Graduate School of Life and Environmental Sciences, Osaka Prefecture University, 1-1 Gakuen-cho, Sakai, Osaka 599-8531, Japan.
Biochem J. 2012 Sep 1;446(2):279-89. doi: 10.1042/BJ20120324.
L-PGDS [lipocalin-type PG (prostaglandin) D synthase] is a multi-functional protein, acting as a PGD₂-producing enzyme and a lipid-transporter. In the present study, we focus on the function of L-PGDS as an extracellular transporter for small lipophilic molecules. We characterize the binding mechanism of human L-PGDS for the molecules, especially binding affinity stoichiometry and driving force, using tryptophan fluorescence quenching, ICD (induced circular dichroism) and ITC (isothermal titration calorimetry). The tryptophan fluorescence quenching measurements revealed that haem metabolites such as haemin, biliverdin and bilirubin bind to L-PGDS with significantly higher affinities than the other small lipophilic ligands examined, showing dissociation constant (K(d)) values from 17.0 to 20.9 nM. We focused particularly on the extra-specificities of haem metabolites and L-PGDS. The ITC and ICD data revealed that two molecules of the haem metabolites bind to L-PGDS with high and low affinities, showing K(d) values from 2.8 to 18.1 nM and from 0.209 to 1.63 μM respectively. The thermodynamic parameters for the interactions revealed that the contributions of enthalpy and entropy change were considerably different for each haem metabolite even when the Gibbs energy change was the same. Thus we believe that the binding energy of haem metabolites to L-PGDS is optimized by balancing enthalpy and entropy change.
L-PGDS（脂联素型 PG（前列腺素）D 合酶）是一种多功能蛋白，既能作为 PGD₂产生酶，又能作为脂质转运蛋白。在本研究中，我们专注于 L-PGDS 作为小分子脂溶性分子的细胞外转运蛋白的功能。我们使用色氨酸荧光猝灭、ICD（诱导圆二色性）和 ITC（等温滴定量热法）来表征人 L-PGDS 与这些分子的结合机制，特别是结合亲和力、配体计量和驱动力。色氨酸荧光猝灭测量表明，血红素代谢物，如血红素、胆红素和胆红素，与 L-PGDS 的结合亲和力明显高于其他研究的小分子脂溶性配体，其解离常数（K(d)）值为 17.0 至 20.9 nM。我们特别关注血红素代谢物和 L-PGDS 的额外特异性。ITC 和 ICD 数据表明，两种血红素代谢物以高亲和和低亲和两种方式与 L-PGDS 结合，K(d)值分别为 2.8 至 18.1 nM 和 0.209 至 1.63 μM。相互作用的热力学参数表明，即使吉布斯自由能变化相同，每个血红素代谢物的焓变和熵变的贡献也有很大差异。因此，我们认为血红素代谢物与 L-PGDS 的结合能通过平衡焓变和熵变来优化。
