Epimer separation is crucial in the field of analytical chemistry, separation science, and the pharmaceutical industry. No reported methods could separate simultaneously epimers or even isomers and remove other unwanted, co-existing, interfering substances from complex systems like herbal extracts. Herein, we prepared a heptapeptide-modified stationary phase for the separation of 1R,2S -(-)-ephedrine [(-)-Ephe] and 1S,2S -(+)-pseudoephedrine [(+)-Pse] epimers from Ephedra sinica Stapf extract and blood samples. The heptapeptide stationary phase was comprehensively characterized by scanning electron microscopy, X-ray photoelectron spectroscopy, and Fourier transform infrared spectroscopy. The separation efficiency of the heptapeptide column was compared with an affinity column packed with full-length β 2 -AR functionalized silica gel (β 2 -AR column). The binding affinity of the heptapeptide with (+)-Pse was 3-fold greater than that with (-)-Ephe. Their binding mechanisms were extensively characterized by chromatographic analysis, ultraviolet spectra, circular dichroism analysis, isothermal titration calorimetry, and molecule docking. An enhanced hydrogen bonding was clearly observed in the heptapeptide-(+)-Pse complex. Such results demonstrated that the heptapeptide can recognize (+)-Pse and (-)-Ephe epimers in a complex system. This work, we believe, was the first report to simultaneously separate epimers and remove non-specific interfering substances from complex samples. The method was potentially applicable to more challenging sample separation, such as chiral separation from complex systems.
Microcin C7 (McC), widely distributed in enterobacteria, is a promising antibiotic against antibiotic resistance. Previous studies have demonstrated that the heptapeptide of McC is only responsible for recognizing the inner membrane transporterYejABEFto deliver McC into microbial cells, but lacks the capacity for inhibiting microbial cell growth. In this study, the effect of the heptapeptide (MR) and two analogues,N-formylated heptapeptide (f-MR) andN-aceylated heptapeptide (a-MR), on microbial cell growth were examined. It is surprising to find that MR not only inhibits the activity of intracellular β-galactosidase, respiratory chain dehydrogenases, and 6-phosphogluconate dehydrogenases (6PGDH), but it is also able to inhibitEscherichia coligrowth, and eventually leads to cell death at the lethal concentration of 5.34 mM within 10 min. The modification of MR results in a slight increase in the lethal concentration. Cell membrane integrity at the lethal concentration confirms that MR undergoes the inhibition effect, but not by destroying the cell membrane integrity. The novel property of MR provides a new insight into the Trojan horse strategy of McC and opens a new route for antibiotics design.Keywords:microcin C7;heptapeptide;cell growth;membrane integrity
Shu Bai,Jie Zhang,Liyan Zhu,Xiaoxing Gong,Linling Yu,Yan Sun
Oriented immobilization of antibodies is important for the effective recognition of target antigens. In this paper, a heptapeptide ligand, HWRGWVC (HC7), was modified onto non-porous monosized poly(glyceryl methacrylate) (pGMA) microspheres (named pGMA-HC7) to explore the antibody immobilization behaviors. Characterization of the microspheres by particle size analyzer, scanning electron microscopy, Fourier transform infrared spectroscopy, and reversed-phase chromatography proved the success of each fabrication step. The capacity and activity of antibody immobilization through HC7 were studied using immunoglobulin G (IgG) as a model antibody and horseradish peroxidase (HRP) as a model antigen. Additionally, IgG immobilizations on pGMA microspheres by nonspecific adsorption and covalent coupling through carbodiimide chemistry were conducted for comparison. pGMA-HC7 exhibited an IgG adsorption capacity of 3–4 mg/g in 10 min by the specific binding of HC7 without nonspecific interactions. Notably, the ligand HC7 showed a by two orders of magnitude stronger affinity for IgG than its original hexapeptide ligand HWRGWV. Moreover, the capacity and activity of the immobilized anti-HRP antibody on pGMA-HC7 were 1.6-fold and 3-fold higher than those of the covalent coupling, respectively. The results proved the superior role of HWRGWVC in the affinity binding of antibody and the potential of pGMA-HC7-25 in immunoassay and immunodiagnostic applications.
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