N-succinyl-alanyl-methionyl-S-benzylcysteine p-nitroanilide as a sensitive substrate for assaying activity of cysteine proteinases

N-Succinyl-alanyl-methionyl-S-benzylcysteine p-nitroanilide has been found to be a very sensitive chromogenic substrate for the assay of cysteine proteinase papain, ficin and bromelain. N-Succinyl-alanyl-S-benzylcysteine p-nitroanilide and N-succinyl-alanyl-alanyl-S-benzylcysteine p-nitroanilide are also suitable for this purpose. These substrates were hydrolyzed only very slightly or not hydrolyzed at all by trypsin.     

Potentiometric determination of cysteine with thiol sensitive silver-mercury electrode

A potentiometric procedure for cysteine thiol group concentration monitoring in media generating free radicals was developed using a thiol specific silver-mercury electrode. Electrolytic deposition of mercury on a silver wire and treatment with 20 mM cysteine in 0.5 M NaOH were used to produce the electrode. A silver-chloride electrode in saturated KCl was the reference. A glass capillary with 1 M KNO3 in 1% agarose gel was the liquid junction. The electrode responded to cysteine concentration in the range from 0.01 to 20 mM yielding a perfect linear relationship for the dependence of log [cysteine] versus electrode potential [mV], with b0 (constant) = -373.43 [mV], b1 (slope) = -53.82 and correlation coefficient r2 = 0.97. The electrode potential change per decade of cysteine concentration was 57 mV. The minimal measurable signal response was at a cysteine concentration of 0.01 mM. The signal CV amounted to 4-6% for cysteine concentrations of 0.01 to 0.05 mM and to less than 1% for cysteine concentrations of 0.5 to 20 mM. The response time ranged from about 100 s for cysteine concentrations of 0.01 to 0.1 mM to 30 s at higher cysteine concentrations. The standard curve reproducibility was the best at cysteine concentrations from 0.1 to 20 mM. In a reaction medium containing cysteine and copper(II)-histidine complex ([His-Cu]2+) solution in 55 mM phosphate buffer pH 7.4 the electrode adequately responded to changes in cysteine concentration. Beside cysteine, the silver-mercury electrode responded also to thiol groups of homocysteine and glutathione, however, the Nernst equation slope was about half of that for cysteine.     

The reaction of cysteine with ceruloplasmin copper

The kinetics of decay in absorbance at 610 nm in the reaction of cysteine with ceruloplasmin was biphasic under anaerobic conditions. Admission of oxygen to the bleached ceruloplasmin restored the blue color to about 75 % of the original value. However, under aerobic or anaerobic conditions an initial bleaching corresponded to a 25 % decrease in blue color. This change was irreversible and remained after removal of excess cysteine from the reaction mixture by dialysis. There was no correlation between transient and steady-state kinetic parameters. Circular dichroism measurements showed a characteristic reduction in the negative band at 450 nm, which is specific for type 1b copper. Isolation and further studies on cysteine-modified ceruloplasmin with a lower A₆₁₀/A₂₈₀ ratio showed < 10% reduction in enzyme activity toward p-phenylenediamine and o-dianisidine. Evidence is also presented that ceruloplasmin catalyzes the oxidation of cysteine with a one-electron reduction of oxygen and the formation of superoxide ion, which is then converted to H₂O₂ by ceruloplasmin. The effect of superoxide dismutase and catalase also confirms the presence of superoxide and H₂O₂. In sum, these data show that a permanent reduction of type 1b copper occurred when cysteine was used as a substrate. We conclude that there is a single electron transfer from cysteine directly to oxygen using one specific copper of ceruloplasmin, type 1b.     

A reaction for the simple sensitive fluorimetric assay of heparin and 2-amino sugars

1. 3,5-Diaminobenzoic acid reacted rapidly with the product from HNO(2) deamination of heparin, heparan sulphate and 2-amino-2-deoxyhexoses under very mild conditions (pH3.0 and 37 degrees C) to give stable fluorescent derivatives. 2. The fluorescence yield was rectilinearly related to the concentration of heparin etc. Less than 0.1mug of 2-amino-2-deoxyhexose was easily measurable in standard cuvettes. 3. The deamination products of glucosamine and (particularly) galactosamine were labile in the HNO(2) reagent, with half-lives of 20-40min at room temperature. At 0 degrees C they were much more stable. The analogous product from heparin was not so labile. 4. Under the standard conditions, and at room temperature, relative fluorescence yields (d-glucosamine=1.0) were: d-galactosamine, 0.75; d-gulosamine, 0.38; d-mannosamine, approx. 0.20. 5. Neutral sugars, chondroitin sulphates, DNA and N-acetylneuraminic acids did not react, nor did N-acetylamino sugars or non-deaminated hexosamines. 6. It is suggested that the Dische-Borenfreund [Dische & Borenfreund (1950) J. Biol. Chem.184, 517-522] indole method, the Kissane-Robins [Kissane & Robins (1962) J. Biol. Chem.233, 184-188] DNA assay and the proposed amino sugar method are all examples of simple aldehyde reactions. The specificity of the proposed method is considerably greater than that of the Dische-Borenfreund procedure, partly because of the much milder reaction conditions. 7. The proposed method is very reproducible, about 50-100 times as sensitive as the Elson-Morgan reaction, and 10-50 times as sensitive as the Dische-Borenfreund procedures. It is also convenient; acid hydrolysates of amino sugar-containing compounds can be directly neutralized with sodium acetate solution.     

A sensitive assay for the reverse reaction of the first histidine biosynthetic enzyme.

A method capable of specifically detecting low levels of adenosine triphosphate phosphoribosyltransferase (EC 2.4.2.17) is given. It is based on conversion of the usual product of the enzyme, N¹-(5′-phospho-d-ribosyl)adenosine triphosphate to ATP, which is detected by luciferase luminescence utilizing a liquid scintillation spectrophotometer. The assay is linear in enzyme concentration down to a lower limit of at least 55 pg enzyme/0.2 ml.     

A single-tube nested real-time polymerase chain reaction for sensitive contained detection of Cryptosporidium parvum

Aim:  A new real-time polymerase chain reaction (PCR) was developed for sensitive contained detection of Cryptosporidium parvum .
Methods and Results:  The method is a nested PCR targeting a specific region of rDNA of C. parvum , which takes place in one tube, using different annealing temperatures to control the first and the second rounds of PCR, with real-time fluorogenic probe-based detection of the second round of PCR. The DNA-based detection limit of the method was 2 fg, which corresponds to approx. one genome per reaction. The detection level determined using diluted samples of C. parvum oocysts was ten oocysts per millilitre.
Conclusions:  The method facilitates sensitive detection of C. parvum thanks to the nested format, while reducing the risk of laboratory contamination thanks to the single-tube, real-time fluorimetric format.
Significance and Impact of the Study:  The developed method may be useful for sensitive contained detection of C. parvum in environmental and food samples, after appropriate separation of oocysts.     

A highly sensitive one-step method for silver intensification of the nickel-diaminobenzidine endproduct of peroxidase reaction

We have developed a new technique which makes silver intensification of the oxidatively polymerized diaminobenzidine (DAB), the endproduct of peroxidase reaction, less laborious without any loss in selectivity or sensitivity. The new technique is based on two strategies: (a) increasing the argyrophilia of the DAB by modifying its polymerization with Ni ions, and (b) decreasing tissue argyrophila by using a mildly acidic physical developer instead of the alkaline one previously presented. Because the nickel modification takes place in the DAB substrate solution, i.e., in the final step of the peroxidase reaction, only one additional step, the physical development, must be carried out if intensification is needed.     

A new bromine-containing reagent for cysteine modification

5-Bromo-2[(2-iodoacetyl)amino]benzenesulfonic acid (AIBSA), a reagent for modification of free of cysteine thiol groups in proteins and peptides, was synthesized. Rate constants of its interaction with thiol groups were determined. The presence of a bromine atom allows an easy identification of the AIBSA-labeled peptides in mass spectra due to the characteristic isotope distribution. The compound is stable in solution and under exposure to light.     

A new bromine-containing reagent for cysteine modification

5-Bromo-2[(2-iodoacetyl)amino]benzenesulfonic acid (AIBSA), a reagent for modification of free of cysteine thiol groups in proteins and peptides, was synthesized. Rate constants of its interaction with thiol groups were determined. The presence of a bromine atom allows an easy identification of the AIBSA-labeled peptides in mass spectra due to the characteristic isotope distribution. The compound is stable in solution and under exposure to light.     

A new and sensitive Co-operational polymerase chain reaction for rapid detection of Ralstonia solanacearum in water

Three primers from 16S rRNA were successfully assayed simultaneously in one reaction for sensitive detection of Ralstonia solanacearum in watercourses. The protocol is a modification of the Co-operational polymerase chain reaction (Co-PCR), which allows the simultaneous and co-operational action of the primers. It specifically amplified R. solanacearum strains belonging to biovars 1, 2 and 4. No products were obtained from any of the 162 unidentified isolates from river water. The sensitivity of the assay was <1 cfu/ml as determined by analysis of heat-treated water samples spiked with R. solanacearum, also containing indigenous microbiota up to 10(5) cfu/ml. The developed Co-PCR assay was more sensitive than other standard PCR assays in the analysis of 51 Spanish environmental water samples. Namely 31.3% of the samples were positive using the newly developed assay, whereas 13.7% or less positive samples were found with the other protocols. The Co-PCR improves the detection sensitivity of R. solanacearum and provides an important tool for its routine detection from environmental water samples and for epidemiological studies.     

Highly sensitive reaction of tryptophan with p-phenylenediamine

A simple and sensitive spectrophotometric method for the determination of tryptophan (TRP) is described. The method is based on the coupling reaction of tryptophan with diazotized p-phenylenediamine dihydrochloride (PPDD) in sulfuric acid medium to give the colored product having an absorption maximum at 520 nm. The coupled product was stable for 2h. Beer's law is obeyed in the tryptophan concentration range of 0.25-11 microg/ml. The method is applied for the analysis of pharmaceutical preparations of tryptophan and also in protein samples for tryptophan. Common excipients used as additives in pharmaceutical preparations do not interfere in the proposed method and the significant feature of the method is that most of the amino acids do not interfere in the determination of tryptophan.     

A sensitive estimator for crosscorrelograms

We present models based on transmitter gating and competition for SUSTAINED and ON-OFF units located in the lamina of the fly visual system. Predictions of the models are compared with data in several experimental paradigms. The overshoot and the plateau of the response are explained by the adaptation process of transmitter dynamics. Furthermore, it is demonstrated that this transmitter adaptation when coupled with fast competition can explain response features arising from different combinations of ON and OFF field inputs.Supported in part under Grant 89004RIG by University of Houston, Grant A-9731 by NSERC (Canada), and Grant EQ-2830 by FCAR (Quebec)     

A sensitive assay for allantoin

Allantoin is separated by thin-layer chromatography (TLC) and sprayed with an acidic solution ofp-dimethylaminobenzaldehyde. The yellow color produced is read in a densitometer and compared with that of a standard. The lower limit of quantitation is 0.1 μg per spot (0.5 mg/100 ml). The method can be utilized for the estimation of allantoin in serum, lymph, and urine.     

Characterization of reaction conditions providing rapid and specific cysteine alkylation for peptide-based mass spectrometry

Alkylation converts Cys thiols to thioethers and prevents unwanted side reactions, thus facilitating mass spectrometric identification of Cys-containing peptides. Alkylation occurs preferentially at Cys due to its high nucleophilicity, however reactions at other such sites are possible. N-ethylmaleimide (NEM) shows rapid reaction kinetics with Cys and careful definition of reaction conditions results in little reactivity at other sites. Analysis of a protein standard alkylated under differing reaction conditions (pH, NEM concentrations and reaction times) was performed using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) and selected reaction monitoring (SRM) of NEM-modified and unmodified peptide pairs. Mis-alkylation sites at primary and secondary amines were identified and limited to one equivalent of NEM. No evidence for hydroxyl or thioether alkylation was observed. Improved specificity was achieved by restricting the pH below neutral, NEM concentration below 10 mM and/or reaction time to below 5 min. Maximal removal of Cys activity was observed in tissue homogenates at 40 mM NEM within 1 min, dependent upon efficient protein denaturation. SRM assays identified peptide-specific levels of mis-alkylation, indicating that NEM-modified to unmodified ratios did not exceed 10%, with the exception of Cys alkylation that proceeded to 100%, and some Lys residues that resulted in tryptic missed cleavages. High reactivity was observed for His residues considering their relatively low abundance. These data indicate that rapid and specific Cys alkylation is possible with NEM under relatively mild conditions, with more abrasive conditions leading to increased non-specific alkylation without appreciable benefit for MS-based proteomics.     

Targeted glycomics by selected reaction monitoring for highly sensitive glycan compositional analysis

The development of glycomics increasingly requires the detection and quantification of large numbers of glycans, which is only partially achieved by current glycomics approaches. Taking advantage of selected reaction monitoring to enhance both sensitivity and selectivity, we report here a strategy termed targeted glycomics that enables highly sensitive and consistent identification and quantification of diverse glycans across multiple samples at the same time. In this proof-of-principle study, we validated the method by analyzing global N-glycans expressed in different systems: single proteins, cancer cells, and serum samples. A dynamic range of three orders of magnitude was obtained for the detection of all five glycans released from ribonuclease B. The limit of detection of 80 attomole for Man(9) GlcNAc(2) demonstrated the excellent sensitivity of the method. The capability of the strategy to identify diverse glycans was demonstrated by identification and detection of 162 different glycans and isomers from pancreatic cancer cells. The sensitivity of the method was illustrated further by the ability to detect eight glycans from 250 cancer cells and five glycans released from 100 cancer cells. In serum obtained from rabbits fed control diet or diet enriched with 2% cholesterol, differences to 42 glycans were accurately measured and this indicates that this strategy might find use in studies of biomarker discovery and validation.