Journal of Protein Chemistry, Vol. 18, No. 1, 1999 Chemical Cleavage of Bovine B-Lactoglobulin by BNPS- Skatole for Preparative Purposes: Comparative Study of Hydrolytic Procedures and Peptide Characterization Veronique Rahali 1 and Jacques Gueguen 1,2 Received July 6, 1998 A comparative study of various procedures for tryptophanyl peptide bond cleavage by BNPS- skatole [2-(2-nitrophenyl)-3-methyl-3-bromoindolenine] was carried out on native and on reduced and alkylated bovine B-lactoglobulin (BLG). The reaction yield and the composition of the derived products were studied in acetic acid, trifluoroacetic acid (TFA), and ethanol/TFA. For BNPS- skatole removal, extraction by water or ethyl ether was compared with dialysis and gel filtration. The three expected peptides (1-19, 20-61, 62-162) and incomplete cleaved fragments (1-61, 20- 162) were separated and characterized by electrophoresis, reverse-phase high-performance liquid chromatography, and mass spectrometry. The highest hydrolysis yield (67.4%) occurred with native BLG cleaved in 88% acetic acid at 47DC for 60 min. Subsequent water extraction and gel filtration led to total recovery of the material, but reagent elimination was only quantitative after gel filtra- tion. Cleavage specificity was ensured by mass spectrometry and the amino acid composition of peptides 1-19 and 62-162. The chemical side reactions identified are discussed. KEY WORDS: B-Lactoglobulin; BNPS-skatole; chemical cleavage; peptide characterization. 1. INTRODUCTION Some enzymatic studies have dealt with the influence of various protein domains on enzyme activities. Specific cleavage methods have generally been developed to gen- erate partially split enzymes and study the role of the different protein regions on interactions with substrates (Yamashita et al., 1986). Similar approaches have pro- duced specific polypeptides and allowed basic studies of the relationships between their structure and biological or technological functionality as well as consideration of their therapeutic interest (Feeney, 1986). For example, some peptides derived by hydrolysis from wheat or milk proteins have proved to be very efficient emulsifiers (Chobert et al., 1989; Touati et al., 1990; Popineau et 1 Laboratoire de Biochimie et Technologie des Proteines, Institut Na- tional de la Recherche Agronomique, BP71627 44316 Nantes Cedex 03, France. 2 To whom correspondence should be addressed. al, 1990; Turgeon et al., 1992; Thoumy, 1995). How- ever, for basic approaches, enzymatic cleavage proce- dures generally generate too many peptides, which are then rather difficult to purify. Conversely, chemical cleavage methods at methionyl, tyrosyl, cysteyl, and tryptophanyl peptide bonds are more specific (Witkop, 1961; Han et al., 1983; Lundblad and Noyes, 1984; Fon- tana and Gross, 1986), but also more difficult to apply, particularly for preparative purposes. The low amount of tryptophan (Trp) residues in proteins is of particular in- terest since it allows limited hydrolysis and the release of large fragments. BNPS-skatole [2-(2-nitrophenylsul- fenyl)-3-methyl-3-bromoindolenine] is the most widely used reagent for Trp-Xaa bond cleavage via oxidative halogenation (Fontana, 1972; Omenn et al., 1970; Mar- tenson et al., 1977; Houghten and Li, 1978a, b; Zeitler and Eulitz, 1978; Fontana et al., 1980; Hunziker et al., 1980; Fontana and Gross, 1986; Mahboub et al., 1986; Crimmins et al., 1990; Vestling et al., 1994). However, 0277-8033/99/0100-0001$16.00/0 © 1999 Plenum Publishing Corporation 1