Proteins from pliable cuticle of locusts, Schistocerca gregaria, and silk m
oth larvae, Hyalophora cecropia, were studied in solution by means of a flu
orescent probe, 8-anilinonaphthalene-1-sulphonic acid (ANS), which is much
more fluorescent in nonpolar media than in polar media. An intense ANS-fluo
rescence was observed in the presence of the cuticular proteins at pH-value
s close to their acidic isoelectric points, and the fluorescence decreased
markedly when pH was increased to neutrality or when small amounts of denat
urants were added. Aggregation and eventual precipitation of both H. cecrop
ia and locust proteins were obtained by addition of neutral salts, and the
aggregation was accompanied by an increased ANS-fluorescence intensity. A d
ecreased ANS-fluorescence was observed at salt concentrations too low to ca
use visible aggregation of the H. cecropia proteins, probably due to weaken
ed electrostatic interactions between chain segments, but such a decrease w
as not observed for the locust proteins. The changes in intensity of ANS-fl
uorescence induced by addition of small amounts of denaturants or salts to
solutions of the proteins indicate that more hydrophobic residues are expos
ed to the solvent, when either hydrophobic interactions or electrostatic at
tractions between chain segments are weakened. The result is a less compact
protein structure, where fewer and smaller hydrophobic clusters are availa
ble for protecting ANS-molecules from the quenching effects of water. The e
ffects of denaturants on ANS-fluorescence in the presence of the cuticular
proteins are different from those observed for globular proteins, such as h
en egg albumen, and the differences can be explained by the suggestion that
the cuticular proteins do not have a precisely folded and densely packed h
ydrophobic core comparable to that present in native globular proteins, and
that accordingly they do not undergo a process of denaturation correspondi
ng to that of globular proteins. The behaviour of the cuticular proteins re
sembles that decribed for unordered, randomly coiled, thermally agitated po
lymer chains, whose hydrodynamic volumes depend upon the composition of the
medium. It is proposed that the major part of the peptide chains of the cu
ticular proteins are in an unordered, random structure both when the protei
ns are in solution and when present in the intact cuticle; probably only th
e chain regions involved in binding the proteins to chitin will have a well
-defined spatial organisation. (C) 2001 Elsevier Science Ltd. All rights re
served.