Triazoyl-phenyl linker system enhancing the aqueous solubility of a molecular probe and its efficiency in affinity labeling of a target protein for jasmonate glucoside.

In methods employing molecular probes to explore the targets of bioactive small molecules, long or rigid linker moieties are thought to be critical factors for efficient tagging of target protein. We previously reported the synthesis of a jasmonate glucoside probe with a highly rigid linker consisting of a triazoyl-phenyl (TAzP) moiety, and this probe demonstrated effective target tagging. Here we compare the TAzP probe with other rigid or flexible probes with respect to target tagging efficiency, hydrophobic parameters, aqueous solubility, and dihedral angles around the biaryl linkage by a combination of empirical and calculation methods. The rigid biaryl linkage of the TAzP probe has a skewed conformation that influences its aqueous solubility. Such features that include rigidness and good aqueous solubility resulted in highly efficient target tagging. These findings provide a promising guideline toward designing of better linkers for improving molecular probe performance.

Ethylene-vinyl alcohol copolymer dialyzer membrane reduces protein oxidation in hemodialysis patients.

BACKGROUND: Oxidative stress has been implicated in the cardiovascular complications that affect hemodialysis (HD) patients. Ethylene-vinyl alcohol copolymer (EVAL) dialyzer membrane induces less production of reactive oxygen species as compared to conventional dialyzers. We evaluated the impact of EVAL membrane on plasma protein oxidation in HD patients. METHODS: HD patients treated with cellulose triacetate (CTA) dialyzers were selected. In the first study performed in a 2-month crossover design alternating between CTA and EVAL, nonmercaptalbumin and advanced oxidation protein products levels were measured in the predialysis blood from 10 subjects. In the second study, predialysis plasma myeloperoxidase levels were measured before and after a 2-week EVAL treatment on 12 patients. RESULTS: Plasma advanced oxidation protein products levels were reduced after a 2-month EVAL treatment and increased again after CTA treatment, although the nonmercaptalbumin proportions were not affected significantly by the change in dialyzer membranes. The following study, a 2-week EVAL treatment, showed the decrease in myeloperoxidase levels immediately before HD. CONCLUSION: The frequent use of EVAL dialyzers has been shown to reduce protein oxidation, possibly through the suppression of circulating phagocytes. This novel biocompatible dialyzer is expected to protect cardiovascular mortality in HD patients.

The high performance of 3XFLAG for target purification of a bioactive metabolite: a tag combined with a highly effective linker structure.

Affinity purification using immunoprecipitation (IP) is an extremely useful method for target profiling of bioactive natural products. We examined IP purification of CMetE, which is a molecular target for potassium isolespedezate (1), a leaf-opening factor of Cassia plant. We studied IP efficiency using a panel of FLAG-connected molecular probes (2-8), including probes with varying structures and lengths of the linker moiety. The results suggest that not only the length, but the chemical nature of the linker moiety, strongly affect the IP efficiency. 3XFLAG, a tag combined with a linker moiety of charged amino acids, gave the best results and was most useful for IP purification of the molecular target.

FLAG tagging by CuAAC and nanogram-scale purification of the target protein for a bioactive metabolite involved in circadian rhythmic leaf movement in Leguminosae.

We report a stepwise FLAG-tagging strategy for the purification of target proteins for bioactive metabolites. This method realizes the microscale purification and identification of target protein from as few as 1 x 10(5) differentiated cells. Using this method, we isolated and identified MetE as a cytosolic target protein of potassium isolespedezate, a metabolite controlling plant nyctinasty.