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Physical Characterization of hFSH and Its Desialylation Products by Isoelectric Focusing and Electrophoresis in Polyacrylamide Gel

Reproduction Research Branch, National Institute of Child Health and Human Development, National Institutes of Health Bethesda, Maryland 20014

Abstract

Iodinated and tritiated derivatives of highly purified hFSH were characterized by polyacrylamide gel electrophoresis (PAGE) at _pH 7.8 and 10.2, 0 C, and by isoelectric focusing in polyacrylamide gel (IFPA). ¹²⁵I— and ³HhFSH appear homogeneous in PAGE, as evidenced by single, linear Ferguson plots at the two pH values, and by a single peak in IFPA. ¹³¹I—hFSH in contrast appears heterogeneous. The physical characteristics of hFSH, recognized most comprehensively by the parameters K_R (retardation coefficient, a measure of molecular size) and Y_o (a measure of molecular net charge) in PAGE, differed significantly between ³H— and ¹²⁵I—hFSH: it appeared that the tritiated hormone was in an aggregated form at ^pH 10.2. However, the molecular size of ³H—hFSH appeared the same at pH 7.8 as that of ¹²⁵I—hFSH at _pH 10.2: both values correspond to a mol wt of 36,000, in agreement with literature values. Size discrepancy between 2 variously labeled preparations under a single set of PAGE conditions implies that recognition of various hFSH species in biological systems is feasible only when a single type of isotopic labeling is used.

The _pH value associated with ³H—hFSH and ¹²⁵I—hFSH in IFPA was 4.0 ± 0.2 (SD), in agreement with unlabeled hormone. Progressive desialylation of urinary hFSH resulted in species of hFSH that were characterized by _pH values increasing from 4 to 6.

The estimate of molecular size based on PAGE and the estimate of pi based on IFPA are in reasonable agreement with previous data obtained by other methods. PAGE and IFPA were demonstrated to be sensitive, rapid and reliable methods for the detection of hFSH and the various desialohFSH species. (Endocrinology 92: 1135, 1973

Received August 25, 1972.