Romanowsky dyes and the Romanowsky-Giemsa effect. 3. Microspectrophotometric studies of Romanowsky-Giemsa staining. Spectroscopic evidence of a DNA-azure B-eosin Y complex producing the Romanowsky-Giemsa effect

The Romanowsky-Giemsa staining (RG staining) has been studied by means of microspectrophotometry using various staining conditions. As cell material we employed in our model experiments mouse fibroblasts, LM cells. They show a distinct Romanowsky-Giemsa staining pattern. The RG staining was performed with the chemical pure dye stuffs azure B and eosin Y. In addition we stained the cells separately with azure B or eosin Y. Staining parameters were pH value, dye concentration, staining time etc. Besides normal LM cells we also studied cells after RNA or DNA digestion. The spectra of the various cell species were measured with a self constructed microspectrophotometer by photon counting technique. The optical ray pass and the diagramm of electronics are briefly discussed. The nucleus of RG stained LM cells, pH congruent to 7, is purple, the cytoplasm blue. After DNA or RNA digestion the purple respectively blue coloration in the nucleus or the cytoplasm completely disappeares. Therefore DNA and RNA are the preferentially stained biological substrates. In the spectrum of RG stained nuclei, pH congruent to 7, three absorption bands are distinguishable: They are A1 (15400 cm-1, 649 nm), A2 (16800 cm-1, 595 nm) the absorption bands of DNA-bound monomers and dimers of azure B and RB (18100 cm-1, 552 nm) the distinct intense Romanowsky band. Our extensive experimental material shows clearly that RB is produced by a complex of DNA, higher polymers of azure B (degree of association p greater than 2) and eosin Y. The complex is primarily held together by electrostatic interaction: inding of polymer azure B cations to the polyanion DNA generates positively charged binding sites in the DNA-azure B complex which are subsequently occupied by eosin Y anions. It can be spectroscopically shown that the electronic states of the azure B polymers and the attached eosin Y interact. By this interaction the absorption of eosin Y is red shifted and of the azure B polymers blue shifted. The absorption bands of both molecular species overlap and generate the Romanowsky band. Its strong maximum at 18100 cm-1 is due to the eosin Y part of the DNA-azure B-eosin Y complex. The discussed red shift of the eosin Y absorption is the main reason for the purple coloration of RG stained nuclei. Using a special technique it was possible to prepare an artificial DNA-azure B-eosin Y complex with calf thymus DNA as a model nucleic acid and the two dye stuffs azure B and eosin Y.(ABSTRACT TRUNCATED AT 400 WORDS)     

Romanowsky dyes and the Romanowsky-Giemsa effect. 4. Binding of azure B to DNA

We investigated the binding of azure B to DNA (calf thymus) over a wide range of concentrations of the dye (CF) and the nucleic acid (CN) using absorption spectroscopy [CF and CN represent the total concentrations of the ye (F) and the mononucleotide units (N) of the DNA, respectively]. The binding isotherms of the dye to DNA in aqueous solutions were determined. In addition, we analysed the composition of insoluble DNA/azure B precipitates that are formed in presence of an excess of azure B. These precipitates are of particular interest, because Giemsa staining is usually performed using high dye concentrations. Azure B easily forms dimers in aqueous solutions. When determining the binding isotherms, the equilibrium between free monomers and dimers must be taken into account. Therefore, we determined the dimerisation constant (Kd) of azure B from the concentration dependency of its absorption spectra in water at the standard temperature T = 298 K (25 degrees C), Kd = 6.5 X 10(3) M-1 (experimental conditions: tris buffer, pH 7.2; concentration of Na ions, CNa = 0.002 M). As the CNa value increases, the dimerisation constant rises rapidly. When the azure B concentration is very low and there is an excess of DNA, ordinary Scatchard and Langmuir isotherms are observed. Monomer dye cations are bound to DNA, these cations being in equilibrium with free monomers in the solution. In order to obtain the Scatchard binding constant (Ks) and the binding parameter (n) spectroscopically, it is necessary to determine the extinction coefficient (epsilon Fb) of the monomer bound (b) dye molecules (F) at one analytical wave number (upsilon a). The three constants can be determined simultaneously using an iterative technique that combines Scatchard isotherms and the Benesi-Hildebrand extrapolation, CN----infinity. We obtained Ks = 1.8 X 10(5) M-1 and n = 0.18 (25 degrees C; tris buffer, pH 7.2; CNa = 0.002 M). At very low dye (CF) and competitor (CNa) concentrations, only 18% of the anionic binding sites of the DNA are capable of binding the dye cations. With increasing CNa values the concentration of bound azure B cations decreases rapidly. The Na cations displace the bound dye cations and act as a competitor. The Ks value also greatly depends on the competitor concentration (CNa).(ABSTRACT TRUNCATED AT 400 WORDS)     

Model investigations on the structure of the purple dye complex of Giemsa staining

Nuclei of Giemsa stained cells show a purple coloration, which is generated by a complex of DNA, azure B (AB) and eosin Y (EY). The structure of this complex is unknown. Its absorption spectrum shows a sharp and strong band at 18,100 cm-1 (552 nm), the so called Romanowsky band (RB). It is possible to produce the complex outside of the cell, but it is cubersome to handle. Easier to handle is a purple complex composed of chondroitin sulfate (CHS), AB and EY, which also shows a sharp and strong RB at 18,100 cm-1 in the absorption spectrum. This CHS-AB-EY complex is a model for the DNA-AB-EY complex of Giemsa stained cell nuclei. We tried to investigate its structure. In the first step of the staining procedure CHS binds AB cations forming a stable CHS-AB complex. In the case of saturation each anionic SO4- and COO- -binding site of CHS is occupied by one dye cation and the complex has 1:1 composition. It has a strong and broad absorption band with its maximum at ca. 18,000 cm-1 (556 nm). In the second step the CHS-AB complex additionally binds EY dianions forming the purple CHS-AB-EY complex with its RB at 18,100 cm-1. This band can be clearly distinguished from the broad absorption of the bound AB cations. RB is generated by the EY chromophore, whose absorption is shifted to longer wavelength by the interaction with the CHS-AB framework.     

Oxygen binding to dithionite-reduced chloroperoxidase

Both the kinetics of ferric chloroperoxidase reduction by dithionite and the binding of molecular oxygen to ferrous chloroperoxidase have been studied. The oxyferrous chloroperoxidase decays spontaneously to the ferric enzyme. In addition the corresponding rapid-scan spectra have been recorded. The reduction reaction is caused by SO-.2 with a rate constant of (7.7 +/- 1.0) X 10(4) M-1 S-1. Oxygen binding occurs with a rate constant of (5.5 +/- 1.0) X 10(5) M-1 S-1 over the pH range 3.5-6. Oxyferrous chloroperoxidase has a Soret absorption peak at 428 nm and two partially resolved peaks at 555 nm and 588 nm. Isosbestic points occur at the following wavelengths: between ferrous and oxyferrous chloroperoxidase at 419, 545, 555 and 580 nm; between oxyferrous and ferric chloroperoxidase at 419, 487, 540, 609 and 682 nm.     

Thermodynamics of the binding of Streptomyces subtilisin inhibitor to alpha-chymotrypsin

The binding of Streptomyces subtilisin inhibitor (SSI) to alpha-chymotrypsin (CT) (EC 3.4.21.1) was studied by isothermal and differential scanning calorimetry at pH 7.0. Thermodynamic quantities for the binding of SSI to the enzyme were derived as functions of temperature from binding constants (S. Matsumori, B. Tonomura, and K. Hiromi, private communication) and isothermal calorimetric experiments at 5-30 degrees C. At 25 degrees C, the values are delta G degrees b = -29.9 kJ mol-1, delta Hb = +18.7 (+/- 1.3) kJ mol-1, delta S degrees b = +0.16 kJ K-1 mol-1, and delta C p,b = -1.08 (+/- 0.11) kJ mol-1. The binding of SSI to CT is weak compared with its binding to subtilisin [Uehara, Y., Tonomura, B., & Hiromi, K. (1978) J. Biochem. (Tokyo) 84, 1195-1202; Takahashi, K., & Fukada, H. (1985) Biochemistry 24, 297-300]. This difference is due primarily to a less favorable enthalpy change in the formation of the complex with CT. The hydrophobic effect is presumably the major source of the entropy and heat capacity changes which accompany the binding process. The unfolding temperature of the complex is about 7 degrees C higher than that of the free enzyme. The enthalpy and the heat capacity changes for the unfolding of CT were found to be 814 kJ mol-1 and 17.3 kJ K-1 mol-1 at 49 degrees C. The same quantities for the unfolding of the SSI-CT complex are 1183 kJ mol-1 and 39.2 kJ K-1 mol-1 at 57 degrees C.     

Mechanisms of chromosome banding

A thorough understanding of the mechanisms of R-, C-and G-banding will come only from studies of the binding of Giemsa dyes to isolated and characterized preparations of heterochromatin and euchromatin. Since such studies require an exact knowledge of the optical characteristics of Giemsa, the spectral adsorption curves and extinction coefficients of Giemsa and its component dyes at various concentrations in the presence and absence of DNA were determined. — Although Giemsa is a complex mixture of thiazin dyes plus eosin; methylene blue, and azure A, B or C alone gave good banding. Thionin, with no methyl groups, gave poor or no banding. Eosin was not a necessary component for banding. — The most striking characteristic of the thiazin dyes is that they are strongly metachromatic, i.e., their adsorption spectra and extinction coefficients change as the concentration of the dye increases or as they bind to positively charged compounds (chromotropes). These changes, especially for methylene blue, are described in detail and allow a distinction between concentration dependent binding to DNA by intercalation and binding by side stacking.     

Romanowsky dyes and romanowsky-Giemsa effect. 1. Azure B, purity and content of dye samples, association (author's transl)

Azure B is the most important Romanowsky dye. In combination with eosin Y it produces the well known Romanowsky-Giemsa staining pattern on the cell. Usually commercial azure B is strongly contaminated. We prepared a sample of azure B-BF4 which was analytically pure and had no coloured impurities. The substance was used to redetermine the molar extinction coefficient epsilon (v)M of monomeric azur B in alcoholic solution. In the maximum of the long wavelength absorption at v = 15.61 kK (lambda = 641 nm) the absorptivity is epsilon (15.61)M = (9.40 +/- 0.15) x 10(4)M-1 cm-1. This extinction coefficient may be used for standardization of dye samples. In aqeuous solution azur B forms dimers and even higher polymers with increasing concentration. The dissociation constant of the dimers, K = 2,2 x 10(-4)M (293 K), and the absorption spectra of pure monomers and dimers in water have been calculated from the concentration dependence of the spectra using an iterative procedure. The molar extinction coefficient of the monomers at 15.47 kK (646 nm) is epsilon (15.47)M = 7.4 x 10(4)M-1 cm-1. The dimers have two long wavelength absorption bands at 14.60 and 16.80 kK (685 and 595 nm) with very different intensities 2 x 10(4) and 13.5 x 10(4)M-1 cm-1. The spectrum of the dimers in aqueous solution is in agreement with theoretical considerations of F?rster (1946) and Levinson et al. (1957). It agrees with an antiparallel orientation of the molecules in the dimers. It may be that dimers bound to a substrate in the cell have another geometry than dimers in solution. In this case the weak long wavelength absorption of the dimers can increase.     

DNA-protein binding in interphase chromosomes

The metachromatic dye, azure B, was analyzed by microspectrophotometry when bound to DNA fibers and DNA in nuclei with condensed and dispersed chromatin. The interaction of DNA and protein was inferred from the amount of metachromasy (increased β/α-peak) of azure B that resulted after specific removal of various protein fractions. Dye bound to DNA-histone fibers and frog liver nuclei fixed by freeze-methanol substitution shows orthochromatic, blue-green staining under specific staining conditions, while metachromasy (blue or purple color) results from staining DNA fibers without histone or tissue nuclei after protein removal. The dispersed chromatin of hepatocytes was compared to the condensed chromatin of erythrocytes to see whether there were differences in DNA-protein binding in "active" and "inactive" nuclei. Extraction of histones with 0.02 N HCl, acidified alcohol, perchloric acid, and trypsin digestion all resulted in increased dye binding. The amount of metachromasy varied, however; removal of "lysine-rich" histone (extractable with 0.02 N HCl) caused a blue color, and a purplish-red color (µ-peak absorption) resulted from prolonged trypsin digestion. In all cases, the condensed and the dispersed chromatin behaved in the same way, indicating the similarity of protein bound to DNA in condensed and dispersed chromatin. The results appear to indicate that "lysine-rich" histone is bound to adjacent anionic sites of a DNA molecule and that nonhistone protein is located between adjacent DNA molecules in both condensed and dispersed chromatin.     

Induction of metachromasia and circular dichroism in the dye 1,9-dimethyl methylene blue by S. aureus wall teichoic acid

Teichoic acid (TA) isolated from the Gram-positive bacteria Staphylococcus aureus induces blue shifted metachromasia in the dye 1,9-dimethyl methyleneblue (DMMB). Results of conductometric titrations show that DMMB binds to TA with 1:1 stoichiometry. Unlike DNA, this TA does not induce a definite metachromatic band in the dye acridine orange. S. aureus TA also induces strong circular dichroism (CD) in this dye with molar ellipticity value of the order of 10(4). This induction of CD in DMMB supports our earlier inference that TA in dilute solution at neutral pH has a helical conformation, and is not a random coil polymer.     

Biochemical characterization of positive cooperativity in the binding of 1 alpha, 25-dihydroxyvitamin D3 to its chick intestinal chromatin receptor

We have characterized a positive cooperativity mechanism in the binding of 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) to its chick duodenum chromatin receptor. The Hill plot which can take account of the possibility of cooperativity resulted in a much better fitting of the experimental data than the Scatchard model (r = +0.998 versus r = -0.94). Concentrating the chromatin receptor preparation from 10 to 40% resulted in an increase of the Hill coefficient (nH) from 1.09 +/- 0.08 to 1.46 +/- 0.08 (S.D.). Increasing the temperature of incubation from 1 degree C to 40 degrees C resulted in a decrease of nH from 1.46 +/- 0.08 to 1.10 +/- 0.02 (S.D.). The calculation of the thermodynamics of the interaction of 1,25-(OH)2D3 with the second binding site of the receptor (from a Van't Hoff plot) showed that this process occurred spontaneously (delta G0 = -11.6 kcal X mol-1 at 1 degree C), was entropy-driven (delta S0 = +26 cal degree-1 mol-1), and was energy-requiring (delta H0 = -4.37 kcal X mol-1). The temperature controlled reversibility of the cooperativity demonstrates that this phenomenon is not an artifact. Finally, in a study of the rate of dissociation of [3H]1,25-(OH)2D3 from the duodenal receptor preparation, we have found two slopes (k-1 = 32 X 10(-3) min-1; k-2 = 3.2 X 10(-3) min-1); this suggests the existence of two species of receptor. These receptor species could result possibly from either a monomer-dimer system or from a conformational change of a monomer via site-site interactions. In conclusion, the positive cooperativity in the binding of 1,25-(OH)2D3 to the two binding sites of its intestinal receptor is an entropy-driven process and requires energy, is reversible with temperature, and has been shown to take place in concentrated chromatin aggregates.     

Histological, Chromatographic and Spectrophotometric Studies of Toluidine Blue

Chromatographic analysis of commercial batches of toluidine blue shows these to be dye mixtures. Histologically, some samples were found to be poor metachromatic dyes. These unsatisfactory stains contained blue dyes with little or no metachromatic properties as well as a metachromatic fraction. On the other hand, contaminating dyes in histologically satisfactory samples had poor staining qualities and hence did not interfere with the color produced by the metachromatic fraction.

Chromatographic fractionation of different commercial batches of toluidine blue yielded identical, homogeneous metachromatic dyes. These purified dyes had a peak absorption at 615 mμ in contrast to that of purified azure A whose peak absorption was at 622.5 mμ.     

Investigation of Thiazin Dyes as Biological Stains I. The Staining Properties of Thionin and its Derivatives as Compared with Their Chemical Formulae

An investigation has been made of the staining properties of eight dyes of the thionin group. The dyes studied are as follows: tetra-ethyl thionin, asymmetrical di-ethyl thionin, tetra-methyl thionin (methylene blue), tri-methyl thionin (azure B), asymmetrical di-methyl thionin (azure A), symmetrical di-methyl thionin, mono-methyl thionin (azure C), and unsubstituted thionin. The staining properties were tested on sections of paraffin embedded material following five different methods of fixation. No counterstain was employed. It was shown that there was a general correlation between the extent of ethylation or methylation of the dyes and their staining properties. As one passes from tetra-ethyl thionin down the series to thionin itself, there is a progressive decrease in the amount of green showing in the preparations, and an increase in the amount of red present, also an increase in the metachromatic effects, and in the intensity of nuclear staining. There seems, also, to be a similar relation between staining qualities on the one hand and the color and solubility of the dye base on the other.     

Investigation of Thiazin Dyes as Biological Stains I. The Staining Properties of Thionin and its Derivatives as Compared with Their Chemical Formulae

An investigation has been made of the staining properties of eight dyes of the thionin group. The dyes studied are as follows: tetra-ethyl thionin, asymmetrical di-ethyl thionin, tetra-methyl thionin (methylene blue), tri-methyl thionin (azure B), asymmetrical di-methyl thionin (azure A), symmetrical di-methyl thionin, mono-methyl thionin (azure C), and unsubstituted thionin. The staining properties were tested on sections of paraffin embedded material following five different methods of fixation. No counterstain was employed. It was shown that there was a general correlation between the extent of ethylation or methylation of the dyes and their staining properties. As one passes from tetra-ethyl thionin down the series to thionin itself, there is a progressive decrease in the amount of green showing in the preparations, and an increase in the amount of red present, also an increase in the metachromatic effects, and in the intensity of nuclear staining. There seems, also, to be a similar relation between staining qualities on the one hand and the color and solubility of the dye base on the other.     

Liposome formation of egg lecithin and its interaction with iodine

The sonicated dispersion of egg lecithin (phosphatidylcholine) in water forms 1:1 molecular complex with iodine, when its concentration is above 1.6 X 10(-5) M. The thermodynamic and spectrophotometric properties of this complex have been determined. The thermodynamic values are: K (25 degrees C) = 1.6 X 10(3) 1 X mol-1, delta G degrees = -18.4 KJ X mol-1, delta H degrees = -27.4 KJ X mol-1 and delta S degrees = -30.0 J X mol-1 X deg-1. The complex shows two absorption bands: one at 293 nm, which is the charge transfer band and the other at 370 nm, which is the blue shifted visible iodine band at 460 nm in water.     

The thermodynamics of calcium binding to thermolysin

Calcium binding isotherms were determined for thermolysin in the range pH 5.6-10.5, and from 5 to 45 degrees C. An extensive statistical analysis of the binding data suggests that at least two of the four binding sites bind Ca2+ with complete positive cooperativity and independently of the other two. Nonlinear regression analysis of the binding data was used to calculate cooperative (K1) and independent (K2) binding constants for the four calcium sites. Thermodynamic parameters obtained from a van't Hoff analysis indicate that calcium binding to both cooperative and independent sites is an entropy-driven process. At pH 7.0, delta H1 = 90.4 kJ/mol; delta H2 = 97.5 kJ/mol; delta S1 = 456 J K-1 mol-1; delta S2 = 262 J K-1 mol-1. These results are compared to those obtained for other calcium-binding proteins. An analysis of the pH dependence of the calcium binding constants indicates that the binding of four protons at the cooperative site and one to two protons at the independent sites, modulates the calcium affinity. This confirms an earlier structural assignment of the double-site as the locus of the two cooperatively binding Ca2+. Calcium binding to thermolysin is enhanced in the presence of an active site directed inhibitor, suggesting that there may be positive cooperativity between substrate and calcium binding.     

Zinc Chloride Methylene Blue. I. Biological Stain History, Physical Characteristics and Approximation of Azure B Content of Commercial Samples

Zinc chloride methylene blue appeared on the market almost contemporaneously with the zinc-free medicinal form. The former has rarely been reported as being used in blood stains. Recent suspension of manufacture of medicinal methylene blue by it. principal American producer has excited interest in the use of the zinc chloride form for the preparation of blood stains. According to Lillie (1944a,b) the azure B content of zinc chloride methylene blue may have varied from 5 to 30% in the samples studied. Taking the Merck Index (1968, 1976) figures for the spectroscopic absorption maximum (λmax) of 667.8 and 668 nm as standard, recent samples of zinc chloride methylene blue are calculated to contain 6-8% azure B. These figures are baaed on 1) the shift of λmax after exhaustive pH 9.5 chloroform extraction, 2) evaluation of the actual ratio of the observed TiCl₂ dye content to the theoretical for pure zinc chloride methylene blue, 3) comparison of spectroscopic and staining effects of graded hot dichromate oxidation products with those of highly purified azure B-methylene blue mixtures of known proportions.

As far as can be found, medicinal methylene blue is almost the exclusive source of cosin polychrome methylene blue blood stains. Lillie (1944c) included a short series comparing 5 zinc chloride methylene blues with a dozen medicinal methylene blue samples; all were oxidized with hot dichromate to produce successful Wright stains. No effort was made to remove the zinc Exhaustive pH 9.5 chloroform extraction of zinc chloride methylene blue (lot MCB 12-H-29) yielded a small amount of red dye which when extracted into 0.1 N HCI gave λmax = 650. The extraction moved the absorption peak of the zinc chloride methylene blue from 667 to 668 nm and the midpoint of the 90% maximum absorption band, 18 nm wide, from 666.5 to 667.5 nm.     

ON THE NATURE OF THE DYE PENETRATING THE VACUOLE OF VALONIA FROM SOLUTIONS OF METHYLENE BLUE

When uninjured cells of Valonia are placed in methylene blue dissolved in sea water it is found, after 1 to 3 hours, that at pH 5.5 practically no dye penetrates, while at pH 9.5 more enters the vacuole. As the cells become injured more dye enters at pH 5.5, as well as at pH 9.5. No dye in reduced form is found in the sap of uninjured cells exposed from 1 to 3 hours to methylene blue in sea water at both pH values. When uninjured cells are placed in azure B solution, the rate of penetration of dye into the vacuole is found to increase with the rise in the pH value of the external dye solution. The partition coefficient of the dye between chloroform and sea water is higher at pH 9.5 than at pH 5.5 with both methylene blue and azure B. The color of the dye in chloroform absorbed from methylene blue or from azure B in sea water at pH 5.5 is blue, while it is reddish purple when absorbed from methylene blue and azure B at pH 9.5. Dry salt of methylene blue and azure B dissolved in chloroform appears blue. It is shown that chiefly azure B in form of free base is absorbed by chloroform from methylene blue or azure B dissolved in sea water at pH 9.5, but possibly a mixture of methylene blue and azure B in form of salt is absorbed from methylene blue at pH 5.5, and azure B in form of salt is absorbed from azure B in sea water at pH 5.5. Spectrophotometric analysis of the dye shows the following facts. 1. The dye which is absorbed by the cell wall from methylene blue solution is found to be chiefly methylene blue. 2. The dye which has penetrated from methylene blue solution into the vacuole of uninjured cells is found to be azure B or trimethyl thionine, a small amount of which may be present in a solution of methylene blue especially at a high pH value. 3. The dye which has penetrated from methylene blue solution into the vacuole of injured cells is either methylene blue or a mixture of methylene blue and azure B. 4. The dye which is absorbed by chloroform from methylene blue dissolved in sea water is also found to be azure B, when the pH value of the sea water is at 9.5, but it consists of azure B and to a less extent of methylene blue when the pH value is at 5.5. 5. Methylene blue employed for these experiments, when dissolved in sea water, in sap of Valonia, or in artificial sap, gives absorption maxima characteristic of methylene blue. Azure B found in the sap collected from the vacuole cannot be due to the transformation of methylene blue into this dye after methylene blue has penetrated into the vacuole from the external solution because no such transformation detectable by this method is found to take place within 3 hours after dissolving methylene blue in the sap of Valonia. These experiments indicate that the penetration of dye into the vacuole from methylene blue solution represents a diffusion of azure B in the form of free base. This result agrees with the theory that a basic dye penetrates the vacuole of living cells chiefly in the form of free base and only very slightly in the form of salt. But as soon as the cells are injured the methylene blue (in form of salt) enters the vacuole. It is suggested that these experiments do not show that methylene blue does not enter the protoplasm, but they point out the danger of basing any theoretical conclusion as to permeability on oxidation-reduction potential of living cells from experiments made or the penetration of dye from methylene blue solution into the vacuole, without determining the nature of the dye inside and outside the cell.     

Site-specific enthalpic regulation of DNA transcription at bacteriophage lambda OR.

Binding of cI repressor to DNA fragments containing the three specific binding sites of the right operator (OR) of bacteriophage lambda was studied in vitro over the temperature range 5-37 degrees C by quantitative footprint titration. The individual-site isotherms, obtained for binding repressor dimers to each site of wild-type OR and to appropriate mutant operator templates, were analyzed for the Gibbs energies of intrinsic binding and pairwise cooperative interactions. It is found that dimer affinity for each of the three sites varies inversely with temperature, i.e., the binding reactions are enthalpy driven, unlike many protein-DNA reactions. By contrast, the magnitude of the pairwise cooperativity terms describing interaction between adjacently site-bound repressor dimers is quite small. This result in combination with the recent finding that repressor monomer-dimer assembly is highly enthalpy driven (with delta H degrees = -16 kcal mol-1) [Koblan, K. S., & Ackers, G. K. (1991) Biochemistry 30, 7817-7821] indicates that the associative contacts between site-bound repressors that mediate cooperativity are unlikely to be the same as those responsible for dimerization. The intrinsic binding enthalpies for all three sites are negative (exothermic) and nearly temperature-invariant, indicating no heat capacity changes on the scale of those inferred in other protein-DNA systems. However, the three operator sites are affected differentially by temperature: the intrinsic binding free energies for sites OR1 and OR3 change in parallel over the entire range, delta H0OR1 = -23.3 +/- 4.0 kcal mol-1 and delta H0OR3 = -22.7 +/- 1.2 kcal mol-1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Microspectrophotometric studies of Romanowsky stained blood cells. IV. Maturation of myeloid and erythroid cell lines in bone marrow

A quantitative characterization has been made of azure B/eosin stained cells from bone marrow. Two cell lines were followed: the myeloid line (white cell blast, promyelocyte, neutrophilic myelocyte, neutrophilic metamyelocyte, neutrophilic band, neutrophilic segmented) and the erythroid line (rubriblast, prorubricyte, rubricyte, metarubricyte, diffusely basophilic erythrocyte, erythrocyte). A consensus scheme was used to obtain the "true" classification of the cells. Cell types were characterized by three methods: absorbance spectra, dye binding, and chromaticities. Within both cell lines nuclear maturation is accompanied by an overall increase in peak absorbance with little shift in the position of the maximum. Generally, binding of azure B and eosin increases; azure B dimer/monomer ratios show a slight downward trend during maturation. Changes in chromaticities are to bluish purples of increasing saturation. Cytoplasmic changes accompanying maturation are much more striking than nuclear changes, and again the two cell lines show similarities. Generally, there is decreased binding of azure B during maturation. In the erythroid line, the Soret band of hemoglobin becomes increasingly prominent. Chromaticities change from bluish purples to purplish pinks, particularly in the erythroid line.     

The suitability of further thiazine and quinoline dyes for topo-optical reactions on the plasmalemma (author's transl)]

The present studies prove that the thiazin dyes, azure B, azure C and thionin, and the quinolin dyes pinacyanol and its hydrochloride, are suitable for topo-optical staining of the plasmalemma. On the membrane surface the orientated bound dye molecules become stabilized, and with subsequent precipitation the anisotropic effect is reinforced. On optical analysis, the thiazin dye molecules (azure B, AZURE C and Thionin) are bound radially on the membrane. The molecules of the previously studied quinolin dye, N,N'-diethylpseudoisocyanide chloride are bound parallel to the membrane, while pinacyanol and its hydrochloride, like the thiazin dyes, are bound in the radial position.