Heparin mediates fundamental biological mechanisms through interaction with proteins. Previously, we have shown that standard heparin binds to granulocyte colony-stimulating factor (G-CSF) with an affinity of 4.8 x 10(5) M-1. To further study the structural features in heparin that are responsible for this interaction, we studied the bindings of G-CSF and N-desulfated and 2,3-O-desulfated heparin by CZE. Results showed that the N-desulfated heparin had a similar affinity for G-CSF ((5.4 +/-0.9) x 10(5) M-1), but the 2,3-O-desulfated heparin had a 1000-fold lower affinity ((3.4 +/- 1.2) x 10(2) M-1) in comparison to standard heparin. The results showed that 2,3-O-sulfate groups are more important than N-sulfate groups in heparin-G-CSF interaction.