Ovine luteinizing hormone. VI. Analysis of the misclassification errors in the separation of intrapituitary isohormones by chromatofocusing.
Luteinizing hormone (LH) in extracts of the ovine (o) anterior pituitary gland elutes as eight or more distinct peaks when analyzed by chromatofocusing on pH 10.5-7 gradients [Keel et al., Biol. Reprod., 36 (1987) 1102]. In order to examine the efficacy of this approach to identify the distinct charge isomers of oLH, a pool of pituitary extracts was de-salted by flow dialysis and chromatofocused on a pH 10.5-7 gradient. The immunoreactive oLH eluted in nine distinct peaks which were coded with letters beginning with the most basic form. The fractions corresponding to each peak were pooled, dialyzed and lyophilized. Each peak was then re-chromatofocused on a pH 10.5-7 gradient except for the immunoreactive oLH eluting in peak A' because of the small amount present in this peak. Each peak, except for F and H, also consisted of a small percentage of immunoreactive oLH associated with adjacent peaks. This was plausible because chromatofocusing does not generally yield baseline resolution of peaks. Peak H eluted in a broad manner and was contaminated with significant amounts of isohormones F, G and Z. In contrast, peaks B, E, F, G and Z almost completely eluted in the anticipated regions. Thus, it appears that analysis of oLH charge isomers by chromatofocusing yields minimal misclassification errors and that the misclassification errors observed are associated with molecular forms which comprise a relatively small percentage of the oLH in pituitary extracts.