Practical Protein Chemistry - A. Darbre 1989

Analytical Methods
Glycoproteins
Introduction

In collaboration with J. Klamp (J. Clamp)

Glycoproteins are biopolymer molecules that are widely distributed in living organisms. They can be intracellular or extracellular, present as membrane components or in solution, and exist as either fibrillar structures or typical Globular Proteins. More than half of all proteins studied in detail contain covalently attached sugars; therefore, when investigating a novel protein, one should always keep in mind that it may be a glycoprotein.

METABOLISM/18.html">The Influence of carbohydrate components on The properties of a protein molecule depends on their content, which varies over a wide range from ≤1% to ≥80%. The attachment of one or two small oligosaccharide chains to an average-sized peptide chain has almost no effect on the typical “protein” Properties of the compounds. At the same time, glycoproteins with a high sugar content essentially behave like Polysaccharides. However, most glycoproteins are characterized by a carbohydrate content intermediate between these two extremes. Any protein may be suspected of being a glycoprotein, especially if it exhibits anomalous behavior during Gel filtration, ultracentrifugation, staining, ultraviolet absorbance measurements, etc.

The simplest way to confirm this suspicion, used by many researchers in the field, is to screen the sample for hexosamines using Amino acid analysis, with glucosamine and galactosamines being the most common. For most amino acid analyzers, the supplied manuals include data on The behavior of these two hexosamines in the system used; if such data are not provided, it is easy to obtain them experimentally. It should be borne in mind, however, that not all glycoproteins contain hexosamine, and a significant amount of CARBOHYDRATES is destroyed under the harsh Hydrolysis conditions employed in amino acid analysis.

Most commonly, typical mammalian glycoproteins contain the following Monosaccharides: L-fucose (6-deoxy-L-galactose), D-mannose, D-galactose, N-acetyl-D-glucosamine (2-acetamido-2-deoxy-D-glucosamine), and sialic acids (various derivatives of neuraminic acid). Some glycoproteins also incorporate D-glucose or N-acetyl-D-galactosamine (2-acetamido-2-deoxy-D-galactosamine). The best results for the Separation, identification, and quantification of these seven monosaccharides are achieved using Gas-Liquid Chromatography. There are two main Methods for sugar modification prior to gas-liquid chromatography analysis: the alditol acetate method and the Methyl Glycoside Trimethylsilyl method. The former involves aqueous acid hydrolysis followed by the reduction of the resulting aldoses to their corresponding alditols and subsequent Acetylation; the latter is based on methanolysis, which yields methyl Glycosides and methyl esters of sialic acids that are then converted into trimethylsilyl derivatives.

It should be noted that proteins purified using Sephadex columns may contain glucose as a contaminant.