N-Propyl-N'-2-pyridylurea-modified silica as mixed-mode stationary phase with moderate weak anion exchange capacity and pH-dependent surface charge reversal.

Herein, we present a novel silica-based stationary phase modified with N-propyl-N'-2-pyridylurea selector. Due to the weakly basic properties of the pyridine selector and the presence of residual silanols after selector immobilization, a zwitterionic surface with a pI observed at approximately pH 5.5 was measured by electrophoretic light scattering in pH-dependent ζ-potential determinations. The capability of the new N-propyl-N'-2-pyridylurea-modified silica to serve as mixed-mode stationary phase was investigated. For this purpose, it was characterized under RP and HILIC conditions using test mixtures. Subsequent classification of this stationary phase in comparison to in-house and commercial benchmarks was carried by principal component analysis of resultant retention factors from chromatographic tests. The results show a relatively unique mixed-mode character amongst the tested stationary phases. The chromatographic retention characteristics of acidic compounds matched well the ζ-potential determinations. The application of anion-exchange at low pH values (e.g. pH 5) and ion exclusion chromatography at pH 7 for the separation of uridine 5'-mono-, di- and triphosphate demonstrated a pH-dependent umpolung of the stationary phase surface. The combination of these separation principles in a pH gradient from 5 to 7 gave rise to weak anion-exchange selectivity with a charge-inducted elution due to repulsive interactions at higher pH and resulted in a significant faster separation with improved peak shape under mild elution conditions.

Identification of common and unique peptide substrate preferences for the UDP-GalNAc:polypeptide alpha-N-acetylgalactosaminyltransferases T1 and T2 derived from oriented random peptide substrates.

A large family of UDP-GalNAc:polypeptide alpha-N-acetylgalactosaminyltransferases (ppGalNAc Ts) catalyzes the first step of mucin-type protein O-glycosylation by transferring GalNAc to serine and threonine residues of acceptor polypeptides. The acceptor peptide substrate specificity and specific protein targets of the individual ppGalNAc T family members remain poorly characterized and poorly understood, despite the fact that mutations in two individual isoforms are deleterious to man and the fly. In this work a series of oriented random peptide substrate libraries, based on the GAGAXXXTXXXAGAGK sequence motif (where X = randomized positions), have been used to obtain the first comprehensive determination of the peptide substrate specificities of the mammalian ppGalNAc T1 and T2 isoforms. ppGalNAc T-glycosylated random peptides were isolated by lectin affinity chromatography, and transferase amino acid preferences were determined by Edman amino acid sequencing. The results reveal common and unique position-sensitive features for both transferases, consistent with previous reports of the preferences of ppGalNAc T1 and T2. The random peptide substrates also reveal additional specific features that have never been described before that are consistent with the x-ray crystal structures of the two transferases and furthermore are reflected in a data base analysis of in vivo O-glycosylation sites. By using the transferase-specific preferences, optimum and selective acceptor peptide substrates have been generated for each transferase. This approach represents a relatively complete, facile, and reproducible method for obtaining ppGalNAc T peptide substrate specificity. Such information will be invaluable for identifying isoform-specific peptide acceptors, creating isoform-specific substrates, and predicting O-glycosylation sites.

Enzymatic synthesis of UDP-galactofuranose and an assay for UDP-galactopyranose mutase based on high-performance liquid chromatography.

A method to prepare UDP-galactofuranose (UDP-Galf) free of UDP-galactopyranose (UDP-Galp) is described. The UDP-Galf is synthesized enzymatically from UDP-Galp using the enzyme UDP-galactopyranose mutase. Treatment of UDP-Galp with the enzyme yields an equilibrium mixture of UDP-Galp and UDP-Galf in which UDP-Galf is approximately 7%. In spite of its low yield, the UDP-Galf is readily purified from starting UDP-Galp using a Dionex PA-100 ion exchange HPLC column. The purified UDP-Galf was characterized by chemical degradations, by electrospray mass spectrometry, and by several nuclear magnetic resonance techniques. In addition, an HPLC assay for the enzyme UDP-galactopyranose mutase is presented that requires 0.5 microgram of UDP-Galf per assay and can be used for both qualitative and quantitative measurements of the enzyme activity. These procedures should thus aid in the characterization of the enzymes involved in galactofuranosyl biosynthesis for the cell walls of Mycobacteria, for the lipophosphoglycan of Leishmania, and for other microorganisms where galactofuranosyl residues are found.

Structure determination of the UDP-disaccharide fragment of cytoplasmic cofactor isolated from Methanobacterium thermoautotrophicum.

The methylcoenzyme M methylreductase reaction has an absolute requirement for 7-mercaptoheptanoylthreonine phosphate or component B, which is the active component of the intact molecule previously referred to as cytoplasmic cofactor. A hydrolytic fragment of cytoplasmic cofactor has been purified and identified as uridine 5'-(O-2-acetamido-2-deoxy-beta-manno-pyranuronosyl acid (1----4)-2-acetamido-2-deoxy-alpha-glucopyranosyl diphosphate) by high resolution NMR and fast atom bombardment mass spectro-metry. It is postulated that UDP-disaccharide may function to anchor 7-mercaptoheptanoyl threonine phosphate at the active site of the methyl-reductase enzyme complex.

Measurement of tissue purine, pyrimidine, and other nucleotides by radial compression high-performance liquid chromatography.

A high-performance liquid-chromatographic (HPLC) method for the rapid separation of purine and pyrimidine nucleotides, NAD+, NADP+, FAD, FMN, UDP-Glc, UDP-glucuronate, and ADP-ribose found in neutralized perchloric acid extracts of rat liver is described. Separation was achieved within 26 min on a radially compressed column of Partisil 10-SAX. The column was eluted with a gradient of sodium phosphate and sodium chloride. The sodium phosphate was purified by passage through tandem columns of anion- and cation-exchange resins to remove uv-absorbing impurities. The sensitivity of this procedure is such that an amount of ATP contained in 10 micrograms of liver can be measured. The recoveries of all nucleotides were between 87 and 107%. In extracts of rat liver interfering substances were found to elute with GDP, and UDP eluted with NADP. Consequently, the tissue contents of UDP and GDP were determined in a second run by measuring the increase in UTP and GTP, respectively, following sample pretreatment with pyruvate kinase (PK). The tissue level of NADP+ was calculated as the difference between the total UDP and NADP+ peak and the increase in UTP following PK treatment. In those nucleotides amenable to enzymatic analysis, namely NAD+, AMP, UDP-Glc, UTP, and ATP, the tissue contents measured enzymatically were not significantly different from those determined by HPLC. However, ADP as measured with PK was found to be 15% higher compared to the HPLC determination.