Abnormal behaviour of proline in the isothiocyanate degradation.

It has been observed that proline residues often initiate overlaps during sequenator analysis. The cause has been shown to be an abnormally slow cleavage reaction. The kinetics of the cleavage reaction has been studied and found to obey pseudo-first-order kinetics. There are considerable differences in reaction rates depending on the position of proline in the sequence, as demonstrated for the four prolines in the N-terminal section of the H2B histone from chicken.     

Sustained degradation of n-pentane and isobutane in a gas-phase bioreactor

Summary Microorganisms were able to remove hydrocarbons (pentane and isobutane) from air by biological action in a columnar bioreactor with ceramic packing. The reactor was operated in a liquid continuous mode with gas recirculation and a slow addition of the organic-containing air. After a period of acclimation, the reactor has operated for 12 months with only pentane and isobutane as carbon sources. The gaseous hydrocarbons have been degraded throughout this period. The hydrocarbon removal rates measured between 1 and 2 g h^–1 m^–3. The microbes were shown to be able to degrade these gaseous hydrocarbons completely in a closed bioreactor without any additional nutrients.Research supported by the Advanced Industrial Concepts Division-Biological and Chemical Technologies Research. U.S. Department of Energy, under contract DE-AC05-84OR21400 with Martin Marietta Energy Systems. Inc.     

Microsequence analysis of peptides and proteins. IX. Manual gas-phase microsequencing of multiple samples

A novel apparatus for performing manual gas-phase Edman chemistry on protein and peptide samples is described. Edman chemistry is performed in 6 to 10 Teflon continuous flow reactors (CFR), previously described by J.E. Shively et al. (1987) Anal. Biochem. 163, 517-529). The CFRs are packed with 10-15 mg of Polybrene-coated spherical silica (Porasil B, Waters Associates). The gas-phase coupling reagent and cleavage reagent are 5% aqueous triethylamine and anhydrous trifluoroacetic acid, respectively, delivered by a stream of argon gas. The delivery of the gas-phase reagents is manually controlled with Hamilton 3-way valves and 2-way valves, and that of the solvents, ethyl acetate and butyl chloride, by syringe pipetting. The average cycle time is 15-20 min for 6 to 10 samples run simultaneously. Conversion of the anilinothiazolinone to phenylthiohydantoin (PTH) amino acid derivatives is accomplished manually with 25% aqueous trifluoroacetic acid. The PTH amino acids are analyzed by reversed-phase HPLC using an autosampler for handling multiple samples. Excellent results were obtained in the 100-200 pmol range. Protein samples can be sequenced from 15-20 cycles, and peptide samples usually to the COOH terminus. Initial yields ranged from 30 to 60% and repetitive yields ranged from 90 to 96%. The sample washout and size of background peaks are significantly reduced, compared to older methods of manual sequence analysis. The yields and background signal to noise are comparable to automated gas-phase Edman chemistry. The improved manual Edman described represents a low cost alternative to automated sequence analysis, and has the advantage being able to process multiple samples simultaneously.     

Heterokaryon identification through simultaneous fluorescence of tetramethylrhodamine isothiocyanate and fluorescein isothiocyanate labelled protoplasts

Protoplasts were separately stained with the fluorescent dyes fluorescein isothiocyanate (FITC) and tetramethylrhodamine isothiocyanate (TRITC). Following fusion, doubly stained heterokaryons were identified under fluorescence microscopy by using the Zeiss filter set 48 77 05 (excitation filter 450-490 nm, dichroic reflector 510 nm, and barrier filter 520 nm) which allowed simultaneous fluorochrome emissions. Previously, either emission spectrum, but not both, was possible for any single filter set.     

Genotoxic effects of allyl isothiocyanate (AITC) and phenethyl isothiocyanate (PEITC)

Two isothiocyanates (ITCs) commonly found in human diet, allyl isothiocyanate (AITC) and phenethyl isothiocyanate (PEITC), were tested for genotoxic effects in a battery of assays: Salmonella/microsome assay with TA 98 and TA 100, differential DNA repair assay with E. coli and micronucleus (MN) induction assay with human derived Hep G2 cells. Albeit to a different degree, both ITCs induced genotoxic effects in all test systems. AITC was more genotoxic in bacterial test systems than in Hep G2 cells; in contrast, the effect of PEITC was stronger in Hep G2 cells. In in vivo assays with E. coli indicators in which mice were exposed to relatively high doses of the compounds (90 and 270 mg/kg), AITC induced moderate but significant effects; PEITC failed to induce significant effects in any of the organs. To find out the reason for the weak genotoxicity of AITC and PEITC under in vivo test conditions, we exposed E. coli indicator cells to the test substances in the absence or presence of rat liver homogenate (with and without cofactors), bovine serum albumin (BSA) and human saliva. All of them markedly attenuated the genotoxicity of AITC and PEITC, implying that the test substances are detoxified by direct non-enzymatic binding to proteins. Additional experiments carried out on the mechanistic aspects of AITC and PEITC-induced genotoxicity showed that the compounds induce the formation of thiobarbituric acid reactive substances (TBARS) in Hep G2 cells. Furthermore, in in vitro assays with E. coli, radical scavengers reduced the differential DNA damage induced by AITC and PEITC. The latter two findings give a clue that reactive oxygen species might be involved in the genotoxic effect of the ITCs. Although ITCs have been repeatedly advocated as very promising anticancer agents, the data presented here indicate that the compounds are genotoxic, and probably carcinogenic, in their own right.     

Genotoxic effects of methyl isothiocyanate

Aim of the study was to investigate the genotoxic effects of methyl isothiocyanate (MITC), a compound widely distributed in the environment as a constituent of certain vegetables, a soil fumigant and breakdown product of carbamate pesticides. MITC caused only marginal mutation induction in reversion assays with Salmonella strains TA100 and TA98 and, the maximum effect (<2-fold increase over the background rate) was seen at 100microg/ml. In differential DNA-repair assays with E. coli (strains 343/763 uvrB/recA and 343/765 uvr(+)/rec(+)), a pronounced dose-response effect (induction of repairable DNA-damage) was seen at low concentrations (>/=4microg/ml). In both bacterial assays, addition of activation mix (rat liver S-9) led to a reduction of the genotoxic effects. In micronucleus assay and in single cell gel electrophoresis assay with human hepatoma cells (HepG2), clear cut positive results were obtained at exposure concentrations of <5microg/ml. On the contrary, only marginal effects were seen in differential DNA-repair host-mediated assays where E. coli indicator cells were recovered from different inner organs of mice that had been treated orally with a high dose (90mg/kg bw) of the test compound. Further in vitro experiments showed that MITC is inactivated by body fluids (saliva, gastric juice) and that its DNA-damaging properties are attenuated by non-enzymatic protein binding. Since exposure of HepG2 cells to MITC led to formation of thiobarbituric acid reactive substances, it is likely that its DNA-damaging effects involve lipid peroxidation processes. Overall, our findings show that MITC induces only marginal effects at extremely high (almost lethal) doses in inner organs in vivo, but it causes DNA-damage at low concentrations in vitro.     

Utility of the gas-phase sequencer for both liquid- and solid-phase degradation of proteins and peptides at low picomole levels

The utility of the commercially available gas-phase sequencer for complete analysis of peptide samples was investigated. Using the program supplied with the instrument, significant extractive loss of samples in Polybrene was observed, even at input levels up to 500 pmol. In order to reduce this loss, the sequencer program was modified by increasing the phenylisothiocyanate (PITC)-coupling steps from two to three and lengthening the duration of ethyl acetate (S2) delivery while reducing the delivery rate. These changes gave improved results with peptides, e.g., all eight residues of angiotensin II were identified at the 25-pmol level. In addition, background contamination was decreased and repetitive yields were increased. The instrument was also found to function well with samples coupled to solid supports; however, some of the methodologies that work adequately for covalent attachment of peptides to solid supports at the level 1-10 nmol were found to give unacceptable coupling/sequenceable yields at or below the 100-pmol level. The coupling methods tried were (1) reaction of homoserine lactone with aminopropyl (AP)-glass, (2) reaction of alpha- and epsilon-NH2 groups with p-phenylenediisothiocyanate (DITC)-glass, and (3) reaction of alpha-COOH groups with aminoaryl (AA)-glass via EDAC (1-ethyl-3,3'-dimethylaminopropyl-carbodiimide). Of these, the first method gave combined yields of 42-94% while the latter two were only 9-35% efficient. The covalently bound samples provided sequence information even at the resulting low levels, e.g., 9/13 residues of dynorphin including Lys-13 at 11 pmol. In general, sequencer runs on solid-phase samples gave "cleaner" analyses and slightly higher repetitive yields (1-2%). Sequence information has also been obtained on peptides made by solid-phase synthesis prior to cleavage from the polystyrene support. With improved coupling efficiencies, solid-phase techniques would provide an alternative to immobilization of peptides in Polybrene films for low picomole level gas-phase sequencing.     

Modification of actin with fluorescein isothiocyanate

Reaction of rabbit skeletal muscle G-actin at pH 8.5 with fluorescein isothiocyanate (FITC) resulted in incorporation of up to 1.20 mol FITC/mol actin. At pH 8.8, the level of incorporation was raised to 1.98 mol FITC/mol actin. When excited with ultraviolet light, the FITC-actin samples fluoresced strongly with an emission maximum near 517 nm. Tryptic digests of FITC-actin containing about 1.0 mol FITC/mol actin could be separated into a nonfluorescent 33.5 kDa trypsin-resistant core protein and a fluorescent pool of small peptides. Chromatography on DEAE-Bio-Gel or two-dimensional separation on cellulose TLC plates of the peptide pool revealed that FITC was highly selective in the site of its reaction with actin, resulting in a single highly fluorescent peptide after tryptic digestion. NH2-terminal and amino acid analyses demonstrated this peptide to be derived from residues 51 to 62, with Lys-61 proposed as the major FITC-sensitive site on actin. FITC-actin is similar to G-actin in gross conformation; circular dichroism spectra of actin before and after labelling are identical. FITC-actin is also able to interact strongly with deoxyribonuclease I. However, FITC-actin solution viscosities and fluorescence properties are not altered by the addition of KCl or MgCl2. Therefore, either a localized conformational change near Lys-61 or steric hindrance due to the FITC attached to Lys-61 blocks the polymerization of actin.     

Amino-terminal analysis of polypeptide using dimethylaminoazobenzene isothiocyanate

A new method for qualitative and quantitative N-terminal analysis of polypeptide using dimethylaminoazobenzene-isothiocyanate is presented. The method can recover all naturally occurring N-terminal amino acids, including asparagine, glutamine, and tryptophane in a nearly quantitative yield. Less than 1 nmol of polypeptide is required for qualitative N-terminal analysis and 5 to 10 nmol of polypeptide is used for quantitative N-terminal analysis. Applications and expected limitations of this new N-terminal method are described.     

Interaction of oxidized glutathione with allyl isothiocyanate

Isothiocyanates formed from glucosinolates in Brassica species have a strong affinity for amino acids and proteins, especially for their thiol, sulphide and terminal amino groups. To investigate the action of isothiocyanate on cystine residues in proteins and peptides, the present study on the interaction between allyl isothiocyanate and oxidized glutathione under physiological conditions was undertaken. Oxidized glutathione was oxidatively cleaved to some modified glutathiones by the attack of allyl isothiocyanate on its disulphide bond. Two new modified products were isolated from the reaction mixture by gel chromatography and HPLC, and their structures were determined by NMR and mass spectral analyses as glutathionyl N-allyldithiocarbaramate and its allyl thiohydantoin derivative. The formation of these products indicated oxidative cleavage of the disulphide bond in the cystine residue; the electrophilic attack of the isothiocyanate on the sulphur atom must cleave the disulphide bond oxidatively to dithiocarbamate and sulphenate, as in the case of cystine.     

Cyanine dye labeling reagents containing isothiocyanate groups

New isothiocyanate derivatives of cyanine dyes were synthesized as fluorescent covalent labeling reagents for proteins and other biomolecules. These dyes have maximum absorbance in the red and near infrared regions of the spectrum, have high extinction coefficients and have adequate quantum yields. Incorporating two alkyl sulfonate groups in the dye structures increases their water solubility, which is beneficial for labeling biological molecules in aqueous solution. Reactivities of proteins with these new cyanines are similar to their reactivities with fluorescein isothiocyanate. These new labeling reagents are complementary to the fluorescein and rhodamine reagents, expanding the possibilities of multicolor analyses. Sheep anti-mouse-IgG antibody was labeled with a pentamethine cyanine dye (CY5.8-ITC) and used with a fluoresceinated antibody as a second reagent for detecting human T-cell subsets by flow cytometry.     

Allyl isothiocyanate is mutagenic in Salmonella typhimurium

Allyl isothiocyanate, a naturally occurring compound, component of oil of mustard and human food plants such as cabbage, cauliflower and horseradish, has up to now been regarded as nonmutagenic in bacterial mutagenicity testing systems. Recently, however, it was found to cause transitional-cell papillomas in the urinary bladder of male F344 rats. Contrary to earlier reports, in this study allyl isothiocyanate showed clear mutagenicity for Salmonella typhimurium TA100 in the preincubation assay after longer, non-standard preincubation times (greater than 20 min). The mutagenicity is expressed only in the presence of a rat-liver homogenate metabolising system, i.e. it is indirect. However, high concentrations of rat-liver homogenate suppress the mutagenicity of allyl isothiocyanate. SKF525, inhibitor of microsomal oxygenase, reduces the mutagenic potential which on the other hand is increased in the presence of 1,1,1-trichloropropene-2-oxide, inhibitor of epoxide hydrolase. This indicates the occurrence of an epoxide intermediate in allyl isothiocyanate metabolism. Another metabolic pathway, namely hydrolysis to allyl alcohol and oxidation to acrolein, a known mutagen, also seems possible as cyanamide, inhibitor of aldehyde dehydrogenase, can slightly increase the mutagenic potential. The reason(s) for allyl isothiocyanate's requirement for long preincubation times to express mutagenicity still requires elucidation, and the question arises: is allyl isothiocyanate a single, exceptional case or not?