Glycoprotein hydrolysate

Fig. 3-119. Separation of the monosaccharide components present in glycoprotein hydrolysates. — Separator column CarboPac PA-1 eluent 0.022 mol/L NaOH flow rate 1 mL/min detection pulsed amperometry at a Au working electrode after post-column addition of 0.3 mol/L NaOH injection volume 50 pL solute concentrations 2.5 nmol each of fucose (1), 2-deoxyglucose (2), galactosamine (3), glucosamine (4), galactose (5), glucose (6), and mannose (7) (taken from [111]).

Polystyrenesulfonate cation-exchange resin, 55% DVB, H + -form Acetonitrile-water (23 2) separates neutral and amino sugars in glycoprotein hydrolysates None 30 Postcolumn reaction with 2- cyanoacetamide UV(280nm)... 

Figure 3.223 Comparison between CarboPac PA1 and PA10 for monosaccharide analysis in a glycoprotein hydrolysate at high sensitivity. Chromatographic conditions see Figure 3.222 ...

Figure 3.225 Separation of the six monosaccharides found in glycoprotein hydrolysates on CarboPac PA20. Eluent 10 mmol/L KOH (EG) flow rate 0.5 mL/min detection pulsed...

Fig. 3-168. Effect of hydroxide concentration on elution times for the six common monosaccharides found in glycoprotein hydrolysates on CarboPac PA20. - Eluant 8-20 mmol/L NaOH flow rate 0.5 mU/min detection pulsed amperometry on a gold working electrode peaks (1) fucose, (2) galactos-amine, (3) glucosamine, (4) galactose,...

Tissue-type plasminogen activator (tPA) is a glycoprotein (68 kDa), synthesized by endothelial and tumor-cells. As a serine protease, tPA hydrolyses Arg561-Val562 peptide bond in plasminogen, resulting in plasmin formation. It needs cofactors for efficient plasminogen activation. 

There are several steps in the absorption of vitamin B. In the stomach and lumen of the small intestine it is hydrolysed from its (peptide) links with the proteins of which it is a component. It then attaches to gastric intrinsic factor, which is a glycoprotein of molecular mass about 50 000 kDa, to form a complex. This protects the vitamin from being damaged by acid in the stomach. The complex is carried into the ileum, where it binds to a receptor on the surface of the absorptive cells and is released from the intrinsic factor within the absorptive cell, hi the portal venous blood, it is transported to the liver bound to the vitamin B 12-binding protein, which also protects the vitamin. 

Carbohydrate Analysis Following glycoprotein hydrolysis, dilute sample 1 1 with DIW and speed vacuum dry well to remove residual TEA. To acheive the required pH 5, add 100 pi of 20 mM sodium acetate (pH 5) and if necessary, adjust pH between 4 and 5 with NaOH. The hydrolysate containing monosaccharides is transfered to a pre-wetted Microcon-SCX and centrifuged for 30 sec at high speed to remove free amino acids and unhydrolyzed peptides for HPAE-PAE analysis by electrochemical detection. 

The analysis of cell culture media and supernatants, as well as non-standard protein hydrolysates such as collagens and glycoproteins, has created the demand for techniques that accurately quantify additional amino acids not normally found in hydrolyzed samples, Methods of amino acid analysis (AAA) based on precolumn derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) have previously been shown to quantify hydrolyzed samples with a high degree of accuracy (1,2). The AQC-based method has also been shown to derivatize effectively in the presence of salts and lipids (3). Taking into account the above strengths, the excellent stability of the derivatives, and the unique fluorescence properties that allow for direct injection of the reaction mixture without cleanup, the AQC methodology represents an ideal choice for the analysis of complex samples. 

Due to an increased interest in analysis of physiological samples, we wanted to establish analyzer methods which would allow us to choose between our standard protocol for protein and peptide hydrolysates and a separate protocol for an expanded number of amino acids, to include the most important free amino acids found in physiological samples. A study of common analysis requirements in our facility indicated that only a limited number of the possible free physiological amino acids is needed for most unknown samples. These additional amino acids of interest are a-amino butyric acid, citrulline, y-amino butyric acid (GABA), hydroxyproline, hydroxylysine, ornithine, taurine, and tryptophan. Other amino acids of interest to us are phosphoserine, phosphothreonine, phosphotyrosine and carboxy-amino acids since they are released from glycoprotein or glycopeptide hydrolysates. 

The enzymes which hydrolyse the GlcNAc residue attached to Ser or Thr of glycoproteins have no sequence similarity to GH 20, but it appears that GH 84,... 

---