APPLICATION OF A SENSITIVE HPLC-BASED FLUOROMETRIC ASSAY TO DETERMINETHE SIALIC-ACID CONTENT OF HUMAN GONADOTROPIN ISOFORMS

The human pituitary gonadotropins, follitropin (hFSH) and lutropin (hL H) are glycoproteins which are microheterogeneous in terms of their ch arge and molecular size, as well as their in vitro and in vivo bioacti vities. The aim of this study was to determine the contribution of var iations in sialic acid (N-acetyl neuraminic acid) content to the struc tural heterogeneity of these glycoproteins. Sialic acid (Neu5Ac) was r eleased by partial acid hydrolysis (0.1 M TFA, 80 degrees C, 1 h) and derivatised with the fluorescent label DMB (1,2-diamino-4,5-methylened ioxybenzene) in conjunction with an internal standard (N-glycoyl-neura minic acid). The derivatives were then separated by reversed-phase HPL C. This method allowed quantitation of the sialic acid content over a range of 5-100 pmol with between assay variation of <6% for sialic aci d released from approximately 100 ng (3 pmol) of hFSH or hLH. Comparis on of the sialic acid contents of standard sialylated glycoproteins by either DMB-derivatisation or high-performance anion-exchange chromato graphy with pulsed amperometric detection yielded similar results, con firming the reliability of the fluorescence detection method. The sial ic acid contents of 9 hFSH isoforms varied between 1.5-13.7 mot Neu5AC /mol FSH, whilst a range of 1.1-9.1 mol Neu5AC/mol LH was observed for 12 hLH isoforms. Tile sialic acid content of the hFSH isoforms was al so observed to be related to the hormonal specific activity in a radio receptor assay, confirming that alterations in the carbohydrate struct ure can influence the FSH-receptor interaction. In contrast, the siali c acid content of the hLH isoforms was found to be not related to spec ific activity at the receptor level.