力与琼脂糖-L-赖氨酸的能力。纤溶酶原对琼脂糖-ε-氨基己酸没有亲和力，仅被琼脂糖-正亮氨酸、琼脂糖-尸胺等微弱吸引。琼脂糖-鸟氨酸对该蛋白质有更大的吸引力，但与琼脂糖-精氨酸的结合最强。对于琼脂糖-赖氨酸类似物，I型纤溶酶原的亲和力总是小于II型，并且从琼脂糖-鸟氨酸和琼脂糖-精氨酸中回收的II型大于I型。纤溶酶原的亲和力顺序为琼脂糖-精氨酸＞琼脂糖-赖氨酸＞琼脂糖-鸟氨酸。然而，由于目前从琼脂糖-精氨酸中回收纤溶酶原存在困难，在兔纤溶酶原的亲和色谱中使用琼脂糖-赖氨酸仍然无可替代。得出的结论是，兔纤溶酶原与赖氨酸及其类似物结合物的结合取决于游离羧基和游离氨基的同时存在，并且这些基团之间的距离至关重要。
