scholarly journals Specific Non-Reducing Ends in Heparins from Different Animal Origins: Building Blocks Analysis Using Reductive Amination Tagging by Sulfanilic Acid

Molecules ◽  
2020 ◽  
Vol 25 (23) ◽  
pp. 5553
Author(s):  
Pierre A. J. Mourier
Keyword(s):  
Building Block ◽  
Reductive Amination ◽  
Building Blocks ◽  
Anion Exchange Chromatography ◽  
Sulfanilic Acid ◽  
Exchange Chromatography ◽  
Sulfated Polysaccharides ◽  
Liquid Chromatography Mass Spectrometry ◽  
Anticoagulant Drugs ◽  
Strong Anion Exchange

Heparins are linear sulfated polysaccharides widely used as anticoagulant drugs. Their nonreducing-end (NRE) has been little investigated due to challenges in their characterization, but is known to be partly generated by enzymatic cleavage with heparanases, resulting in N-sulfated glucosamines at the NRE. Uronic NRE (specifically glucuronic acids) have been isolated from porcine heparin, with GlcA-GlcNS,3S,6S identified as a porcine-specific NRE marker. To further characterize NRE in heparinoids, a building block analysis involving exhaustive heparinase digestion and subsequent reductive amination with sulfanilic acid was performed. This study describes a new method for identifying heparin classical building blocks and novel NRE building blocks using strong anion exchange chromatography on AS11 columns for the assay, and ion-pair liquid chromatography-mass spectrometry for building block identification. Porcine, ovine, and bovine intestine heparins were analyzed. Generally, NRE on these three heparins are highly sulfated moieties, particularly with 3-O sulfates, and the observed composition of the NRE is highly dependent on heparin origin. At the highest level of specificity, the isolated marker was only detected in porcine heparin. However, the proportion of glucosamines in the NRE and the proportion of glucuronic/iduronic configurations in the NRE uronic moieties greatly varied between heparin types.

scholarly journals Heparin Isomeric Oligosaccharide Separation Using Volatile Salt Strong Anion Exchange Chromatography

2016 ◽  
Vol 88 (23) ◽  
pp. 11542-11550 ◽  
Author(s):  
Rebecca L. Miller ◽  
Scott E. Guimond ◽  
Maitreyi Shivkumar ◽  
Jemma Blocksidge ◽  
James A. Austin ◽  
...  
Keyword(s):  
Anion Exchange ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Strong Anion Exchange

Determination of uronic acids and neutral carbohydrates in pulp and biomass by hydrolysis, reductive amination and HPAEC-UV

Holzforschung ◽  
2017 ◽  
Vol 71 (10) ◽  
pp. 767-775 ◽  
Author(s):  
Dominic Lorenz ◽  
Ron Janzon ◽  
Bodo Saake
Keyword(s):  
Enzymatic Hydrolysis ◽  
High Performance ◽  
Reductive Amination ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Kraft Pulps ◽  
Uronic Acids ◽  
Sulfite Pulp ◽  
The Individual

AbstractThe exact quantification of all carbohydrate constituents in wood and pulp is a challenge because of the various glycosidic linkages of the polysaccharides with different stabilities. The individual detector responses for the compounds in the hydrolysates additionally complicate the quantification as pure standards for 4-O-methyl-α-D-glucuronic acid (meGlcA) and related oligosaccharides are not commercially available for calibration. In the present paper, a new analytical procedure is presented, based on the reductive amination of the carbohydrates obtained via acidic and enzymatic hydrolysis of the polysaccharides before quantification by means of high performance anion exchange chromatography (HPAEC) and UV-detection. This approach was suitable for the analysis of neutral carbohydrates and uronic acids obtained via enzymatic hydrolysis from bleached pulps. In the case of unbleached pulps, the enzymatic hydrolysis was not complete and unhydrolyzed nano-scaled and micro-scaled particles remained in the hydrolysates as detected by dynamic light scattering (DLS) measurements. The new HPAEC-UV methodology was also applied to kraft pulps and a sulfite pulp; six different kinds of wood as well as wheat straw and bagasse. All relevant monosaccharides and the dimer of meGlcA and xylose could be detected in the hydrolysates. Accordingly, significantly higher yields of meGlcA were found compared to literature data.

scholarly journals Amplification and Purification of T4-Like Escherichia coli Phages for Phage Therapy: from Laboratory to Pilot Scale

2013 ◽  
Vol 80 (4) ◽  
pp. 1469-1476 ◽  
Author(s):  
Gilles Bourdin ◽  
Bertrand Schmitt ◽  
Laure Marvin Guy ◽  
Jacques-Edouard Germond ◽  
Sophie Zuber ◽  
...  
Keyword(s):  
High Speed ◽  
Production Systems ◽  
Anion Exchange Chromatography ◽  
Pilot Scale ◽  
Exchange Chromatography ◽  
Cold Chain ◽  
Contamination Level ◽  
Stirred Tank Reactors ◽  
Medium Speed ◽  
Strong Anion Exchange

ABSTRACTWe investigated the amplification and purification of phage preparations with respect to titer, contamination level, stability, and technical affordability. Using various production systems (wave bags, stirred-tank reactors, and Erlenmeyer flasks), we obtained peak titers of 109to 1010PFU/ml for T4-like coliphages. Phage lysates could be sterilized through 0.22-μm membrane filters without titer loss. Phages concentrated by differential centrifugation were not contaminated with cellular debris or bacterial proteins, as assessed by electron microscopy and mass spectrometry, respectively. Titer losses occurred by high-speed pelleting of phages but could be decreased by sedimentation through a sucrose cushion. Alternative phage concentration methods are prolonged medium-speed centrifugation, strong anion-exchange chromatography, and ultrafiltration, but the latter still allowed elevated lipopolysaccharide contamination. T4-like phages could not be pasteurized but maintained their infectivity titer in the cold chain. In the presence of 10 mM magnesium ions, phages showed no loss of titer over 1 month at 30°C.

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