Stanton P G, Shen Z, Kecorius E A, Burgon P G, Robertson D M, Hearn M T
Centre for Bioprocess Technology, Monash University, Victoria, Australia.
J Biochem Biophys Methods. 1995 Feb;30(1):37-48. doi: 10.1016/0165-022x(94)00063-j.
The human pituitary gonadotropins, follitropin (hFSH) and lutropin (hLH) are glycoproteins which are microheterogeneous in terms of their charge and molecular size, as well as their in vitro and in vivo bioactivities. The aim of this study was to determine the contribution of variations in sialic acid (N-acetyl neuraminic acid) content to the structural heterogeneity of these glycoproteins. Sialic acid (Neu5Ac) was released 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-methylenedioxybenzene) in conjunction with an internal standard (N-glycoyl-neuraminic acid). The derivatives were then separated by reversed-phase HPLC. This method allowed quantitation of the sialic acid content over a range of 5-100 pmol with between assay variation of < 6% for sialic acid released from approximately 100 ng (3 pmol) of hFSH or hLH. Comparison of the sialic acid contents of standard sialylated glycoproteins by either DMB-derivatisation or high-performance anion-exchange chromatography with pulsed amperometric detection yielded similar results, confirming the reliability of the fluorescence detection method. The sialic acid contents of 9 hFSH isoforms varied between 1.5-13.7 mol Neu5AC/mol FSH, whilst a range of 1.1-9.1 mol Neu5AC/mol LH was observed for 12 hLH isoforms. The sialic acid content of the hFSH isoforms was also observed to be related to the hormonal specific activity in a radioreceptor assay, confirming that alterations in the carbohydrate structure can influence the FSH-receptor interaction. In contrast, the sialic acid content of the hLH isoforms was found to be not related to specific activity at the receptor level.
人垂体促性腺激素，促卵泡激素（hFSH）和促黄体激素（hLH）是糖蛋白，其电荷、分子大小以及体外和体内生物活性存在微观异质性。本研究的目的是确定唾液酸（N-乙酰神经氨酸）含量变化对这些糖蛋白结构异质性的影响。通过部分酸水解（0.1 M三氟乙酸，80℃，1小时）释放唾液酸（Neu5Ac），并与荧光标记物DMB（1,2-二氨基-4,5-亚甲基二氧基苯）和内标（N-糖酰神经氨酸）一起衍生化。然后通过反相高效液相色谱分离衍生物。该方法能够在5 - 100 pmol范围内定量唾液酸含量，对于从约100 ng（3 pmol）hFSH或hLH释放的唾液酸，测定间变异小于6%。通过DMB衍生化或高效阴离子交换色谱结合脉冲安培检测比较标准唾液酸化糖蛋白的唾液酸含量，得到了相似的结果，证实了荧光检测方法的可靠性。9种hFSH同工型的唾液酸含量在1.5 - 13.7 mol Neu5AC/mol FSH之间变化，而12种hLH同工型的唾液酸含量范围为1.1 - 9.1 mol Neu5AC/mol LH。在放射受体测定中还观察到hFSH同工型的唾液酸含量与激素比活性相关，证实碳水化合物结构的改变可影响FSH - 受体相互作用。相比之下，发现hLH同工型的唾液酸含量与受体水平的比活性无关。
