High enrichment of MMP-9 and carboxypeptidase A by tweezing adsorptive bubble separation (TABS).

Tweezing adsorptive bubble separation (TABS) was used as a method for the enrichment of matrix metalloproteinases (92-kDa type IV, gelatinase B (MMP-9)) and carboxypeptidase A (CPA) from dilute aqueous solutions. The method is based on the chelation of metalloenzymes applying 2-(carbamoylmethyl-(carboxymethyl)amino)acetic acid (ADA) coupled with an octyl part to form a surface active unit. MMP-9 could be enriched with an enrichment ratio of 12.0 and a recovery of 87.3%, and CPA could be enriched 18.8-fold and with 95.3% recovery. Both enzymes were enriched without significant losses of enzymatic activity. To verify that the enzymes were tweezed by ADA-C8 without abstraction of the zinc ions from the active center, TABS trials were additionally conducted with zinc ions in complex with ADA-C8, which revealed only negligible enrichment ratios of the enzymes (2.2 for MMP-9 and 0.2 for CPA). The results obtained impressively demonstrate that zinc-containing proteases can be enriched selectively and efficiently by TABS.

Efficient enrichment of alpha,beta-unsaturated bovine insulins-(C12)n using tweezing adsorptive bubble separation (TABS) with bovine serum albumin.

Alkyl-substituted insulin molecules are depository preparations used in the treatment of diabetes. The isolation and purification of these active compounds is performed on the hand of several complicated separation steps. In this work, nonmodified and new synthesized alpha,beta-unsaturated bovine insulins-(C12)(n) were selectively isolated from aqueous solution using tweezing adsorptive bubble separation (TABS) with BSA, which enabled reversible binding with the unpolar side chains of bovine insulin and its derivates. The bovine insulin/serum albumin complex could be efficiently transferred into the foam phase at pH 8 with an enrichment factor of 2.6 and a recovery of 89.7%. Experiments with alkylated alpha,beta-unsaturated bovine insulins-(C12)(n) performed at pH 9 enriched even quantitatively the complex under equal experimental conditions, accompanied with an enrichment factor of 3.3. Additionally performed radio immunoassay tests revealed that the immunological activity of alpha,beta-unsaturated bovine insulins-(C12)(n) remained unchanged after foaming.

Permissible level of toxaphene residues in fish from the German market based on in vivo and in vitro effects to tumor promotion.

Toxaphene is a chlorinated pesticide consisting of more than 200 congeners that are mainly chlorobornanes and chlorocamphenes. As the congeners exhibit different stability properties in the environment, only between 20 and 30 compounds can be observed in, e.g., fish, which are represented by technical toxaphene as a mixture. In human body, the congeners Parlar #26, #40, #41, #44, #50, and #62 are detected frequently. Three of them, #26, #50, and #62, pose a potential risk to human health due to their persistent characteristic. By using experimental results of a European Union study (MATT, 2000. Investigation into the Monitoring, Analysis and Toxicity of Toxaphene in Marine Foodstuffs, European Union, Brussels, Final report, FAIR CT PL.96.3131. Investigation into the Monitoring, Analysis and Toxicity of Toxaphene in Marine Foodstuffs), a reference dose related to tumor promotion was calculated for these representative persistent toxaphene congeners. In Germany, the sum of the congeners #26, #50, and #62 is defined as the official standard for toxaphene residues in food. In this work, different fish samples obtained from German markets were studied regarding their contamination with toxaphene congeners, presented either in sum, or as single constitutes. The obtained data were used to define the acceptable total concentration of the sum of Parlar #26, #50, and #62 with regard to prevention of tumor promotion in human. The results showed that the currently existing permissible level of the sum of these congeners (0.1 mg/kg) is higher than the acceptable concentration in fish samples determined by this work and calculated at ca. 0.090 mg/kg. It is therefore recommended to improve the permissible level of toxaphene in German food samples.

A proteome analysis of Corynebacterium glutamicum after exposure to the herbicide 2,4-dichlorophenoxy acetic acid (2,4-D).

The herbicide 2,4-dichlorophenoxy acetic acid (2,4-D) induces a wide spectrum of toxic responses in living organisms. In this study, we analyzed the stress-induced responses of Corynebacterium glutamicum cells on protein level upon treatment with 2,4-D. For this, growing C. glutamicum cells were exposed to sublethal concentrations of 2,4-D, and changes of the gene expression profiles in comparison to non-exposed organisms were analyzed by two-dimensional gel electrophoresis and mass spectrometry. 2,4-D induced the over-expression of at least six C. glutamicum proteins, four of which could be identified by MALDI-TOF-MS. One protein (Cg2521; long-chain acyl-CoA synthetase) was related to the energy metabolism, and two proteins were involved in cell envelope synthesis (Cg2410; glutamine-dependent amidotransferase, and Cg1672; glycosyltransferase). The last induced protein was the ABC type transport system (Cg2695, ATPase component). The newly observed proteins, except for the ABC transport system, were not in general stress-related proteins, but were specifically expressed upon 2,4-D exposure and, therefore, can be used as respective biomarkers. Moreover, since these proteins seem to play a pivotal role in the adaptation of the cell to 2,4-D, they may help to gain deeper insight into the damage mechanisms of 2,4-D induced in the living cell.