Department of Chemistry and Massey Cancer Center , Virginia Commonwealth University (VCU) , Richmond , Virginia 23284 , United States.
Reichert Technologies , Depew , New York 14043 , United States.
Inorg Chem. 2018 Mar 19;57(6):3116-3125. doi: 10.1021/acs.inorgchem.7b03043. Epub 2018 Feb 23.
We present spectroscopic and biophysical approaches to examine the affinity of metal-ammine coordination complexes for heparin as a model for heparan sulfate (HS). Similar to nucleic acids, the highly anionic nature of heparin means it is associated in vivo with physiologically relevant cations, and this work extends their bioinorganic chemistry to substitution-inert metal-ammine compounds (M). Both indirect and direct assays were developed. M compounds are competitive inhibitors of methylene blue (MB)-heparin binding, and the change in the absorbance of the dye in the presence or absence of heparin can be used as an indirect reporter of M-heparin affinity. A second indirect assay uses the change in fluorescence of TAMRA-R, a nonaarginine linked to a fluorescent TAMRA moiety, as a reporter for M-heparin binding. Direct assays are surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC). The K values for TriplatinNC-heparin varied to some extent depending on the technique from 33.1 ± 2 nM (ITC) to 66.4 ± 1.3 nM (MB absorbance assay) and 340 ± 30 nM (SPR). The differences are explained by the nature of the technique and the use of heparin of differing molecular weight. Indirect probes using the displacement of ethidium bromide from DNA or, separately, fluorescently labeled oligonucleotide (DNA-Fl) can measure the relative affinities of heparin and DNA for M compounds. These assays showed essentially equivalent affinity of TriplatinNC for heparin and DNA. The generality of these methods was confirmed with a series of mononuclear cobalt, ruthenium, and platinum compounds with significantly lower affinity because of their smaller overall positive charge but in the order [Co(NH)] > [Ru(NH)] > [Pt(NH)]. The results on heparin can be extrapolated to glycosoaminoglycans such as HS, emphasizing the relevance of glycan interactions in understanding the biological properties of coordination compounds and the utility of the metalloglycomics concept for extending bioinorganic chemistry to this class of important biomolecules.
我们提出了光谱和生物物理方法来研究金属氨配合物与肝素的亲和力，肝素是硫酸乙酰肝素（HS）的模型。与核酸类似，肝素的高度阴离子性质意味着它在体内与生理相关的阳离子结合，这项工作将它们的生物无机化学扩展到取代惰性金属氨化合物（M）。我们开发了间接和直接两种测定方法。M 化合物是亚甲蓝（MB）-肝素结合的竞争性抑制剂，在存在或不存在肝素的情况下，染料的吸光度变化可作为 M-肝素亲和力的间接报告。第二种间接测定方法使用与荧光 TAMRA 部分连接的非精氨酸九肽 TAMRA-R 的荧光变化作为 M-肝素结合的报告。直接测定方法是表面等离子体共振（SPR）和等温热力学滴定（ITC）。根据技术的不同，TriplatinNC-肝素的 K 值在一定程度上有所不同，从 33.1 ± 2 nM（ITC）到 66.4 ± 1.3 nM（MB 吸光度测定法）和 340 ± 30 nM（SPR）。差异可通过技术的性质以及使用不同分子量的肝素来解释。使用溴化乙锭从 DNA 中置换或单独使用荧光标记的寡核苷酸（DNA-Fl）的间接探针可测量肝素和 DNA 对 M 化合物的相对亲和力。这些测定表明，TriplatinNC 对肝素和 DNA 的亲和力基本相同。一系列单核钴、钌和铂化合物的结果证实了这些方法的通用性，由于其总正电荷较小，这些化合物的亲和力明显较低，但顺序为 [Co(NH)] > [Ru(NH)] > [Pt(NH)]。肝素的结果可以外推到糖胺聚糖，如 HS，强调糖基相互作用在理解配合物的生物学性质以及将金属糖化学概念扩展到这一类重要生物分子的生物无机化学中的重要性。
