The antioxidant activity of caseins and casein-derived peptides was evaluated by using three free radical producing reactions-the lipoxygenase- and AAPH-catalyzed oxidation of linoleic acid and the hemoglobin-catalyzed oxidation of linoleic acid hydroperoxide. Caseins and casein-derived peptides were able to inhibit enzymatic and nonenzymatic lipid peroxidation, suggesting they were preferred targets for the free radical intermediates. The antioxidative feature was not lost with the dephosphorylation or the proteolysis of the proteins. The fractionation of the tryptic beta-casein digest yielded peptides with antioxidant activity. A structure-function relationship between the amino acid sequence and the antioxidant capacity and effectiveness is proposed. In addition, indirect evidence suggested that the trapping of free radicals by the proteins/peptides was accompanied by the oxidation of proteins/peptides, according to a sequence-specific mechanism.

Whole casein from bovine origin, the different casein subtypes alpha, beta, and kappa, and the related dephosphorylated proteins were assayed as modulators of soybean lipoxygenase 1 activity and were found to inhibit it. To define the lipoxygenase inhibitory domain, whole casein and beta-casein were digested by proteases (trypsin, clostripain, and subtilisin). The beta-casein tryptic digest and the tryptic and subtilisin digests of whole casein retained their inhibitory properties. The tryptic beta-casein digest was the most potent inhibitor of lipoxygenase activity and was further fractionated by FPLC or HPLC. The collected peptides inhibited the lipoxygenase-catalyzed reaction to different extents. The active fractions were analyzed by ESI-MS, and the sequences of several lipoxygenase inhibitory peptides, corresponding mainly to the C-terminal moiety of beta-casein, were identified.

CENTA, a chromogenic cephalosporin, is readily hydrolyzed by ␤-lactamases of all classes except for the Aeromonas hydrophila metalloenzyme. Although it cannot practically be used for the detection of ␤-lactamaseproducing strains on agar plates, it should be quite useful for kinetic studies and the detection of the enzymes in crude extracts and chromatographic fractions.Nitrocefin and, to a lesser extent, PADAC have been used as chromogenic substrates of ␤-lactamases. Such substrates, whose hydrolysis can be directly monitored in the visible wavelength range, are of particular interest for the kinetic characterization of ␤-lactamases. Nitrocefin has, for instance, been widely used as a reporter substrate in the study of the inactivation of ␤-lactamases or of their interactions with poor substrates (3). It also allows the rapid identification of active fractions during ␤-lactamase purification. However, the price of nitrocefin has recently been increased significantly, and PA-DAC is no longer commercially available. Synthesis of compounds is also rather tedious. It is thus surprising that a third chromogenic cephalosporin, CENTA ( Fig. 1), which can be prepared from the commercially available drug cephalothin, has not received more attention, although it was shown to be sensitive to many ␤-lactamases (11). In the study described in this report, we determined the kinetic parameters characterizing the interactions between CENTA and a representative set of ␤-lactamases and some penicillin-binding proteins (PBPs). Although CENTA cannot be used for the direct detection of ␤-lactamase-producing colonies on agar plates, it still represents an interesting alternative to nitrocefin for the kinetic characterization of ␤-lactamases.CENTA was prepared as follows: 3-carboxyl-4-nitrothiophenol (TNB) was obtained by dissolving 5.05 mmol (2 g) of 5,5Ј-dithio-bis-(2-nitrobenzoic acid) in 100 ml of an aqueous solution of 0.5 M Tris base, and the pH was adjusted to 8.0 by addition of 6 M HCl. Dithiothreitol (7.1 mmol, 1.1 g) was added, and the solution turned orange-red. The mixture was stirred for 10 min at 22°C and extracted six times with 25 ml of ethyl acetate before being acidified to pH 1.5 by addition of 6 M HCl. The residual ethyl acetate was eliminated by bubbling nitrogen through the solution, which was thereafter left overnight at 4°C. The precipitate (TNB) was collected by filtration, washed, and dried. The sodium salt of cephalothin (1 g, 2.4 mmol) and 1 equivalent of TNB (478 mg) were dissolved in 19 ml of H 2 O, and the pH was adjusted to 7.0 with 1 M NaOH. The solution was stirred for 6 h at 65°C. The cooled solution was extracted with 10 ml of ethyl acetate, acidified to pH 2.0 with 1 M HCl, and extracted three times with 15 ml of ethyl acetate. The organic phase was washed three times with 15 ml of water, dried over MgSO 4 , and evaporated to dryness in vacuo. The sodium salt of CENTA was obtained by dissolving the dry residue in 25 ml of water containing 1 equivalent of NaHCO 3 , and the solution was freeze-dried,...

We have investigated the influence of substrate binding on the zinc ion affinity of representatives from the three metallo-␤-lactamase subclasses, B1 (BcII from Bacillus cereus and BlaB from Chryseobacterium meningosepticum), B2 (CphA from Aeromonas hydrophila), and B3 (L1 from Stenotrophomonas maltophilia). By competition experiments with metal-free apoenzymes and chromophoric zinc chelators or EDTA, we determined the dissociation constants in the absence and presence of substrates. For the formation of the monozinc enzymes we determined constants of 1.8, 5.1, 0.007, and 2.6 nM in the absence and 13.6, 1.8, 1.2, and 5.7 pM in the presence of substrates for BcII, BlaB, CphA, and L1, respectively. A second zinc ion binds in the absence (presence) of substrates with considerably higher dissociation constants, namely 1.8 (0.8), 0.007 (0.025), 50 (1.9), and 0.006 (0.12) M for BcII, BlaB, CphA, and L1, respectively. We have concluded that the apo form might be the prevailing state of most of the metallo-␤-lactamases under physiological conditions in the absence of substrates. Substrate availability induces a spontaneous self-activation due to a drastic decrease of the dissociation constants, resulting in the formation of active mononuclear enzymes already at picomolar free zinc ion concentrations. In the presence of substrates, the binuclear state of the enzymes only exists at unphysiologic high zinc concentrations and might be of no biological relevance. From the competition experiments with EDTA it is further concluded that the reactivation rate does not depend on the pool of free zinc ions but proceeds via the EDTA-Zn(II)-enzyme ternary complexes.Metallo-␤-lactamases (class B ␤-lactamases) are produced by bacteria as extracellular or periplasmatic enzymes. All known representatives possess two conserved metal binding sites and require zinc ions as enzymatic cofactors. By catalyzing the hydrolysis of ␤-lactams they render the corresponding strains resistant to almost all ␤-lactam antibiotics. Their increasing emergence in pathogenic bacterial strains due to a rapid dissemination by horizontal gene transfer has induced a growing interest in this enzyme family because of the lack of efficient therapies to treat infected patients.The metallo-␤-lactamases constitute a group of heterogeneous proteins that is divided into subclasses B1, B2, and B3 (1). Subclass B1 exhibits a broad substrate profile (2), and its zinc binding sites are composed of His-116, His-118, and His-196 (site 1) and Asp-120, Cys-221, and His-263 (site 2) (numbering according to Ref. 3). In subclass B2 the zinc ligands in site 2 are conserved, whereas His-116 in site 1 is replaced by Asn. Representatives of subclass B2 efficiently hydrolyze only carbapenems (2) and are active as monozinc enzymes, whereas the binding of a second zinc ion causes non-competitive inhibition (4). In subclass B3, Cys-221 is substituted by a Ser and is replaced by His-121 as a zinc ligand in site 2. In the Gob-1 enzyme (Chryseobacterium meningosepticum PINT) an additional H...

Aprotinin is a small serine protease inhibitor used in human health. Spirodela were transformed, via Agrobacterium, with a synthetic gene encoding the mature aprotinin sequence and a signal peptide for secretion which was driven by the CaMV 35S promoter. A total of 25 transgenic Spirodela lines were generated and aprotinin production was confirmed by northern and western blot analyses. Expression levels of up to 3.7% of water soluble proteins were detected in the plant and 0.65 mg/l in the growth medium. In addition, immunoaffinity purification of the protein followed by amino acid sequencing confirmed the correct splicing of the aprotinin produced in Spirodela and secreted into the growth medium.