TD Chem- GAGs

Analysis of HS purification
- Separate with Heparinase 3 or Hep 1
- Depolim techniques
Heparinase 1 > GlcNS+/- 6s - IdoA, 2S
Heparinase 3 > glucNAc+/- 6s- GlcA
Low pH nitrous acid > glucNS +/- 6s - HexA +/- 2S
- Technique: ion change chromatography > anion or cation exchange??? Anion
- To determine composition of disaccharides: After cleaving into disaccharides we
can exchange again with ion exchange chromatography. Can do with 3
heparinases
- Commercially available disacc
- Iduronic and glucuronic acid information is lost
- Orange vs red: orange can bind but not activate (image in slide)
- Conclusion of graph: 6OS is required for the biological activity
- EXERCISE 1.a
Rat HS composition
- 31.4 (1), 18 (2), 6.2 (3), 1.2 (7), 2.1 (*), 5 (4), 14.4 (5), 18.9 (6)
Human HS composition
- 56 (1), 28 (2), 9 (3), 2(7), 2 (4), 5 (5), 2 (6)
- EXERCISE 1.b
Rat N sulfation = 2+4+5+6 = 18+5+14.4+18.9 = 56.3
Human N sulfation = 2+4+5+6 = 28+2+5+2= 37
Rat O sulfation = 3+4+5+6+7 = 6.2+5+14.4+(2x18.9)+1.2 = 64.6

 Human O sulfation = 3+4+5+6 = 9+2+5+(2x2)= 20

Rat 2 sulfation = 5+6+7= 14.4 +18.9+1.2 = 34.5
Human 2 sulfation = 5+6= 5+2 = 7
Rat 6 sulfation = 3+4+6= 6.2+5+18.9= 30.1
Human 6 sulfation = 3+4+6= 9+2+2= 13
- EXERCISE 1.c
Comment asterisk: it could be one of the saccharides for which we don't have a
standardised model. The measurement is not well correlated. Could also be an
artefact (contaminant), you can repeat experiment to make sure. Also: maybe
partial degradation/digestion > do gel filtration to look for tetra or
hexasaccharide, if there is, then do experiment again to get disaccharides.
- EXERCISE 2
It can be octasaccharide > 2 dis + 1 dis + 1 dis = 8 saccharides. In theory it
could be any multiple of 8 BUT here it is not because it would be highly
heterogeneous (look at graph in first slides).
HexUA> GlcNS> IdoA,2S> GlcNS> IdoA,2S > GlcNS> HexUA >
GlucNAc, 6s