An Ehrlich chromogen in collagen cross-links.

A well-characterized three-chain peptide [(Col1)2 X T9] from human type III collagen was a rich source of Ehrlich chromogen. The corresponding two-chain peptide [(Col1)2] was not, implying that the Ehrlich chromogen is a trifunctional cross-link. (Col1)2 X T9 also contained pyridinoline, which is not an Ehrlich chromogen. The 7S domain of type IV collagen also contained an Ehrlich chromogen.     

Collagen crosslinks and their relationship to the thermal properties of calf tendons

The hydrothermal isometric tension and thermal transition temperature of collagen were determined in tendons from three different calf muscles. The levels of the nonreducible collagen crosslink, pyridinoline, and the collagen-associated Ehrlich chromogen were also measured in the three tendons. The reducible collagen crosslinks, hydroxylysinonorleucine, dihydroxylysinonorleucine, and histidinohydroxymerodesmosine were measured in two tendons. The thermal properties and levels of crosslinks were found to vary considerably between the different tendons, and also at different sites in two of the tendons. A strong correlation was observed between the thermal transition temperatures and the hydrothermal isometric tensions of the nine tendon sites examined. Both thermal properties correlated with the concentration of both pyridinoline and Ehrlich chromogen. The analogous behavior of the collagen-associated Ehrlich chromogen and the pyridinoline crosslink supports the role of the Ehrlich chromogen as a nonreducible crosslink.     

Application of Photoshop and Scion Image analysis to quantification of signals in histochemistry, immunocytochemistry and hybridocytochemistry

OBJECTIVE: To describe a simple method to achieve the differential selection and subsequent quantification of the strength signal using only one section. STUDY DESIGN: Several methods for performing quantitative histochemistry, immunocytochemistry or hybridocytochemistry, without use of specific commercial image analysis systems, rely on pixel-counting algorithms, which do not provide information on the amount of chromogen present in the section. Other techniques use complex algorithms to calculate the cumulative signal strength using two consecutive sections. To separate the chromogen signal we used the "Color range" option of the Adobe Photoshop program, which provides a specific file for a particular chromogen selection that could be applied on similar sections. The measurement of the chromogen signal strength of the specific staining is achieved with the Scion Image software program. CONCLUSION: The method described in this paper can also be applied to simultaneous detection of different signals on the same section or different parameters (area of particles, number of particles, etc.) when the "Analyze particles" tool of the Scion program is used.     

A comparison of eight different chromogen protocols for the demonstration of immunoreactive neurofilaments or glial filaments in rat cerebellum using the peroxidase-antiperoxidase method and monoclonal antibodies

Eight different previously described chromogen protocols were evaluated with respect to their sensitivity for the visualization of horseradish peroxidase (HRP) in a peroxidase-antiperoxidase (PAP) complex used with the unlabeled antibody method for immunohistochemistry. The protocols were evaluated in a test system that involved the demonstration of immunoreactive neurofilaments (NF) or glial filaments (GF) in paraffin-embedded sections of rat cerebellum using anti-NF or anti-GF monoclonal antibodies (MA). The chromogens included: amino-ethylcarbazole (AEC), diaminobenzidine (DAB), O-tolidine, paraphenylenediamine-pyrocatechol (PPD-PC), and tetramethylbenzidine (TMB). The incubation medium using DAB as the chromogen was employed at neutral pH, at pH 5.1, or at neutral pH with the addition of either cobalt chloride or imidazole to intensify the reaction product. The relative sensitivity of the chromogen protocols was quantitated by comparing the dilution of the anti-NF or anti-GF MA at which NF or GF immunoreactivity was extinguished using each protocol. The results obtained with both the anti-NF and anti-GF MA indicated that DAB with imidazole was the most sensitive chromogen protocol.     

Amplification of chromogenic staining of proteases within electrophoretic gels

A method is described for amplification of bands generated by chromogenic substrates following their reaction with proteases within electrophoretic gels. Chromogenic substrates, consisting of synthetic peptides containing the chromophore 4-nitroaniline (paranitroaniline, PNA), are applied directly to the surface of agarose or acrylamide gels. Protease activity within the gel results in the enzymatic amidolysis of the chromogenic substrates, releasing free PNA. The yellow PNA chromogen is then derivatized by reaction with p-dimethylaminocinnamaldehyde (DACA) to form a purple Schiff base compound. The resultant complex has a significantly higher molar absorbancy than the original PNA chromogen, thus increasing the sensitivity for low levels of amidolytic activity. The derivatized chromogen is easily visualized for photography.     

Chromatographic separation and automated analysis of flavanols

Flavanols from barley or hops were separated chromatographically and assayed automatically by reaction with the chromogen, 4-dimethylaminocinnamaldehyde. For separating the flavanols on Sephadex G-25, gradient elution with water-methanol mixtures was necessary. The chromogen was specific for flavanols and well suited to AutoAnalyzer methods. The method appears generally applicable in flavanol analysis of plant materials.     

Factors influencing the determination of dNA with indole. II. Recovery of hydrolyzed dNA from protein precipitates

DNA can be measured by the indole method in hydrolyzates containing large concentrations of nonspecific chromogen without exceeding the sufficiency of the color reagents or the efficiency of the chloroform extraction procedure. Since TCA is nonessential for the completeness of the color reaction, there is considerable latitude in the choice of extraction procedures. The action of hot acid hydrolysis to release the indole chromogen from DNA is opposed by two major factors: (1) the capacity of the hot hydrolysis to entrap solubilized but incompletely hydrolyzed DNA in a protein precipitate and (2) the ability of the hydrolysis to destroy the chromogen for the reaction. Because of these factors and since the rate of chromogen release is eventually exceeded by the rate of its destruction, the hydrolysis should be run with internal and external DNA standards and not exceed 20, 40, 50, or 120 min at 90, 85, 80, or 70°C, respectively. If the recovery of internal standard indicates that regnificant amounts of DNA have been left unmeasured, an increase in the volume of the system is preferable to an increase in the strength of the acid or the number of extractions.     

Sensitivity of various visualization methods for peroxidase and alkaline phosphatase activity in immunoenzyme histochemistry

Summary Various chromogen protocols for visualizing peroxidase and alkaline phosphatase activity in immunoenzyme histochemistry were compared with respect to their sensitivity. They were tested on tissue sections of human skeletal muscle and in an antigen spot test using antibodies against slow skeletal muscle myosin. The chromogens included 3-amino-9-ethylcarbazole (AEC), 3, 3-diaminobenzidine (DAB),p-phenylenediamine-pyrocatechol (PPD-PC) and 4-chloro-1-naphthol (CN) in peroxidase histochemistry, and 5-bromo-4-chloro-3-indolyl phosphate-nitro blue tetrazolium salt (BCIP-NBT), BCIP-tetra nitro blue tetrazolium salt (TNBT) and various combinations of substituted naphthol phosphate-diazonium salt in alkaline phosphatase histochemistry. DAB, CN, and PPD-PC were also employed with imidazole and DAB in addition to Co²⁺ and Ni²⁺ ions. The results indicate that DAB-imidazole and DAB-Co²⁺ and Ni²⁺ ions are the most sensitive chromogen protocols for visualizing peroxidase activity. Although no large differences were found between the various chromogen protocols for visualizing alkaline phosphatase activity, the protocol BCIP-TNBT is especially recommended. Furthermore, the various chromogen protocols were evaluated as to stability of chromogen solutions and final precipitates, background staining, localization properties, and enhancement of enzyme activity.     

On the screening of hydrogen peroxide-generating lactic acid bacteria

The published plate methods for the detection of hydrogen peroxide-producing lactic acid bacteria, which employ horseradish peroxidase and a chromogen, clearly fail to detect all the organisms that produce H₂O₂. Whilst keeping the same principle, the use of a novel growth medium and of the chromogen tetramethyl-benzidine allows the detection of H₂O₂ production by strains previously classified as non-producers.     

Spectrophotometric determination of lantadene A, 22β-angeloxy-3-oxoolean-12-en-28-oic acid

The formation of a blue chromogen between sodium borohydride-treated lantadene A (22β-angeloyloxy-3-oxoolean-12-en-28-oic acid) and acetic anhydride-sulfuric acid (9:1) formed the basis of a spectrophotometric method for its quantitation. The chromogen had a broad absorption maximum (λ_max) at 630–645 nm. The optimum amount of sodium borohydride for lantadene A reduction was 1 mg/mg lantadene A in methanolic solution. The chromogen was stable for 5, 7, and 26 min after reaction at 25, 18, and 0°C, respectively. The method is convenient, sensitive, and reproducible. The amount of lantadene A in the leaves of Lantana camara collected in the month of May quantitated by the present method was found to be 13.6 mg/g dry weight of the leaves.     

The potency of tissue-type plasminogen activator (TPA) determined with chromogen and clot-lysis assays

An assessment was made of two methods for determining the potency of tissue-type plasminogen activator (TPA). A chromogenic microtitre plate assay was established which contained TPA, plasminogen, a synthetic plasmin substrate (H-D-valyl-L-leucyl-L-lysyl-p-nitroaniline dihydrochloride, S2251) and any one of the following stimulators: native fibrinogen, enzymatic and chemical digests of fibrinogen, poly-D-lysine (PDL) and chemical derivatives of the latter. The chromogen assay was compared with an automated clot-lysis (turbidimetric) assay for sensitivity, reproducibility and validity for potency determination. Reference preparations of TPA were titrated in both assays: in the chromogen assay the dose-response curves were non-parallel, whereas parallelism was observed in the clot-lysis assay. Thus, the chromogen assay was restricted in its applicability and disqualified from any routine regulatory use. The potency of individual lots of recombinant (r)TPA could only be estimated in International Units (IU) of TPA activity with the automated clot-lysis assay and the potency values obtained (IU/vial) were in remarkably close agreement with the manufacturers' values.     

Determination of laccase activity in mixed solvents: comparison between two chromogens in a spectrophotometric assay

Spectrophotometric determination of laccase activity with ABTS acting as chromogen yields exceedingly low values whenever conducted in a water-organic mixed solvent. Nevertheless, there is firm evidence that laccase is able to oxidize substrates such as phenols and amines quantitatively in these mixed solvents. We show that the apparently small rate of ABTS oxidation by laccase in a mixed solvent, such as buffered water-dioxane 1:1, is not amenable to the denaturation of laccase but rather to the decreased stability of ABTS(.+). We propose HAA as a more reliable chromogen for the determination of laccase activity in mixed solvents.     

A Note on Screening Hydrogen Peroxide-producing Lactic Acid Bacteria Using a Non-toxic Chromogen

An agar plate method for detection of hydrogen peroxide-producing bacteria using a non-carcinogenic chromogen is described.     

The synthesis from vitamin A1 of retinene 1 and of a new 545 m-mu chromogen yielding light-sensitive products

Ball, Goodwin, and Morton (1946) have reported that vitamin A(1) in contact with solid manganese dioxide is transformed slowly into a substance which displays spectroscopic properties of retinene(1). The latter is known to be the precursor of vitamin A(1) in the rhodopsin cycle of the retinal rods. The synthetic product is here referred to as "retinene(1)." In the present experiments this observation is confirmed. The procedure is recast in the form of a chromatographic oxidation. Manganese dioxide is packed in a column, vitamin A(1) solution poured in at the top, and the product drawn off in the filtrate. Depending upon the proportions of manganese dioxide and vitamin A(1), the product is either "retinene(1)," or a new substance which yields with antimony chloride a wine-red product with maximal absorption at 545 mmicro (545 mmicro chromogen). This procedure is an example of a potentially important class of chromatographic reactions. The synthetic "retinene(1)" is virtually identical with the natural substance in absorption spectrum and antimony chloride reaction. It lacks the pH indicator properties of crude natural retinene(1). The 545 mmicro chromogen possesses absorption maxima at 380 and 290 mmicro in chloroform; at 376 and 290 mmicro in ethanol; and at 361 and 277 mmicro in hexane. It is non-fluorescent. It has no acidic character, but on the contrary is mildly basic, being extracted from hexane by sulfuric or hydrochloric acids to form orange-red products. In partition between petroleum ether and aqueous methanol it is highly hypophasic. It is adsorbed strongly on calcium carbonate. Certain peculiarities in spectral behavior indicate the presence of a carbonyl group in the 545 mmicro chromogen, and support Morton's proposal that such a group occurs in retinene(1). Other properties of the 545 mmicro chromogen indicate hydroxyl groups. This substance therefore appears to be a hydroxy-carbonyl derivative of vitamin A(1). The red products which the 545 mmicro chromogen forms with antimony chloride or with sulfuric or hydrochloric acids are all markedly light-sensitive. They appear to be formed by the condensation of two molecules with loss of water; and to bear a close generic relation to the prosthetic groups of the visual photopigments.     

Avidin-biotin-immunoglucose oxidase: use in single and double labeling procedures

We have investigated the use of an avidin-biotin-immunoglucose oxidase (AB-GO) technique for single and double antigen localization in conjunction with the avidin-biotin-immunoperoxidase (AB-P) technique in fixed, embedded specimens, using sequential monoclonal and polyclonal antibodies of the same species. The optimal technique for double labeling requires the first antibody to be applied and localized with the AB-P technique using 3,3'-diaminobenzidine (DAB) as the chromogen, followed by an optional elution step and/or incubation with mild detergent (0.01% Triton). The second antigen is localized with the AB-GO technique with nitro blue tetrazolium (NBT) as a chromogen. Effects of antigen concentration, intermediate elution steps, and the relative efficiency of the two methodologies are described.     

Quantitative immunohistochemistry by measuring cumulative signal strength using commercially available software photoshop and matlab.

Currently available techniques for performing quantitative immunohistochemistry (Q-IHC) rely upon pixel-counting algorithms and therefore cannot provide information as to the absolute amount of chromogen present. We describe a novel algorithm for true Q-IHC based on calculating the cumulative signal strength, or energy, of the digital file representing any portion of an image. This algorithm involves subtracting the energy of the digital file encoding the control image (i.e., not exposed to antibody) from that of the experimental image (i.e., antibody-treated). In this manner, the absolute amount of antibody-specific chromogen per pixel can be determined for any cellular region or structure. (J Histochem Cytochem 48:303-311, 2000)     

Some structural features of the teichuronic acid of Bacillus licheniformis N.C.T.C. 6346 cell walls

A teichuronic acid, containing glucuronic acid and N-acetylgalactosamine, was purified from acid extracts of Bacillus licheniformis 6346 cell walls as described by Janczura, Perkins & Rogers (1961). After reduction of the carboxyl function of glucuronic acid residues in the polysaccharide the reduced polymer contains equimolar amounts of N-acetylgalactosamine and glucose. Methylation of the reduced polysaccharide by the Hakamori (1964) technique showed the glucose residues to be substituted on C-4. A disaccharide, 3-O-glucuronosylgalactosamine, was isolated from partial acid hydrolysates of teichuronic acid. After N-acetylation the disaccharide produces chromogen readily on heating at pH7, in agreement with C-3 substitution of the reducing N-acetylamino sugar. Teichuronic acid also produces chromogen under the same conditions, with concurrent elimination of a modified polysaccharide from C-3 of reducing terminal N-acetylgalactosamine residues of the teichuronic acid chains. The number-average chain lengths of several preparations of teichuronic acid were estimated from the amounts of chromogen produced in comparison with the N-acetylated disaccharide. The values obtained are in good agreement with the weight-average molecular weight determined by ultracentrifugal analysis. The reducing terminals of teichuronic acid are shown to be exclusively N-acetylgalactosamine by reduction with sodium boro[(3)H]hydride. The number-average chain lengths of the teichuronic acid preparations were estimated by the extent of in corporation of tritium and are in agreement with values obtained by the other methods.     

A continuous spectrophotometric assay for the Bacillus cereus phospholipase C using a thiophosphate substrate analog: evaluation of assay conditions and chromogenic agents

A thiophosphate analog of dioctanoylphosphatidylcholine has been used as the substrate in a continuous spectrophotometric assay for the Bacillus cereus phospholipase C. The reaction has been monitored at 412 nm using 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) and at 324 nm using 4,4'-dithiopuridine (DTP) as the respective thiol-reactive chromogenic agents. An optimum pH 6.0 was determined for the phospholipase C-catalyzed reaction which was independent of the chromogen utilized. Although the reaction rates observed when DTP was used were increased over those seen with DTNB, the rates were insensitive to changes in the concentration of the chromogen normally used for the assay. The initial velocities were shown to be linearly dependent upon the amount of enzyme added over at least a 20-fold enzyme concentration range. The dependency of the initial velocity on the concentration of substrate showed a discontinuity at [S] = 40 microM when either DTP or DTNB was used. This was consistent with a value of 56 microM estimated for the substrate critical micelle concentration by an independent measurement. While the substrate data measured using DTP could not be fit to existing equations based on Michaelis-Menten kinetics, the data obtained using DTNB as the chromogen conformed with the model proposed by Wells for enzymes acting upon micelle-forming substrates (M. A. Wells (1974, Biochemistry 13, 2248-2257). This allowed for the estimation of monomer and micelle Michaelis-Menten parameters for the B. cereus phospholipase C-catalyzed reaction with a thiophosphate analog substrate.     

The deoxyribose method: a simple "test-tube" assay for determination of rate constants for reactions of hydroxyl radicals

Hydroxyl radicals, generated by reaction of an iron-EDTA complex with H2O2 in the presence of ascorbic acid, attack deoxyribose to form products that, upon heating with thiobarbituric acid at low pH, yield a pink chromogen. Added hydroxyl radical "scavengers" compete with deoxyribose for the hydroxyl radicals produced and diminish chromogen formation. A rate constant for reaction of the scavenger with hydroxyl radical can be deduced from the inhibition of color formation. For a wide range of compounds, rate constants obtained in this way are similar to those determined by pulse radiolysis. It is suggested that the deoxyribose assay is a simple and cheap alternative to pulse radiolysis for determination of rate constants for reaction of most biological molecules with hydroxyl radicals. Rate constants for reactions of ATP, ADP, and Good's buffers with hydroxyl radicals have been determined by this method.     

Tetramethyl benzidine for horseradish peroxidase neurohistochemistry: a non-carcinogenic blue reaction product with superior sensitivity for visualizing neural afferents and efferents.

Tetramethyl benzidine (TMB) is a presumptively non-carcinogenic chromogen which yields a blue reaction-product at sites of horseradish peroxidase activity. Sixty-six distinct procedures were performed in rats and monkeys in order to determine the optimal incubation parameters for TMB. As a result, a procedure is recommended whose sensitivity greatly surpasses that of a previously described benzidine dihydrochloride method. Indeed, the sensitivity of this new method in demonstrating retrograde transport is markedly superior to that of the previously described benzidine dihydrochloride method. Furthermore, as a consequence of this enhanced sensitivity, many efferent connections of the injection site are also visualized. The injection site demonstrated by this TMB procedure is significantly larger than the one demonstrated when benzidine dihydrochloride or diaminobenzidine is used as a chromogen. Finally, this TMB procedure has been compared to two other TMB procedures and found to provide superior morphology and sensitivity.