The effect of modification of the carboxyl groups of high affinity heparin was investigated. The binding affinity toward antithrombin III decreases in the following order: Heparin greater than heparin methyl ester greater than heparinylglycine greater than heparinylglycine methyl ester. This result agrees qualitatively with the previous studies using unfractionated heparin. Esterification of the carboxyl groups (i.e., HME) does not affect the CD profile of heparin at 210 nm but introduction of a bulkier glycine methyl ester (i.e., HGME) leads to formation of a very intense band at 235 nm. Based on reported CD analyses of uronic acid derivatives and our model building studies, it is concluded that the large difference in CD spectra of HGME as compared to unmodified heparin and HME is due to a change in ring conformation of the uronic acid moiety (i.e., 4C1 to 1C4 or vice versa).