Strong Ion-exchange Chromatography Research Articles

Medium optimization to analyze the protein composition of Bacillus pumilus HR10 antagonizing Sphaeropsis sapinea

A previous study found that a biocontrol bacterium, Bacillus pumilus HR10, inhibited the Sphaeropsis shoot blight disease of pine, and the fermentation broth of HR10 strain contained protein antifungal substances. The optimal formulation of the fermentation medium for the antagonistic substance of B. pumilus HR10 was finally obtained by single-factor test, Packett-Burman test, steepest ascent test and Box-Behnken Design (BBD) response surface test, and the best formulation of the fermentation medium for the antagonistic substance of B. pumilus HR10 was 12 g/L corn meal, 15 g/L beef extract and 13 g/L magnesium sulfate, with a predicted bacterial inhibition rate of 89%. The fermentation filtrate of B. pumilus HR10 cultured with the optimized medium formulation was verified to have an inhibition rate of (87.04 ± 3.2) % on the growth of Sphaeropsis sapinea by three replicate tests. The antagonistic crude protein of B. pumilus HR10 were further isolated and identified using HiTrap Capto Q strong Ion-Exchange Chromatography and LC–MS-MS, and it was speculated that glycoside hydrolase (Ghy), beta-glucanase (Beta), arabinogalactan endonuclease β-1,4-galactanase (Arab), and immunosuppressant A (ImA) are proteins with antagonistic activity against S. sapinea in the B. pumilus HR10.

Partial structural studies of fucosylated chondroitin sulfate (FuCS) using attenuated total reflection fourier transform infrared spectroscopy (ATR-FTIR) and chemometrics

The compound fucosylated chondroitin sulfate from three sea cucumber species Holothuria atra, Stichopus horrens, and Holothuria arenicola was extracted and purified using strong ion exchange chromatography. The structure and sulfation patterns on their fucose branches were partially characterized and compared using liquid, gas chromatography-mass spectrometry, infrared spectroscopy, and chemometrics. The monosaccharide composition was consistently a 1:1 molar ratio of glucuronic acid to N-acetylgalactosamine while the fucose molar ratio was statistically different among the three species. Normal IR spectra exhibited a broad band at the region of 850–830cm−1 assigned to the sulfate COS group. This band was resolved in the second derivative spectra into two or three bands corresponding to the difference of sulfation patterns among sea cucumber species. Principal component analysis (PCA) confirmed the difference in the sulfate substitution region with additional discrimination of the combination amide NH and CO group, sulfate SO region, and skeletal region 1050–980cm−1. The hierarchical clustering analysis (HCA) successfully grouped each sea cucumber FuCS in the same cluster. Nevertheless, the comparison of the FuCS structure was simplified using chemometrics analysis.

Peroxide-Mediated Desulfurization of Phosphorothioate Oligonucleotides and Its Prevention

Desulfurization at the internucleotide phosphorothioate linkage of antisense oligonucleotides (ASOs) in dermatological formulations has been investigated using strong ion exchange chromatography and mass spectroscopy. The formation of phosphate diester linkages appeared to arise from a reaction between the phosphorothioate oligonucleotide and a potent oxidizing agent. Screening of excipients used in the formulation indicated that the cause of desulfurization was related to the presence of polyethylene glycol-derived nonionic surfactants MYRJ 52 or BRIJ 58. Autoxidation of the polyethylene glycol chain is suggested as the probable origin for the observed incompatibility. The ability of various antioxidants to prevent oxidative degradation of ASO-1 in simple test systems and in oil-in-water emulsions is described. It is found that in test systems both lipophilic and hydrophilic antioxidants are effective. However, in cream formulation (oil-in-water emulsions) of ASO-1 the addition of hydrophilic antioxidants L-cysteine or DL-α-lipoic acid has been shown to be superior in protecting the oligonucleotide from desulfurization upon storage.

Separation of small cationic bioactive peptides by strong ion-exchange chromatography

The high resolving power of a Hyper D cation-exchange column was evaluated to achieve the separation of small cationic bioactive peptides derived from tryptic digest of κ-casein: MAIPPKK (p I = 9.9), MAIPPKK (p I = 10.5), KNQDK (p I = 9.6) and NQDK (p I = 6.35). The influence of pH (1.5–6), gradient slope (2–10 m M sodium chloride/min) and elution of the mixture under isocratic conditions was investigated. Although their physico-chemical properties are very similar, these four peptides were readily resolved with an excellent selectivity and recovery. The selectivity of the exchanger was also expressed toward peptides of the same net positive charge; the most hydrophilic peptide always eluted last. It was also shown that the elution order of these molecules depends on pH. From the observed retention times and the elution order, we have established a simple approach to linearization of peptide retention behaviour on the S-Hyper D support.