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JBC, Vol. 251, Issue 20, 6388-6391, Oct, 1976
J. G. Pierce, L. C. Giudice and J. R. Reeve
The five disulfide bonds of isolated alpha subunits of luteinizing hormone
(LH) and thyroid-stimulating hormone (TSH) are completely reduced at pH 8.5
in 15 min with no denaturant required and with only a slight excess of
reducing agent. At pH 7.0, reduction is complete after 6 to 10 h. These
results together with an earlier study concerning the positions of the two
most readily reduced bonds (Cornell J.S., and Pierce, J.G. (1974) J. Biol.
Chem. 249, 4166-4174) show that, in the isolated alpha subunit, all
disulfides are readily accessible, although it is possible that a change in
conformation, after rapid initial reduction of two disulfides, makes the
remaining three more susceptible to reduction. No partially reduced and
S-carboxymethylated intermediates were found at pH 7.0 other than those
seen at pH 8.5, nor were additional intermediates found at pH 8.5 when
reduction was initiated in the presence of alkylating agent. In contrast,
reduction of the beta, hormone-specific, subunits of LH and TSH, while
complete at pH 8.5 after 2 to 6 h, does not proceed to completion at pH 7.0
even after 24 h or upon addition of 6 M urea or large concentrations of
reducing agent, and partially reduced intermediates useful in location of
disulfide bridges can be trapped (e.g. Reeve, J.R., Cheng, K.-W., and
Pierce, J.G. (1975) Biochem. Biophys. Res. Commun. 67, 149-155). Little or
no reduction of the intact hormones is found at pH 7.0 in the absence of
denaturing agents. This protection by the intact structure shows that the
two most readily reduced disulfides of the alpha subunit and the single
most readily reduced sidulfide of the beta subunits are either in regions
of subunit-subunit contact or that these bonds become more reactive in the
isolated subunits because of different influences by neighboring groups. At
pH 8.5, intact LH is completely reduced after 6 h, but intact TSH is more
resistant to reduction, which may reflect a higher affinity between
subunits than exists in LH.
Studies on the disulfide bonds of glycoprotein hormones. Course of reduction of bovine luteinizing hormone, bovine thyroid-stimulating hormone, and their subunits
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