308 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS 2,500 2,000 1,500 1,000 500 X. =370 nm ext HMSC OCTANOL WATER o 400 42o 440 460 480 500 52o 540 Wavelength (nm) Figure 3. Emission spectrum of ANS from isolated human stratum corneum. For comparison the spectra in water and octanol are also included. The excitation wavelength was 370 nm. Note the increase in fluorescence intensity and the shift in •'m•x relative to water. emission at 463 nm) showed a maximum at around 365 nm, similar to the absorption maximum for protein-bound ANS. The blue shift and increased fluorescence intensity suggest that the probe is in a hydrophobic environment similar to octanol. The similarity of ANS emission from corneum with the spectra from other ANS-protein complexes (12) indicates that ANS is bound to proteins in the corneum. ANS binding to proteins is also evidenced by its quenching of the intrinsic protein fluorescence from the.corneum. The fluorescence emissions from an untreated and an ANS-treated human corneum at an excitation wavelength of 295 nm are shown in Figure 4. The fluorescence emission from an untreated corneum at an excitation wavelength longer than 290 nm is primarily from tryptophan, the aromatic acid residue in proteins. Soaking the cor- neum for an hour in a dilute aqueous solution (10 -4 moles/l) of ANS reduced the protein emission by about 60%. At the same time, there was a strong emission (kma x = 465 rim) from the ANS molecules bound to the corneum. Similar short-range quenching of tryptophan emission by ANS bound to it has been observed in other studies of protein binding (11,13, 18). REMOVAL OF ANS FROM CORNEUM BY WATER Soaking the ANS-treated corneum in water for one minute reduced the ANS emission

SURFACTANT-SKIN INTERACTIONS 309 300 25{) 15{) • 100 k =295 nm ext Tryptophan Emission ANS treated ß ß ß ANS Emission 350 400 450 5{)0 Wavelength (nm) Figure 4. Emission spectrum from untreated and ANS-treated isolated human stratum corneum. Excita- tion wavelength = 295 nm. (at }kma x : 463 nm) by 24% (Figure 5A). However, the tryptophan emission changed very little after water treatment. The results suggest that the fraction of ANS that was weakly bound to corneum and located at some distance away from the tryptophan moleties was displaced by a rapid rinse in water. ANS displacement increased to 34% when the corneum was rinsed with a soak solution of pH 9.5 (Figure 5B). The relatively small effect of pH on ANS displacement suggests that the binding strength of ANS to corneum proteins is sufficiently large to overcome the electrostatic effects of a change in the state of ionization of the amino acid residues resulting from a change in pH. Increasing the soaking time to one hour, on the other hand, significantly increased the ANS displacement (Figure 6). This is possibly due to the continued equilibration as well as to soaking-induced structural changes in corneum proteins. INTERACTION OF STRATUM CORNEUM WITH PURE ANIONIC SURFACTANTS In order to further understand the role of actives in influencing their harshness toward skin, spectroscopic as well as direct binding sites with sodium lauroyl isethionate (SLI), the primary anionic surfactant in Bar A, and a representative soap surfactant, sodium laurate (SL), were conducted. In these experiments triethanolamine (TEA) was used to solubilize the soaps in the aqueous phase. TEA was used at a concentration of 0.04 moles/1. The binding data for the two surfactants were also compared to that of sodium lauryl sulfate (SLS), a known harsh anionic surfactant.