Gawinowicz M A, Goodman D S
Biochemistry. 1982 Apr 13;21(8):1899-905. doi: 10.1021/bi00537a030.
Three radioactive retinoid bronoacetates were synthesized as potential retinoid affinity labels for the retinol binding site of human plasma retinol-binding protein (RBP). The compounds synthesized were beta-[9-3H]ionyl bromoacetate (IBA), beta-[11-3H]ionylideneethyl bromoacetate (IEBA), and [15-3H]retinyl bromoacetate (RBA). When excess ligand was incubated with RBP for 5 h at 37 degrees C, IBA and IEBA formed nearly 1:1 molar complexes with RBP, whereas RBA bound only approximately one-third as well. Subsequent addition of retinol to the retinoid-RBP complex resulted in complete displacement of IBA and RBA from the protein, whereas a large proportion (37%) of the [3H]IEBA remained bound to the retinol binding site of RBP. For maximization of covalent bonding of IEBA to RBP, IEBA was incubated with RBP for varying lengths of time, followed, in each instance, by addition of retinol to displace noncovalently bound IEBA. The amount of IEBA remaining bound to RBP increased with increasing incubation time, reaching a maximum of about 0.66 mol/mol of RBP at 18 h. Moreover, at each time point, the binding of retinol to RBP was inhibited to an extent that was equivalent to the amount of [3H]IEBA that was not displaced from RBP by retinol. Only a very small proportion of the bound [3H]IEBA that was not displaced with retinol could be extracted from the protein with chloroform-methanol. Taken together, these several lines of evidence strongly suggest that the IEBA was bound in the retinol binding site of RBP and was attached to the protein in a covalent manner. Thus, IEBA appears to be an effective affinity label for the retinol binding site of RBP.
合成了三种放射性视黄酸溴乙酸酯，作为人血浆视黄醇结合蛋白（RBP）视黄醇结合位点潜在的视黄酸亲和标记物。合成的化合物为β-[9-³H]紫罗酮溴乙酸酯（IBA）、β-[11-³H]亚乙基紫罗酮溴乙酸酯（IEBA）和[15-³H]视黄醇溴乙酸酯（RBA）。当过量配体与RBP在37℃孵育5小时时，IBA和IEBA与RBP形成近1:1摩尔比的复合物，而RBA的结合量仅约为前者的三分之一。随后向视黄酸-RBP复合物中加入视黄醇，导致IBA和RBA从蛋白质上完全被取代，而很大一部分（37%）的[³H]IEBA仍与RBP的视黄醇结合位点结合。为了使IEBA与RBP的共价结合最大化，将IEBA与RBP孵育不同时长，然后在每种情况下加入视黄醇以取代非共价结合的IEBA。与RBP结合的IEBA量随孵育时间增加而增加，在18小时时达到约0.66摩尔/摩尔RBP的最大值。此外，在每个时间点，视黄醇与RBP的结合受到的抑制程度与未被视黄醇从RBP上取代的[³H]IEBA量相当。只有极少量未被视黄醇取代的结合[³H]IEBA能用氯仿-甲醇从蛋白质中提取出来。综合这些多方面的证据有力地表明，IEBA结合在RBP的视黄醇结合位点，并以共价方式与蛋白质相连。因此，IEBA似乎是RBP视黄醇结合位点的有效亲和标记物。
