Polyphenols are largely responsible for the astringency and "mouthfeel" of tea and wine by their interactions with basic salivary proline-rich proteins. Astringency arises from precipitation of polyphenol/peptide complexes, which is an important protective mechanism in animals that consume polyphenols. This paper presents biophysical studies of the interactions between chemically defined polyphenols and peptides. It is shown that intermolecular binding is dominated by stacking of polyphenolic rings onto planar hydrophobic surfaces and is strengthened by multiple cooperative binding of polyphenolic rings. Affinities weaken at higher temperatures and are unaffected by pH between pH 3.8 and 6.0. Measurements of self-diffusion rates for peptides with increasing concentrations of polyphenol demonstrate that peptides become increasingly coated with polyphenol. When the coating is sufficiently extensive to provide cooperative polyphenol bridges, the peptide dimerizes and precipitates. Light scattering measurements and electron microscopy indicate that the insoluble particles fall into two discrete size classes of ca. 80 and 500 nm diameter. The larger particles are favored at higher temperature and pH, suggesting that the particles are in a colloidal state, with the smaller particles being stabilized by charge repulsion between particles, and that precipitation of the complexes may be a phase separation process.

Spores of Bacillus anthracis, the causative agent of anthrax, possess an exosporium. As the outer surface layer of these mature spores, the exosporium represents the primary contact surface between the spore and environment/host and is a site of spore antigens. The exosporium was isolated from the endospores of the B. anthracis wild-type Ames strain, from a derivative of the Ames strain cured of plasmid pXO2 " , and from a previously isolated pXO1" doubly cured strain, B. anthracis UM23Cl2. The protein profiles of SDS-PAGE-separated exosporium extracts were similar for all three. This suggests that avirulent variants lacking either or both plasmids are realistic models for studying the exosporium from spores of B. anthracis. A number of loosely adsorbed proteins were identified from amino acid sequences determined by either nanospray-MS/MS or N-terminal sequencing. Salt and detergent washing of the exosporium fragments removed these and revealed proteins that are likely to represent structural/integral exosporium proteins. Seven proteins were identified in washed exosporium: alanine racemase, inosine hydrolase, ExsF, CotY, ExsY, CotB and a novel protein, named ExsK. CotY, ExsY and CotB are homologues of Bacillus subtilis outer spore coat proteins, but ExsF and ExsK are specific to B. anthracis and other members of the Bacillus cereus group.

The exosporium is the outermost layer of spores of Bacillus cereus and its close relatives Bacillus anthracis and Bacillus thuringiensis. For these pathogens, it represents the surface layer that makes initial contact with the host. To date, only the BclA glycoprotein has been described as a component of the exosporium; this paper defines 10 more tightly associated proteins from the exosporium of B. cereus ATCC 10876, identified by N-terminal sequencing of proteins from purified, washed exosporium. Likely coding sequences were identified from the incomplete genome sequence of B. anthracis or B. cereus ATCC 14579, and the precise corresponding sequence from B. cereus ATCC 10876 was defined by PCR and sequencing. Eight genes encode likely structural components (exsB, exsC, exsD, exsE, exsF, exsG, exsJ, and cotE). Several proteins of the exosporium are related to morphogenetic and outer spore coat proteins of B. subtilis, but most do not have homologues in B. subtilis. ExsE is processed from a larger precursor, and the CotE homologue appears to have been C-terminally truncated. ExsJ contains a domain of GXX collagen-like repeats, like the BclA exosporium protein of B. anthracis. Although most of the exosporium genes are scattered on the genome, bclA and exsF are clustered in a region flanking the rhamnose biosynthesis operon; rhamnose is part of the sugar moiety of spore glycoproteins. Two enzymes, alanine racemase and nucleoside hydrolase, are tightly adsorbed to the exosporium layer; they could metabolize small molecule germinants and may reduce the sensitivity of spores to these, limiting premature germination.Spores of the Bacillus cereus family, which includes Bacillus anthracis and Bacillus thuringiensis, all possess a loose balloonlike exosporium (7). A similar layer is also found on spores of some other bacilli and clostridia. The particular adherence and hydrophobic properties conferred by the exosporium (4, 14) suggest that it may possibly be of significance to spore pathogenicity. Bacillus subtilis, the paradigm of sporeformers, has no such clearly defined exosporial layer, so the exosporium has not been studied in molecular detail. Scanning electron microscopy has revealed a paracrystalline basal layer, with hexagonal periodicity, and a hairlike outer layer (3, 11). There are also pilus-like structures on the surface (15). The exosporium contains protein, lipid, and carbohydrate (43 to 52, 15 to 18, and 23% of dry weight, respectively [3,19]). A spore glycoprotein of B. thuringiensis was purified and partially characterized (10); it was present as two forms according to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)-a 70-kDa monomer and a 205-kDa multimer. Another glycoprotein, BclA, important to the surface hairlike layer, has recently been identified in the B. anthracis exosporium (22). Crude exosporium extracts of B. cereus (5) contain at least 12 major and some minor protein components that can be readily solubilized, including GroEL and a zinc metalloprotease called immune inh...

The complete amino acid sequence of hen ovalbumin, comprising 385 residues, has been determined. The sequence was deduced from the 17 cyanogen bromide fragments and from peptides derived by digestion with a number of proteolytic enzymes. The molecular weight of the polypeptide chain of ovalbumin is 42699.Ovalbumin has four sites of postsynthetic modification ; in addition to the acetylated N terminus, the carbohydrate moiety is located at Asn-292, and the two phosphorylated serines are at residues 68 and 344. The 'signal sequence' of ovalbumin is between residues 234 and 252. The heptapeptide released during the conversion of ovalbumin to plakalbumin by subtilisin digestion corresponds to residues 346 -352. The hen ovalbumin polymorphism characterised by an Asn-tAsp replacement results from a mutation at residue 31 1.The amino acid sequence of ovalbumin deduced from these amino acid sequence studies is in complete agreement with the sequence of mRNA determined by McReynolds et al. [Nature (Lond.) 273, 723 -728 (1978)].Ovalbumin is the most abundant protein in egg white, and is readily purified and crystallised in gram quantities [l].