AN ELECTRON-MICROSCOPE AND ELECTRON-DIFFRACTION STUDY OF THE EFFECT OF CALCOFLUOR AND CONGO RED ON THE BIOSYNTHESIS OF CHITIN IN-VITRO

The structure of chitin made in vitro by chitin synthetase was studied by electron microscopy and electron diffraction. Two different forms of chitin synthetase from the fungus Mucor rouxii were tested: chitoso mes and 16 S particles. The long chitin fibrils produced by chitosomes had a high degree of crystallinity as revealed by electron diffractio n. Specimen regions made of largely parallel microfibril bundles produ ced distinct fiber diagrams, an indication that the chitin chains were aligned along the fibril axis. Microdiffractometry of the shorter chi tin crystals synthesized by 16 S particles also showed a similar align ment of chitin chains. Calcofluor and Congo red were powerful inhibito rs of chitin synthetase and had profound effects on the color, macrosc opic texture, electron microscopic morphology, and crystal structure o f the biosynthesized chitin. Chitin made in the presence of Congo red had a bright red color; the one made in the presence of Calcofluor was strongly fluorescent and had a distinctly blue hue when illuminated b y daylight. The dyes were tightly bound to the chitin and could not be removed by washing with water or ethanol. At low dye concentration, a mixture of two kinds of crystals was produced by 16 S particles: some were of the same dimensions as those made in the absence of dyes, but others were much thinner. At high dye concentration, there were only thin crystals. With increasing concentrations of Calcofluor or Congo r ed, the typical electron diffraction reflections of alpha-chitin, part icularly the strong equatorial band at 0.466 nm became fainter and a n ew additional reflection centered at 0.40 nm arose as the dominant fea ture of the patterns. We regard the gel-like material, synthesized at highly inhibitory dye concentrations, as an apposition complex where t he dye does not form part of the crystal structure of chitin and the b ulk of the complex consists of molecular stacks of dye associated with nascent chitin chains or narrow chitin microfibrils. (C) 1994 Academi c Press, Inc.