Quantitative aspects of the Ninhydrin-Schiff reaction on amino cellulose films and tissue sections

Summary In the ninhydrin-Schiff reaction primary amino groups are converted by oxidation with ninhydrin to aldehyde groups which are subsequently stained with pararosanilin. Amino cellulose films, used as a model, and sections of muscle tissue were submitted to this reaction. The amino groups were stained before and after the ninhydrin reaction with dinitrofluorobenzene and the generated aldehyde groups were stained with dinitrophenyl-hydrazine. The molar extinction coefficients used for the calculation of the molar amounts of chromophores from extinction values, and the conditions for maximal staining intensity were determined on the amino cellulose model. With these data the yields of the different steps in the reaction sequence could be calculated in molar amounts from the extinction measurements. The results showed that from the amino groups originally present in the tissue sections less than 40% were converted by ninhydrin. About 90% of the converted amino groups were found as aldehyde groups and from these only 7% reacted with pararosanilin. On amino cellulose similar data were obtained. Attempts were made by modification of the conditions in the ninhydrin oxidation step to increase the overall yield of the reaction. These were only partially successful, but indicate that further quantitative study of other reaction conditions and different aldehyde generating agents could be promising.     

Calculation of protein extinction coefficients from amino acid sequence data

Quantitative study of protein-protein and protein-ligand interactions in solution requires accurate determination of protein concentration. Often, for proteins available only in "molecular biological" amounts, it is difficult or impossible to make an accurate experimental measurement of the molar extinction coefficient of the protein. Yet without a reliable value of this parameter, one cannot determine protein concentrations by the usual uv spectroscopic means. Fortunately, knowledge of amino acid residue sequence and promoter molecular weight (and thus also of amino acid composition) is generally available through the DNA sequence, which is usually accurately known for most such proteins. In this paper we present a method for calculating accurate (to +/- 5% in most cases) molar extinction coefficients for proteins at 280 nm, simply from knowledge of the amino acid composition. The method is calibrated against 18 "normal" globular proteins whose molar extinction coefficients are accurately known, and the assumptions underlying the method, as well as its limitations, are discussed.     

Statistical determination of the average values of the extinction coefficients of tryptophan and tyrosine in native proteins.

Spectroscopic measurement of protein concentration requires knowledge of the value of the relevant extinction coefficient. If the amino acid composition of a protein is known, however, extinction coefficients can be calculated approximately, provided that the values of the molar absorptivities for tryptophan and tyrosine residues in the protein are known. We have applied a matrix linear regression procedure and a mapping of average absolute deviations between experimental and calculated values to find molar extinction coefficients (epsilon M, 1 cm, 280 nm) of 5540 M-1 cm-1 for tryptophan and 1480 M-1 cm-1 for tyrosine residues in an "average" protein, as defined by a set of experimentally determined extinction coefficients for more than 30 proteins. Use of these values provides a significant improvement in extinction coefficient estimation over that obtained with the commonly used values obtained from solutions of model compounds in guanidine-HCl. The consistency of these results when compared to the large deviations often observed between experimentally determined extinction coefficients suggest that this method may offer acceptable accuracy in the initial estimation of molar absorptivities of globular proteins.     

Histochemical observations of protein-bound amino groups in human developing deciduous teeth

Summary Human developing deciduous teeth were studied by histochemical methods for protein groups; dinitrofluorobenzene (DNFB) H-acid stain for tyrosine histidine and tryptophan, coupled tetrazonium reaction with pre-iodination for histidine, diazotization and Millon's reaction for tyrosine revealed strong staining in the granular precursor of ameloblastic cytoplasma, pre- and young enamel layers. But only alloxan-Schiff stain showed a more intense stainability in the dentin matrix and pre-enamel than the young enamel matrix. The reaction of sulfhydryl and disulfide groups was observed in the matrix-forming ameloblast and Tomes' process with some variation of staining in the enamel matrix. The present histochemical results of protein groups in the enamel matrix were similar to those of the cornified layer of skin.Ameloblasts moderately stained with DNFB H-acid, coupled tetrazonium and alloxanschiff reaction were similarly stained in both matrix-forming stage and reduced stage. Increasing stainability of odontoblasts was limited in the matrix-forming stage.Pronounced changes of staining intensity in the enamel matrix for protein groups were found especially in the intra-rod organic substance with progress of mineralization and growth of deposited mineral crystallites, starting from the region of apex of cusp, parallel to the dentino-enamel junction.The stainability of the dentin matrix for protein groups increases following decalcification, and non-decalcified dentin was weakly stained for the alloxan-schiff method, coupled tetrazonium reaction, sulfhydryl and disulfide method.     

Tetraborate concentration on Morgan-Elson reaction and an improved method for hexosamine determination.

Effect of tetraborate concentration on the determination of hexosamine by Morgan-Elson color reaction according to Levvy and McAllan [(1959) Biochem. J., 73, 127–232] was studied using the three different preparations of the tetraborate solution. The concentration of the previous report during the chromogen formation (0.29 m) was too high comparing to the optimum concentration of the present experiment (0.175 m). An improved method is now proposed which is excellent on the ground of convenience and reproducibility; same readings were obtained with the tetraborate solution from various preparations which were stable at least for two years. It showed same molar extinction coefficient as obtained before.     

New spectrophotometric method for continuous recording of the spleen exonuclease activity

Some of the synthetic chromophoric substrates of various enzymes cannot be used for direct spectrophotometric recording of the reactions, when a difference between the pH optimum of the enzyme reaction and the pH of maximum absorption of the released chromophore exists. In the present paper we describe a new method for following the time course of the spleen exonuclease-catalyzed reaction with thymidine 3'-monophospho-p-nitrophenyl ester as a substrate, based on the difference obtained in the absorbency of the substrate and its products in the far UV (at 330 nm). This difference, not published before, permits direct spectrophotometric recording of the amount of the hydrolyzed chromophoric substrate in acidic pH, whereas the maximum absorption of the product as accepted in the literature, is in alkaline pH. The molar absorption coefficient of the measurement at pH 5.7 is determined to be epsilon = 522 M-1.mm-1.     

Protein determination using bicinchoninic acid in the presence of sulfhydryl reagents

The bicinchoninic acid (BCA) copper reagent, developed for quantification of proteins, was found to react with thiol reagents in a linear and reproducible manner. The reactivity with thiols closely matched the extinction coefficient determined for the Cu(I)-BCA complex [6.6 X 10(3) liters (mol Cu.cm)-1], suggesting that the reaction is quantitative. This reaction interferes with the accurate determination of protein concentrations. A method was developed for determining protein concentrations in the presence of thiol reagents using the BCA protein reagent. The procedure involves preincubation of the protein solution with iodoacetamide prior to addition of the BCA protein reagent. Iodoacetamide does not react with the BCA reagent by itself. In the presence of a 10-fold molar excess of iodoacetamide over thiol equivalents, the reaction of the thiol with the BCA reagent is prevented. The method is simple and allows the assay of solutions of proteins which have been stabilized by the addition of thiol reagents.     

Combined staining procedures for cytophotometry of protein and DNA Feulgen-Naphthol Yellow S and dinitrofluorobenzene-Feulgen

A comparison has been made between dinitrofluorobenzene (DNFB) and Naphthol Yellow S (NYS) as protein stains in combination with the pararosaniline-SO2 Feulgen procedure. Chicken erythrocytes were used as test cells. Cytophotometric measurements were made using a Zeiss scanning stage cytophotometer coupled to a PDP 11/10 minicomputer using the BICOSCAN program to obtain values for protein per cell, protein per "nuclear area' and DNA per nucleus. With 5N HCl as the Feulgen hydrolysis agent, DNFB staining, applied before the Feulgen procedure, was found to be unaffected by hydrolysis conditions required to give optimum Feulgen staining and showed only small losses after longer hydrolysis times. On the other hand measurements of NYS staining, of necessity applied after the Feulgen procedure, seem to be susceptible to the duration of Feulgen hydrolysis. This susceptibility is probably due to the interaction of the DNA phosphates with the basic amino acid residues, potential binding sites for NYS. Since the degree of this interaction may be variable, it is argued that NYS binding will measure the available basicity of proteins at the time of staining but no specific protein fraction. DNFB binding is unaffected by DNA-protein interactions and therefore can give a more reliable measure of "nuclear' protein, particularly in conjunction with Feulgen-DNA measurements.     

A Spectrophotometry Analysis of Tissue Staining

By comparing spectral absorption curves of representative staining solutions and of substances stained with these solutions it is shown that information may be obtained regarding chemical changes associated with the staining process. The stains used in these determinations were acid fuchsin, anilin blue, azo-carmine G, basic fuchsin, eosin Y, orange G, picric acid and Sudan IV. The substrates stained were gelatin, tendon, blood plasma, thymus gland and fat.

Aqueous basic fuchsin and fuchsin-sulfurous reagent to which formalin was added (Setoff reaction) are different stains. The spectral absorption curves for staining solutions and substances stained with the solutions were comparable. Within the limitations of the spectrophotometry methods and stains employed, there was no evidence of significant chemical alteration in the chromophore radicals of the stains associated with the process of tissue staining.     

A spectrophotometric method for quantitation of carboxyl group modification of proteins using Woodward's Reagent K

Reaction of proteins with Woodward's Reagent K in 0.05 ionic strength Tris-HCl, pH 7.8, followed by removal of excess reagent by chromatography on Sephadex G-25 in the same buffer, results in covalently attached chromophores with an absorption maximum at 340 nm and an extinction coefficient of 7000 M-1 cm-1. This absorbance can be used to quantitate the reaction of Woodward's Reagent K with carboxyl groups in proteins, provided sulfhydryl groups do not react. The chromophore also enables specific detection and identification of carboxyl-modified peptides upon separation by chromatography or electrophoresis.     

Dinitrophenylation as a probe for the determination of environments of amino groups in protein. Reactivities of individual amino groups in lysozyme

Dinitrophenylation of hen egg white lysozyme with 2,4-dinitrofluorobenzene (DNFB) was carried out at pH 7-11 and room temperature in order to examine whether dinitrophenylation could be applied to determine the environments of individual amino groups in lysozyme or not. Lightly dinitrophenylated lysozyme was reduced, S-carboxymethylated and then subjected to reversed-phase high-performance liquid chromatography (RP-HPLC). All tryptic peptides, which contained dinitrophenylated amino groups (one alpha-amino group, Lys 1(alpha), and six epsilon-amino groups, Lys 1(epsilon), Lys 13, Lys 33, Lys 96, Lys 97, and Lys 116), could be separated and monitored by absorbance measurement at 360 nm on RP-HPLC. The relative reactivities of individual amino groups, determined from the relative peak areas of dinitrophenylated tryptic peptides at 360 nm, were found to be sensitive to the reaction pH and to the presence of the trimer of N-acetyl-D-glucosamine or NaCl. It was concluded that dinitrophenylation of a protein with DNFB followed by peptide analysis by RP-HPLC with detection at 360 nm is a good method for probing the environments of individual amino groups in the protein.     

The measurement of amino groups in proteins and peptides

A technique is examined for determining amino groups with 2,4,6-trinitrobenzenesulphonic acid, in which the extinction at 420nm of sulphite complexes of the trinitrophenylated amino groups is measured. The sensitivity of the method is 5-200nmol of amino group. The method is especially suitable for checking the extent of blocking or unblocking of amino groups in proteins and peptides, owing to the short time required for reaction (5min at room temperature). The reaction of the reagent with thiol groups has been studied and was found to proceed 30-50 times faster than with in-amino groups of model compounds. The in(420) of a trinitrophenylated thiol group was found to be 2250m(-1).cm(-1). The reaction with several amino acids, peptides and proteins is presented. The in(420) of a typical alpha-amino group was found to be 22000m(-1).cm(-1) and that of an in-amino group, 19200m(-1).cm(-1). Difficulties inherent in the analysis of constituent amino group reactions in proteins are discussed.     

An ultraviolet spectrophotometric assay for measuring lipase activity using long-chain triacyglycerols from Aleurites fordii seeds

In this study, we designed a specific, continuous, and sensitive UV spectrophotometric lipase assay using natural triacylglycerols (TAGs) from the Aleurites fordii seed oil (tung oil). alpha-Eleostearic acid (9,11,13-cis, trans,trans-octadecatrienoic acid) is the main fatty acid component (it accounts for up to 70%) of the TAGs from tung oil. The conjugated triene present in alpha-eleostearic acid constitutes an intrinsic chromophore, which confers strong UV absorption properties on both the free fatty acid and the TAGs from tung oil. The lipase assay is based on the difference between the apparent molar extinction coefficients of the two types of alpha-eleostearic acid present, that which is esterified into TAGs and that which is released into the reaction medium. This difference is responsible for the variations in the UV absorption spectrum of the reaction medium occurring upon enzymatic TAGs hydrolysis. Using the purified lipase from Thermomyces lanuginosa (TLL) and the detergent sodium taurodeoxycholate (NaTDC, 4 mM), it was established that the most suitable method of measuring lipolysis consisted of monitoring the decrease in the OD at 292 nm, which was linear with time and proportional to the amount of lipase added. In order to be able to estimate the specific activity of TLL, we determined an apparent molar extinction coefficient of alpha-eleostearic acid (epsilon = 13,900 M(-1) cm(-1)) under the assay conditions. Amounts of pure TLL as small as 1 ng can be easily detected in the presence of 4 mM NaTDC. Interestingly, the NaTDC concentration can be decreased as far as 0.05 mM. In comparison with other well-known methods of lipase assay, the detection limit of this new method is 100-fold lower than with the pH-stat method and similar to that of a fluorescent assay recently developed at our laboratory.     

Reconstitution of Escherichia coli DNA photolyase with various folate derivatives

DNA photolyase from Escherichia coli contains both flavin and pterin. However, the isolated enzyme is depleted with respect to the pterin chromophore (0.5 mol of pterin/mol of flavin). The extinction coefficient of the pterin chromophore at 360 nm is underestimated by a method used in earlier studies which assumes stoichiometric amounts of pterin and flavin. The extinction coefficient of the pterin chromophore, determined on the basis of its (p-aminobenzoyl)polyglutamate content (epsilon 360 = 25.7 x 10(3) M-1 cm-1), is in good agreement with that expected for a 5,10-methenyltetrahydrofolate derivative. Also consistent with this structure, the pterin chromophore could be reversibly hydrolyzed to yield a 10-formyltetrahydrofolate derivative or reduced to yield a 5-methyltetrahydrofolate derivative. The isolated enzyme could be reconstituted with various folate derivatives to yield enzyme that contained equimolar amounts of pterin and flavin. Similar results were obtained in reconstitution studies with the natural pterin chromophore, with 5,10-methenyltetrahydrofolate, and with 10-formyltetrahydrofolate. The results show that the polyglutamate moiety, previously identified in the natural chromophore, is not critical for binding. Reconstitution with the natural pterin chromophore did not affect catalytic activity. The latter is consistent with our previous studies which show that, although the pterin chromophore acts as a sensitizer in native enzyme, it is not essential for dimer repair which can occur at the same rate under saturating light with flavin (1,5-dihydro-FAD) as the only chromophore.     

Sequence‐specific determination of protein and peptide concentrations by absorbance at 205 nm

Quantitative studies in molecular and structural biology generally require accurate and precise determination of protein concentrations, preferably via a method that is both quick and straightforward to perform. The measurement of ultraviolet absorbance at 280 nm has proven especially useful, since the molar absorptivity (extinction coefficient) at 280 nm can be predicted directly from a protein sequence. This method, however, is only applicable to proteins that contain tryptophan or tyrosine residues. Absorbance at 205 nm, among other wavelengths, has been used as an alternative, although generally using absorptivity values that have to be uniquely calibrated for each protein, or otherwise only roughly estimated. Here, we propose and validate a method for predicting the molar absorptivity of a protein or peptide at 205 nm directly from its amino acid sequence, allowing one to accurately determine the concentrations of proteins that do not contain tyrosine or tryptophan residues. This method is simple to implement, requires no calibration, and should be suitable for a wide range of proteins and peptides.     

A method for determining the individual optical characteristics of posttranslational fluorescent forms of fluorescent proteins with the use of nonlinear laser fluorimetry

A method for determining the individual optical characteristics (fluorescence quantum yield, the rate constant and quantum yield of singlet-triplet conversion, excitation of fluorescence cross-section, extinction coefficient) and concentration correlations between the fluorescent forms of fluorescent proteins arising in the reaction of posttranslational chromophore formation has been developed, which is based on combined application of absorption spectroscopy and classical and nonlinear laser fluorimretry. The method allows one to determine the share of fluorescent forms in the mixture of chromoproteins. The individual optical characteristics of the red form of the fluorescent protein mRFP1 has been determined: the fluorescence quantum yield eta = 0.24 +/- 0.03; the extinction coefficient in the maximum of absorbance band (584 nm) epsilon = 213 +/- 40 mM(-1) cm(-1) (the cross-section of absorbance sigma = (8.2 +/- 1.5).10(-16) cm2); the constant of singlet-triplet conversion rate K32 = (0 +/- 0.6)-10970 s(-1). The part of the red form in the mixture of chromoproteins is 26 +/- 6%.     

A rapid microtitre plate Folin-Ciocalteu method for the assessment of polyphenols

Several methods have been described for the determination of phenolic compounds in animal and plant products using the Folin-Ciocalteu (FC) assay. Most of these methods describe the use of this reagent and sodium carbonate in spectrophotometric methods. The macro FC assay was compared with two micro FC assays carried out on a microplate reader. Excellent correlation was obtained among the three assays with a molar extinction coefficient of 5.228±0.187x10³ M^?1 cm^?1. The micro assay may serve as a high throughput method for the rapid determination of polyphenols in various samples.     

Comparison of Protein Precipitants for the Determination of Free Amino Acids in Plasma

The present study was undertaken to select the most preferred deproteinizing agent for microbiological or chromatographic determination of free amino acids in plasma. The highest quantities of amino acids were obtained from samples treated with picric acid, ethanol or sulfosalicylic acid. It was also found from examining the recoveries of ¹⁴C-labeled amino acids added to plasma following treatment with picric acid, ethanol and sulfosalicylic acid that one kind of deproteinizing agent is not equally effective with all amino acids. Picric acid treatment gave the most reliable results for determination of amino acids except the basic amino acids, alanine and tryptophan. Sulfosalicylic acid was the most recommended deproteinizing agent for basic amino acids and ethanol was good for the assay of alanine and tryptophan. The response of d-amino acids to the deproteinizing agents tested was not always similar to that of their l-isomers.     

Micro method for determination of borohydride with NAD+

A spectrophotometric method for the determination of borohydride is described. It involves the reduction of NAD⁺ to a number of isomeric forms of NADH by borohydride. In the standard assay procedure herewith presented, there is a direct proportionality between the absorption at 340 nm and the amount of borohydride in solution over the range 10–100 nmoles, with an effective “molar extinction coefficient” of 12.2 × 10³. The method is simple, rapid, and sensitive.     

Aqueous solutions of some basic dyes--their use in the cytochemical detection of DNA

The paper contains results of staining DNA-aldehyde molecules with aqueous solutions of brilliant cresyl blue, thionin or neutral red, following Feulgen procedure and also reports on the use of aqueous solutions of these dyes, with primary amino group(s) in their molecules, for staining animal tissue nuclei after extraction of RNA with cold phosphoric acid. The pH of the dye solutions most suitable for optimum staining is 6.0. The time necessary for optimum staining of DNA-aldehydes and DNA-phosphate groups are 10 and 2 min respectively for tissues fixed in formalin, paraformaldehyde or Craf. Tissue fixed in Buin-fluid stain slower. The absorption curves of nuclei stained for DNA-aldehyde molecules and DNA-phosphate groups, stained with each of the three dyes are different from each other. The in vitro absorption curves of aqueous solutions of the three dyes have also been presented. Some implications of the results obtained are discussed.