Studies on a growth-inhibitory peptide derived from alpha-fetoprotein and some analogs

A 34-amino acid synthetic peptide was derived from the third domain of huma n alpha-fetoprotein, and the peptide was shown to inhibit estrogen-stimulat ed growth. Under certain conditions, however, the peptide lost growth-inhib itory activity. A biophysical study of the peptide was undertaken with a go al of obtaining completely reliable preparations. The peptide was studied u sing gel-filtration column chromatography as a function of peptide concentr ation and age of solution, and was found to exhibit complex aggregation beh aviors. During the early period (0-3 h) after dissolving lyophilized peptid e into pH 7.4 buffer, solutions were composed mostly of trimers. At higher peptide concentrations (greater than or equal to3.0 g/L), the trimers aggre gated extensively to a large aggregate (minimum size approximate to 102 pep tides). At 5.0-8.0 g/L, these large aggregates increased in size (up to app roximate to 146 peptides) until trimers were largely exhausted from solutio n. During the later times (>3 h) after sample preparation, the trimeric oli gomer of the peptide dissociated slowly to form dimers for samples at 0.10- 3.0 g/L. After their build-up, a very small number of dimers associated to form hexamers. Disulfide bonds stabilized the dimers as indicated by the co nversion of dimers to trimers upon the addition of a reducing agent, and th e failure of dimers to form in the presence of reducing agent. Reducing age nt did not affect trimer or large aggregate formation. Trimers were found t o be active in an assay monitoring inhibition of estrogen-stimulated growth , whereas dimers and large aggregates were inactive. The two cysteines in t he peptide were modified to either S-methylcysteine or S-(2-aminoethyl)cyst eine, and both derivatives showed significant growth-inhibition activity. A serine analog in which both cysteines were replaced had very different agg regation behavior than the cysteine peptide and lacked its growth inhibitor y ability. Peptide aggregation is critically important in establishing the ability of the peptide to inhibit growth and have anticancer activity, but the state of its two cysteines is of little influence.