ELSEVIER Biochimica et Biophysica Acta 1252 (1995) 43-52 Biochl"PmPic~a et Biophysica A~ta Spectroscopic characterization of the alkylated a-sarcin cytotoxin: analysis of tlhe structural requirements for the protein-lipid bilayer hydrophobic interaction Maria Gasset, Jos6 M. Manchefio, Javier Lacadena, Alvaro Martinez del Pozo, Mercedes Ofiaderra, Jos~ G. Gavilanes * Departmento de Bioqufmica y Biologfa Molecular, Facultad de Qufmica, Universidad Complutense, 28040 Madrid, Spain Received 24 October 1994; revised 21 April 1995; accepted 9 May 1995 Abstract a-Sarcin is a ribosome-inactivating protein that translocates across lipid bilayers, these two abilities explaining its cytotoxic character. This protein is composed of a single polypeptide chain with two disulfide bridges. Reduction and carboxyamidomethylation of a-sarcin results in protein unfolding, based on the results of the spectroscopic characterization of the chemically modified protein. The absorption and fluorescence emission bands of the tryptophan residues of the modified protein appear blue- and red-shifted, respectively. Far-UV circular dichroism analysis reveals the presence of residual secondary structure (/~-strands and turns) in the alkylated protein. This retains its ability to interact with lipid bilayers. It promotes vesicle aggregation, lipid-mixing between bilayers and leakage of the intravesicular aqueous contents. The modified protein tends to abolish the phase transition of acid phospholipids as detected by differential scanning calorimetry and depolarization measurements of fluorescence-labelled vesicles. The protein gain access to vesicle-entrapped trypsin. The fluorescence emission of the tryptophan residues is blue-shifted upon interaction of the protein with the bilayers, and anthracene incorporated into the hydrophobic core of the membranes quenches the tryptophan fluorescence emission of the protein. The secondary structure of the alkylated protein interacting with lipid vesicles has been studied by infrared spectroscopy. An increase in the a-helix and turn contents and a concomilant decrease in the /3-structure content are observed upon interaction with the bilayers. The results obtained are discussed in terms of the structural requirements for the interaction of c~-sarcin with lipid membranes. Keywords: Antitumor protein; Cytotoxin; Protein unfolding; Spectroscopy of protein; Protein-lipid interaction; ot-Sarcin 1. Introduction a-Sarcin is the best known member of a family of proteins produced by several Aspergillus strains, that ex- hibit more than 90% amino-acid sequence similarity [1 ]. It was early described as a potent antitumour agent against several tumours induced in mice [2,3]. Recent studies have Abbreviations: ANTS, 8-aminonaphthalene-l,3,6-trisulfonic acid; CD, circular dichroism; DMPC, dimyristoylphosphatidylcholine; DMPG, dimyristoylphosphatidylglycerol; DMPS, dimyristoylphosphatidylserine; DPH, 1,6-diphenyl-l,3,5-hexatriene; DPX, p-xylene bis(pyridinium bro- mide); DSC, differential scanning calorimetry; FTIR, Fourier transform infrared; NBD-PE, N-(7-nitro-2-1,3-benzoxadiazol-4-yl)di- myristoylphosphatidylethanolamine; PG, phosphatidylglycerol; RET, res- onance energy transfer; Rh-PE, N-(lissamine rhodamine B sulfo- nyl)diacylphosphatidylethanolamine; o~SRC, reduced and car- boxyamidomethylated o~-sarcin. * Corresponding author. Fax: +34 1 3944159. 0167-4838/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 01 67 -4838(95)001 06-9 revealed that a-sarcin is cytotoxic for many human tumour cell lines [4], and it is being studied as anfitumour agent under the form of either free protein or conjugated to monocional antibodies (immunotoxin) [5]. This cytotoxin displays a very specific ribonuclease activity that inacti- vates the ribosomes (see [6] for a review) and inhibits the cellular protein biosynthesis. Therefore, the cytotoxicity of a-sarcin would involve two general steps, i.e., entering the cell membrane and inactivation of the ribosomes. No protein membrane receptors have been so far described for a-sarcin. However, this protein strongly interacts with phospholipid vesicles promoting their aggregation and fu- sion [7-9]. The protein translocates across the membrane of asolectin vesicles in the absence of any permeabilizing agent [10], in a way which may be related to its ability in passing through cell membranes. The interaction of c~-sarcin with lipid vesicles shows an electrostatic component. This may be explained by the net