The first question to be asked about a purified protein is ‘Is it a glycoprotein?’
Glycoprotein bands in gels (e.g. on SDS-polyacrylamide gels) can be stained with
cationic dyes such as Alcian Blue, which bind to negatively charged glycosaminogly-
can side-chains, or by the periodic acid-Schiff reagent (PAS), where carbohydrate is
initially oxidised by periodic acid then subsequently stained with Schiff’s reagent.
However, although they are both carbohydrate specific (i.e. non-glycosylated proteins
are not stained) both methods suffer from low sensitivity. A more sensitive, and
informative, approach is to use the specific carbohydrate-binding proteins known
as lectins. Blots from SDS-PAGE, dot blots of the glycoprotein sample, or the glyco-
protein sample adsorbed onto the walls of a microtitre plate can be challenged with
enzyme-linked lectins. Lectins that bind to the glycoprotein can be identified by the
associated enzymic activity. By repeating the experiment with a range of different
lectins, one can not only confirm the presence of a glycoprotein but also identify
which sugar residues are, or are not, present. Having confirmed the presence of
glycoprotein the following procedures would normally be carried out.- Identification of the type and amount of each monosaccharide: Release of
monosaccharides is achieved by hydrolysis in methanolic HCl at 80C for 18 h. The
released monosaccharide can be separated and quantified by gas chromatography. - Protease digestion to release glycopeptide: A protease is chosen that cleaves the
glycoprotein into peptides and glycopeptides of ideally 5–15 amino acid residues.
Glycopeptides are then fractionated by HPLC and purified glycopeptides subjected
to N-terminal sequence analysis to allow identification of the site of glycosylation. - Oligosaccharide profiling: Oligosaccharide chains are released from the polypeptide
backbone either chemically, for example by hydrazinolysis to release N-linked
oligosaccharide, or enzymatically using peptide-N-glucosidase F (PNGase F), which cleaves
sugars at the asparagine link, or using endo-a-N-acetylgalactosaminidase (O-glycanase),
which cleaves O-linked glycans. These released oligosaccharides can then be separated
either by HPLC or by high performance anion exchange chromatography (HPAEC). - Structure analysis of each purified oligosaccharide: This requires the determination
of the composition, sequence and nature of the linkages in each purified
oligosaccharide. A detailed description is beyond the scope of this book, but would
involve a mixture of complementary approaches including analysis by FAB-MS, gas
chromatography-MS, lectin analysis following partial release of sugars and nuclear
magnetic resonance (NMR) analysis.
8.4.5 Tertiary structure
The most commonly used method for determining protein three-dimensional structure
is X-ray crystallography. A detailed description of the theory and methodology is
beyond the scope of this book, requiring a detailed mathematical understanding of the
process and computer analysis of the extensive data that are generated. The following is
therefore a brief and idealised description of the overall process, and ignores the multitude
of pitfalls and problems inherent in determining three-dimensional structures.- Clearly the first step must be to produce a crystal of the protein (a crystal should
be thought of as a three-dimensional lattice of molecules). Protein crystallisation is
336 Protein structure, purification, characterisation and function analysis