Biochimica et Biophysica Acta, 998 (1989) 21-24 Elsevier 21 BBAPRO 33432 Unusual far-ultraviolet circuIar dichroism of wheat germ agglutinin and hevein originated from cystine residues Adela Rodriguez-Romero ~, Barbarin Arreguin 2 and AndrOs Hernhndez-Arana ! Depart~'nento de Quimica, Universidad Aut[moma Metropolitana-htapalapa and z Instituto de Qulmica, Unioersidad Nacional Autbnoma de Mdxico, M~xico D.F. (M~xico) (Received 21 February 1989) (Revised manuscript received 15 May 1989) Key words: Hevein; Wheat germ agglutinin; Circular dichroism; Disulfide bridge The conformation of wheat germ agglutinin and hevein was studied by means of circular dichroism. The spectra of these proteins were remarkably similar below 250 nm, indicating the presence of common structural characteristics in their polypeptide chains. Besides, both spectra displayed a positive extremum around 221-223 nm which is rather unusual among globular proteins, this dichroic signal was only slightly modified in the presence of 6 M guanidine hydrochloride, but it was completely cancelled in 0.05 M dithiothreitol. Thus, it seems that in both proteins disulfide bonds are responsible for this singular circular dichroism band. Kinetic studies of disulfide-bridge reduction showed different reactivities of these groups for herein and wheat germ agglutinin. Introduction The large number of protein structures determined by X-ray diffraction has revealed the existence of differ- ent folding patterns for the polypeptide chain [1]. One of these patterns, the so-,called ' toxin-agglutinin fold' [2] is represented by the beckbone structures of the four isostructural domains (43 amino acid residues each) of wheat germ agglutinin (WGA) [3,4], and the erabutox- ins [2,5]. The domain or molecular structures of these proteins are characterized by several randomly coiled loops which are held together by four disulfide bridges [21. On the basis of the alignment of half cystines, Drenth et al. [2] have proposed that ragweed pollen allergen Ra5 and hevein, a 43-residue protein found in rubber- tree latex, may posses folding patterns similar to that of WGA domains. Furthermore, Wright et al. [6] have stressed the high sequence homology (approx. 50%) that exists between hevein and each of the four domains of WGA. In this work we stu4;_ed the conformations in solu- tion of WGA and hevein, by means of circular dichro- Abbreviation: WGA, wheat germ agglutinin. Correspondence: A. Rodrlguez-Romero, Departamento de Quimica, Universidad Autbnoma Metropolitana-lztapalapa, Apartado Postal 55-5340, Iztapalapa, D. F., 0934, M6xico. ism (CD). In the far-ultraviolet region, these proteins showed very similar spectra whose salient feature was an intense, positive band centered around 221-223 nm. This band, which is certainly unusual among globular proteins, was not affected by addition of guanidine (6 M); however, when the proteins were incubated in 0.05 M dithiothreitol, the positive band was completely abolished. These observations suggest that the CD spec- tra of WGA and hevein are dominated to a large extent by contributions from cystine residues of the molecule. Materials and Methods Materials Hevein was purified from rubber-tree (Hevea brasili- ensis) latex by the procedure described previously [7]. Highly purified, salt-free wheat germ agt~lutinin (from Triticum vulgaris), grade I guanidine hydrochloride, and Dbdithiothreitol were purchased from Sigma Chemical Co. Protein solutions were prepared on a weight basis using samples dryed over silica gel in a vacuum desicca- tor. For CD measurements of native proteins, samples were dissolved in 0.022 M Na2HPO4/0.028 M NaH2PO 4 buffer (pH 7.0). To test the effect ~'~f guani- dine hydrochloride and ditlfiothreitol, protein solutions were prepared in 0.05 M phosphate buffer (pH 7.0) that contained either 6 M guanidine hydrochloride or 0.05 M dithiothreitol; after incubation for 12 h at 25 *C, the 0167-4838/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)