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The calcium-depleted form of ␣-lactalbumin (␣-LA) at neutral pH can be induced to adopt a partly folded state or molten globule upon moderate heating, by dissolving the protein in aqueous TFE or by adding oleic acid. This last folding variant of the protein, named HAMLET, can induce apoptosis in tumor cells. The aim of the present work was to unravel from circular dichroism (CD) measurements and proteolysis experiments structural features of the molten globule of apo-␣-LA at neutral pH. CD spectra revealed that the molten globule of apo-␣-LA can be obtained upon mild heating at 45°C, as well as at room temperature in the presence of 15% TFE or by adding to the protein solution 7.5 equivalents of oleic acid. Under these various conditions the far-and near-UV CD spectra of apo-␣-LA are essentially identical to those of the most studied molten globule of ␣-LA at pH 2.0 (A-state). Proteolysis of the 123-residue chain of apo-␣-LA by proteinase K at 4°C occurs slowly as an all-or-none process leading to small peptides only. At 37°C, proteinase K preferentially cleaves apo-␣-LA at peptide bonds Ser34-Gly35, Gln39-Ala40, Gln43-Asn44, Phe53-Gln54, and Asn56-Asn57. All these peptide bonds are located at level of the ␤-subdomain of the protein (chain region 34-57). Similar sites of preferential cleavage have been observed with the TFE-and oleic acid-induced molten globule of apo-␣-LA. A protein species given by the N-terminal fragment 1-34 linked via the four disulfide bridges to the C-terminal fragment 54-123 or 57-123 can be isolated from the proteolytic mixture. The results of this study indicate that the same molten globule state of apo-␣-LA can be obtained at neutral pH under mildly denaturing conditions, as indicated by using a classical spectroscopic technique such as CD and a simple biochemical approach as limited proteolysis. We conclude that the molten globule of ␣-LA maintains a native-like tertiary fold characterized by a rather well-structured ␣-domain and a disordered chain region encompassing the ␤-subdomain 34-57 of the protein. Proteins 2002;49:385-397.Abbreviations: ␣-LA, ␣-lactalbumin; A-state and apo-␣-LA, low pH and calcium-depleted form of ␣-LA, respectively; CD, circular dichroism; des␤-LA, protein species of ␣-LA with the ␤-subdomain deleted; DTT, dithiothreitol; EDTA, ethylenediaminetetraacetic acid; E/S, enzyme to substrate ratio; ESI, electrospray ionization; gapped or truncated ␣-LA, protein species with excision of a chain segment at the middle of 123-residue chain of the protein; HAMLET, human ␣-lactalbumin made lethal to tumor cells; HPLC, high-performance liquid chromatography; MALDI, matrix-assisted laser-desorption ionization; MG, molten globule; MS, mass spectrometry; nicked ␣-LA, disulfidecrosslinked protein species of ␣-LA with a single peptide bond cleaved;

The partly folded states of protein members of the lysozyme (LYS)/␣-lactalbumin (LA) superfamily have been analyzed by circular dichroism (CD) measurements and limited proteolysis experiments. Hen, horse, dog, and pigeon LYSs and bovine LA were used in the present study. These are related proteins of 123-to 129-amino-acid residues with similar three-dimensional structures but low similarity in amino acid sequences. Moreover, notable differences among them reside in their calcium-binding properties and capability to adopt partly folded states or molten globules in acid solution (A-state) or on depletion of calcium at neutral pH (apo-state). Far-and near-UV CD measurements revealed that although the structures of hen and dog LYS are rather stable in acid at pH 2.0 or at neutral pH in the absence of calcium, conformational transitions to various extents occur with all other LYS/LA proteins herewith investigated. The most significant perturbation of tertiary structure in acid was observed with bovine LA and LYS from horse milk and pigeon egg-white. Pepsin and proteinase K were used as proteolytic probes, because these proteases show broad substrate specificity, and therefore, their sites of proteolysis are dictated not by the specific amino acid sequence of the protein substrate but by its overall structure and dynamics. Although hen LYS at pH 2.0 was fully resistant to proteolysis by pepsin, the other members of the LYS/LA superfamily were cleaved at different rates at few sites of the polypeptide chain and thus producing rather large protein fragments. The apo-form of bovine LA, horse LYS, and pigeon LYS were attacked by proteinase K at pH 8.3, whereas dog and hen LYSs were resistant to proteolysis when reacted under identical experimental conditions. Briefly, it has been found that the proteolysis data correlate well with the extent of conformational transitions inferred from CD spectra and with existing structural informations regarding the proteins herewith investigated, mainly derived from NMR and hydrogen exchange measurements. The sites of initial proteolytic cleavages in the LYS variants occur at the level of the ␤-subdomain (approximately chain region 34-57), in analogy to those observed with bovine LA. Proteolysis data are in agreement with the current view that the molten globule of the LYS/LA proteins is characterized by a structured ␣-domain and a largely disrupted ␤-subdomain. Our results underscore the utility of the limited proteolysis approach for analyzing structure and dynamics of proteins, even if adopting an ensemble of dynamic states as in the molten globule.Keywords: Lactalbumin; lysozyme; circular dichroism; limited proteolysis; molten globule; protein folding Reprint requests to: Angelo Fontana, CRIBI Biotechnology Centre, University of Padua, Viale G. Colombo 3, I-35121 Padua, Italy; e-mail: fontana@civ.bio.unipd.it; fax: 39-049-8276159.Abbreviations: LA, bovine ␣-lactalbumin; LYS, lysozyme; A-state, lowpH form of LA; apo-LA and apo-LYS, calcium-depleted forms of LA and

The current analytical technology for the determination of New Psychoactive Substances in biological samples is still largely inadequate, because the immunoassays are unsuitable for the detection of most of these compounds and the use of traditional gas chromatography-mass spectrometry techniques is hampered by the lack of chromatographic standards and mass fragmentation patterns. Taking advantage of the molecular recognition capability of high-resolution mass spectrometry, the present work aimed to apply liquid chromatography-quadrupole-time of flight mass spectrometry for the rapid identification of New Psychoactive Substances in the hair, a peculiar tissue which "keeps memory" of the recent history of drug intake of the subject. All the samples were screened for the presence of 50 different New Psychoactive Substances (synthetic cannabinoids, cathinones and phenethylamines), substances that had been reported officially by the National Early Warning System in the period 2009-2011. Among the 435 samples analyzed, 8 were found "positive" for the following compounds: JWH-018, JWH-073, JWH-081, JWH-250, JWH-122, in a broad range of concentrations (0.010-1.28 ng/mg). Results strongly support the use of hair analysis to monitor the diffusion of new psychoactive drugs in the community.