New investigations on hematoxylin,hematein, and hematein-aluminium complexes

Summary Analytically pure hematoxylin (Htx), penta-acetylhematoxylin (PAHtx), and hematein (Hm) were isolated and characterized by ¹H-NMR spectroscopy. The VIS/UV spectra of Htx and Hm were recorded in MeOH and in H₂O at various pH values. The molar extinction coefficients of the long wavelength absorption bands are reported. The pK _a value for the 1st acidic dissociation step of Hm has been determined from the pH dependency of the absorption spectra of Hm in aqueous buffer solutions. Finally, the absorption spectra are qualitatively discussed.     

New investigations on hematoxylin, hematein, and hematein-aluminium complexes. II. Hematein-aluminium complexes and hemalum staining.

The absorption spectra of hematein-aluminium solutions have been recorded at various concentrations and pH values; the solutions were prepared using analytically pure hematein and potassium alum as aluminium source. In aqueous solution, four different hematein-aluminium complexes could be distinguished by absorption spectroscopy. In weakly acidic media we observed the violet 1:1 and 1:2 complexes HmAl (VII) and HmAl2(3) (VIII), and in strongly acidic solution the red 1:1 complex HmAl2 (IX). Whereas, in weakly alkaline solution the blue 1:1 complex HmAl0 (X) was detected. By change of the pH value the complexes were mutual interconverted. The dye complexes were characterized by their absorption spectra and molar extinction coefficients. We have stained HeLa cells with the complex solutions under different experimental conditions. In all cases the nuclear staining was intense whereas the staining of the cytoplasm was weak. The microspectra of the stained nuclei were recorded and compared with the absorption spectra of the complexes in solution. Thus it was possible to identify the bound dye species. After staining in acidic media, the cells were red to red-violet depending on the reaction conditions. The three cationic dye species VII, VIII, and IX were bound in varying amounts. After blueing in weakly acidic media or in water, only the violet dye complex VII was detected whereas, after blueing in weakly alkaline media, only the blue complex X has been observed. Enzymatic digestion experiments have shown that the dye complexes in the nuclei were bound to DNA while those in the cytoplasm and nucleoli were bound to RNA. The binding between the dye complexes and the nucleic acids is discussed.     

Romanowsky dyes and Romanowsky-Giemsa effect. 2. Eosin Y, erythrosin B, tetrachlorofluorescein, spectroscopic characterization of pure dyes, association of eosin Y

Analytically pure samples of the Romanowsky dyes eosin y, erythrosin b and tetrachlorofluorescein are prepared. DC of the dye samples shows no contaminations. We measured the absorption spectra of the dye dianions in alkaline aqueous solution and of the dye acids in 95% ethanol at very low dye concentrations. The molar extinction coefficients of the long wavelength absorption of the monomeric dye species are determined (Table 1). The extinction coefficients may be used for standardisation of dye samples. The absorption spectra of eosin y in aqueous solution are dependent on concentration. Using a new very sensitive method it was possible to identify two association equilibria from the concentration dependency of the spectra. Dimers are formed even in very dilute solutions, at higher concentrations tetramers. The dissociation constant of the dimers D in monomers M at 293 K, pH = 12, is K21 = 2,9 X 10(-5) M; of the tetramers Q in dimers D K42 = 2,4 X 10(-3) M. From the experimental spectra of eosin solutions at various concentrations, pH = 12, and the equilibrium constants K21, K42 the absorption spectra of the pure monomers, dimers and tetramers are calculated. M has one long wavelength absorption band, VM = 19300 cm-1, epsilon M = 1,03 X 10(5) M-1 cm-1; D also one absorption band, VD = 19300 cm-1, epsilon D = 1,74 X 10(5) M-1 cm-1; Q two absorption bands, VQ1 = 19100, VQ2 = 20200 cm-1, epsilon Q1 = 1,65 X 10(5), epsilon Q2 = 1,96 X 10(5) M-1 cm-1. The absorption spectrum of the dimers is discussed by quantum mechanics.     

Nucleic acid vibrational circular dichroism, absorption, and linear dichroism spectra. I. A DeVoe theory approach.

Infrared (IR) vibrational circular dichroism (VCD), absorption, and linear dichroism (LD) spectra of four homopolyribonucleotides, poly(rA), poly(rG), poly(rC), and poly(rU), have been calculated, in the 1750-1550 cm-1 spectral region, using the DeVoe polarizability theory. A newly derived algorithm, which approximates the Hilbert transform of imaginaries to reals, was used in the calculations to obtain real parts of oscillator polarizabilities associated with each normal mode. The calculated spectra of the polynucleotides were compared with previously measured solution spectra. The good agreement between calculated and measured polynucleotide spectra indicates, for the first time, that the DeVoe theory is a useful means of calculating the VCD and IR absorption spectra of polynucleotides. For the first time, calculated DeVoe theory VCD and IR absorption spectra of oriented polynucleotides are presented. The calculated VCD spectra for the oriented polynucleotides are used to predict the spectra for such measurements made in the future. The calculated IR spectra for the oriented polynucleotides are useful in interpreting the linear dichroism of the polynucleotides.     

Raman and IR study on copper binding of histamine

A comparative Raman and FTIR study of histamine (Hm), a small hormone present in a wide selection of living organisms, and its complexes with copper(II) at different pH values was carried out. Both the Raman and IR spectra present some marker bands useful for the identification of the structure of the species predominating in the Cu(II) aqueous and alcoholic systems. In particular, Raman spectroscopy appears to be a useful tool for analyzing the tautomeric equilibrium of the imidazole ring of Hm, because some bands (i.e., nuC(4)dbond;C(5)) appear at different wavenumbers, depending on whether the imidazole moiety is in the N(tau)-H (tautomer I) or N(pi)-H (tautomer II) protonated form. In aqueous solutions the manner in which Hm binds to Cu(II) depends on the pH. At basic pH the most relevant species formed are a dimer, [Cu(2)L(2)H(-2)](2+), and a monomeric complex, [CuL](2-) or [CuL(2)](+). On the contrary, by decreasing the pH, Hm acts as a mono- or bidentate ligand, giving rise to two types of monomeric complexes, [CuLH](2-) and [CuL](2-) or [CuL(2)](+). With respect to the Cu(II)-Hm alcoholic system, both the aminic group and the imidazole ring (tautomer I) take part in the Cu(II) coordination, leading to the formation of the [CuL](2-) or [CuL(2)](+) monomeric complex.     

1H and 13C NMR spectroscopic studies of the ferriheme resonances of three low-spin complexes of wild-type nitrophorin 2 and nitrophorin 2(V24E) as a function of pH

The ferriheme resonances of the low-spin (S = 1/2) complexes of wild-type (wt) nitrophorin 2 (NP2) and its heme pocket mutant NP2(V24E) with imidazole (ImH), histamine (Hm), and cyanide (CN⁻) as the sixth ligand have been investigated by NMR spectroscopy as a function of pH (4.0–7.5). For the three wt NP2 complexes, the ratio of the two possible heme orientational isomers, A and B, remains almost unchanged (ratio of A:B approximately 1:6 to 1:5) over this wide pH range. However, strong chemical exchange cross peaks appear in the nuclear Overhauser effect spectroscopy/exchange spectroscopy (NOESY/EXSY) spectra for the heme methyl resonances at low pH (pH* 4.0–5.5), which indicate chemical exchange between two species. We have shown these to be two different exogenous ImH or Hm orientations that are denoted B and B′, with the ImH plane nearly parallel and perpendicular to the ImH plane of the protein-provided His57, respectively. The wt NP2–CN complex also shows EXSY cross peaks due to chemical exchange, which is shown to be a result of interchange between two ruffling distortions of the heme. The same ruffling distortion interchange is also responsible for the ImH and Hm chemical exchange. For the three NP2(V24E) ligand complexes, no EXSY cross peaks are observed, but the A:B ratios change dramatically with pH. The fact that heme favors the A orientation highly for NP2(V24E) at low pH as compared with wt NP2 is believed to be due to the steric effect of the V24E mutation. The existence of the B′ species at lower pH for wt NP2 complexes and the increase in A heme orientation at lower pH for NP2(V24E) are believed to be a result of a change in structure near Glu53 when it is protonated at low pH. ¹H{¹³C} heteronuclear multiple quantum coherence (HMQC) spectra are very helpful for the assignment of heme and nearby protein side chain resonances.     

Prototropic equilibria, tautomerization and electronic absorption properties of dibenzofluorescein in aqueous solution related to its capability as a fluorescence probe

The protolytic equilibria of 1,2,7,8-dibenzofluorescein in aqueous solution have been characterized by visible absorption and fluorescence spectra. The species involved are identified as dianion, monoanion, neutral form and cation. The neutral form includes both the quinoid and lactone structures. The pK(a)s were calculated by an improved procedure to be 3.14, 4.04 and 6.28, respectively. The absorption spectra for each protolytic form were resolved. The absorption maxima (molar absorption coefficient, x10(5), M(-1) cm(-1)) are 532 nm (0.87) for the dianion, 510 nm (0.39) for the monoanion, 500 nm (0.16) for the neutral form, and 494 nm (0.19) for the cation, respectively. Contrary to the assumption in the literature, we found that the monoanion is highly fluorescent (Phi(f) = 0.66, compared to Phi(f) = 0.25 for dianion) and its molar ratio can reach 50% at neutral pH. It is therefore concluded that under physiological pH conditions the monoanion plays a major role when it is used as a fluorescence probe.     

Effect of pH on Absorption Spectra of Pea 114 and 121 Kilodalton Phytochromes during and after Red-Light Irradiation

Absorption spectra of pea 114 and 121 kDa phytochromes weremeasured at pH 6.8, 7.8 and 8.8 using a custom-made transientmultichannel spectrum analyzer. The absorption spectra of 114kDa phytochrome as P_R and P_FR were least affected by mediumpH. The absorption spectra at photostationary state under redlight, however, were different under the three different pHconditions, and were different from those obtained 55 s afterred-light irradiation, owing to rapid pH-dependent absorbanceincrease in both red and far-red regions in the dark. In contrast,the absorption spectra of 121 kDa phytochrome were significantlyless affected by medium pH. The absorption spectra measuredat the photostationary state showed a lower P_FR peak at higherpH. The absorption spectra obtained 55 s after the irradiationwere similar under the three pH conditions since the rapid absorbanceincrease in the far-red region in the dark was small. Possibleaccumulation of 114 kDa phytochrome population(s) with low absorbanceat red-light-induced photostationary state at pH 8.8, and theprotective role of the 7 kDa polypeptide at the amino terminusagainst the pH effect in 121 kDa phytochrome are discussed. (Received February 1, 1986; Accepted April 1, 1986)     

Anthocyanin from strawberry (Fragaria ananassa) with the novel aglycone, 5-carboxypyranopelargonidin

An anthocyanin, 1, with the novel 4-substituted aglycone, 5-carboxypyranopelargonidin, was isolated in small amounts from the acidified, methanolic extract of strawberries, Fragaria ananassa Duch., by preparative HPLC after purification by partition against ethyl acetate, Amberlite XAD-7 and Sephadex LH-20 column chromatography. It was identified mainly by 2D NMR spectroscopy and electrospray LC-MS as the 3-O-beta-glucopyranoside of 5-carboxy-2-(4-hydroxyphenyl)-3,8-dihydroxy-pyrano[4,3,2-de]-1-benzopyrylium, an anthocyanidin which is homologous to 5-carboxypyranomalvidin (vitisidin A) reported in red wines and 5-carboxypyranocyanidin recently isolated from red onions. By comparison of UV-Vis absorption spectra, 1 showed in contrast to 2, pelargonidin 3-O-beta-glucopyranoside, a local absorption peak around 360 nm, a hypsochromic shift (8 nm) of the visible absorption maximum, and lack of a distinct UV absorption peak around 280 nm. The similarities between the absorption spectra of 1 in various acidic and neutral buffer solutions implied restricted formation of the instable colourless equilibrium forms, which are typical for most anthocyanins in weakly acidic solutions. The molar absorptivity (epsilon) of 1 varied little with pH contrary to similar values of for instance the major anthocyanin in strawberry, 2. However, 2 revealed higher epsilon-values than 1 at all pH values except 5.1. At pH 5.1, the epsilon-value of 1 (6250) was nearly four times the corresponding value of 2 (1720), which showed the potential of 5-carboxypyranopelargonidin derivatives as colorants in solutions with pH around 5. The colours of 1 and 2 in buffered solutions with pH 1.1 and pH 6.9 have been described by the CIELAB coordinates h(ab) (hue angle), C* (chroma), and L* (lightness).     

Axial ligand complexes of the<Emphasis Type="Italic"> Rhodnius</Emphasis> nitrophorins: reduction potentials,binding constants,EPR spectra,and structures of the 4-iodopyrazole and imidazole complexes of NP4

Previously, we utilized 4-iodopyrazole (4IPzH) as a heavy atom derivative for the initial solution of the crystal structure of the nitrophorin from Rhodnius prolixus, NP1, where it was found to bind to the heme with the iodo group disordered in two positions. We have now determined the structure of the 4IPzH complex of NP4 at pH 7.5 and find that the geometry and bond lengths at the iron center are extremely similar to those of the imidazole (ImH) complex of the same protein (structure determined at pH 5.6), except that the G–H loop is not in the closed conformation. 4IPzH binds to the heme of NP4 in an ordered manner, with the iodo substituent pointed toward the opening of the heme pocket, near the surface of the protein. In order to understand the solution chemistry in terms of the relative binding abilities of 4IPzH, ImH, and histamine (Hm, a physiological ligand for the nitrophorins), we have also investigated the equilibrium binding constants and reduction potentials of these three ligand complexes of the four Rhodnius nitrophorins as a function of pH. We have found that, unlike the other Lewis bases, 4IPzH forms less stable complexes with the Fe(III) than the Fe(II) oxidation states of NP1 and NP4, and similar stability for the two oxidation states of NP2 and NP3, suggesting that this ligand is a softer base than ImH or Hm, for both of which the Fe(III) complexes are more stable than those of Fe(II) for all four nitrophorins. Surprisingly, in spite of this and the much lower basicity of 4IPzH than imidazole and histamine, the EPR g-values of all three ligand complexes are very similar.Abbreviations NP1–4 nitrophorins 1–4 from Rhodnius prolixus - 4IPzH 4-iodopyrazole - ImH imidazole - Hm histamine - NO nitric oxide - NOS nitric oxide synthase     

Nucleic acid vibrational circular dichroism, absorption, and linear dichroism spectra. II. A DeVoe theory approach.

The DeVoe polarizability theory is used to calculate vibrational circular dichroism (VCD) and infrared (IR) absorption spectra of four polyribonucleotides: poly(rA) x poly(rU), poly(rU) x poly(rA) x poly(rU), poly(rG) x poly(rC), and poly(rC+) x poly(rI) x poly(rC). This is the first report on the use of the DeVoe theory to calculate VCD, oriented VCD, IR absorption, and IR linear dichroism (LD) spectra of double- and triple-stranded polyribonucleotides. Results are reported for DeVoe theory calculations--within the base-stretching 1750-1550 cm(-1) spectral region--on several proposed multistranded polyribonucleotide geometries. The calculated spectra obtained from these proposed geometries are compared with previously reported measured and calculated VCD and IR spectral results. Base-base hydrogen-bonding effects on the frequencies and magnitudes of the base carbonyl stretching modes are explicitly considered. The good agreements found between calculated and measured spectra are proposed to be further evidence of the usefulness of the DeVoe theory in drawing three-dimensional structural conclusions from measured polyribonucleotide VCD and IR spectra.     

The green hemoproteins of bovine erythrocytes. II. Spectral, ligand-binding, and electrochemical properties

The two green hemoproteins isolated from bovine erythrocytes (form I and form II) have been characterized as to spectral, electrochemical, and chemical properties. The absorption spectra of the isolated hemoproteins are typical of high spin ferric states. Reduction of the hemoproteins yields high spin ferrohemoproteins. Complexation of the ferrohemoproteins with CO and the ferrihemoproteins with cyanide yields low spin complexes, demonstrating the presence of an exchangeable weak field ligand in both the ferrous and ferric states of the hemoproteins. The differences in position and intensity of the absorption peaks of the visible spectra allow the two forms to be distinguished from one another. The midpoint potential of forms I and II were found to be +0.075 and +0.019 V, respectively, at pH 6.4 and +0.038 and -0.005 V, respectively, at pH 7.0. This is consistent with the gaining of 1 proton/electron during the reduction. The Nernst plot reveals an unusual 0.5-electron transfer, whereas a quantitative titration demonstrates a 1-electron transfer. Form I binds cyanide more tightly than form II (KD of 84 and 252 micrometer, respectively). The observed spectral, electrochemical, and ligand-binding differences between forms I and II can be explained in terms of a greater electron-withdrawing ability of the side chains of the heme of form I relative to the heme of form II.     

Survey of genome sequences in a wild sweet potato,Ipomoea trifida (H. B. K.) G. Don

Ipomoea trifida (H. B. K.) G. Don. is the most likely diploid ancestor of the hexaploid sweet potato, I. batatas (L.) Lam. To assist in analysis of the sweet potato genome, de novo whole-genome sequencing was performed with two lines of I. trifida, namely the selfed line Mx23Hm and the highly heterozygous line 0431-1, using the Illumina HiSeq platform. We classified the sequences thus obtained as either ‘core candidates’ (common to the two lines) or ‘line specific’. The total lengths of the assembled sequences of Mx23Hm (ITR_r1.0) was 513 Mb, while that of 0431-1 (ITRk_r1.0) was 712 Mb. Of the assembled sequences, 240 Mb (Mx23Hm) and 353 Mb (0431-1) were classified into core candidate sequences. A total of 62,407 (62.4 Mb) and 109,449 (87.2 Mb) putative genes were identified, respectively, in the genomes of Mx23Hm and 0431-1, of which 11,823 were derived from core sequences of Mx23Hm, while 28,831 were from the core candidate sequence of 0431-1. There were a total of 1,464,173 single-nucleotide polymorphisms and 16,682 copy number variations (CNVs) in the two assembled genomic sequences (under the condition of log₂ ratio of >1 and CNV size >1,000 bases). The results presented here are expected to contribute to the progress of genomic and genetic studies of I. trifida, as well as studies of the sweet potato and the genus Ipomoea in general.     

Anthocyanin,flavonol copigments,and pH responsible for larkspur flower color

The anthocyanin and flavonol glycosides in Larkspur flowers (cv. Dark Blue Supreme) are delphinidin 3-di(p-hydroxybenzoyl)glucosylglucoside, kaempferol 3-robinobioside-7-rhamnoside (robinin), kaempferol 3-rutinoside, kaempferol 7-rhamnoside, and kaempferol 3-(caffeylgalactosylxyloside)-7-rhamnoside. As young flowers age the pH of epidermal tissue increases from 5·5 to 6·6 and the color of many of the cells changes from moderate reddish-purple to light purplish-blue. Many of the older cells also contain blue crystals. Visible absorption spectra of moderate reddish-purple and light purplish-blue cells were simulated with a solution of the anthocyanin (10⁻² M) plus robinin (5 × 10⁻³ M) at pH 5·6 and 7·1, respectively. Changes in the absorption spectra of living tissue with heating or cooling and of concentrated solutions of the anthocyanin with dilution or moderate heat, indicate that in the natural state the pigment is present in an associated form.     

Polynucleotides. XLVI. 1 Synthesis and properties of poly (2''-amino-2''-deoxyadenylic acid).

Poly (2'-amino-2'-deoxyadenylic acid) [poly (Aa)] was prepared from chemically synthesized 2'-amino-2'-deoxy-ADP by the catalysis of polynucleotide phosphorylase. Poly (Aa) showed a similar UV absorption spectra to poly (A), but quite different CD spectra at pH 7.0 and 5.7. At the former pH it showed a single negative Cotton band and at the latter a curve with a large splitting of bands. Acid titration of poly (Aa) suggested protonated form below pH 7.0. Temperature absorption profiles and their dependency on sodium ion concentration suggested an ordered structure for poly (Aa) which is stabilized by stacking of bases and intrastrand interaction between 2'-amino and internucleotidic phosphate groups. Poly (Aa) forms a 1:2 complex with poly (U) at neutrality and its Tm was 45 degrees in the presence of 0.15M sodium ion.     

Development of new strains and related SCAR markers for an edible mushroom, Hypsizygus marmoreus

New fast-growing and less bitter varieties of Hypsizygus marmoreus were developed by crossing monokaryotic mycelia from a commercial strain (Hm1-1) and a wild strain (Hm3-10). Six of the better tasting new strains with a shorter cultivation period were selected from 400 crosses in a large-scale cultivation experiment. We attempted to develop sequence characterized amplified region (SCAR) markers to identify the new strain from other commercial strains. For the SCAR markers, we conducted molecular genetic analysis on a wild strain and the eight most cultivated H. marmoreus strains collected from various areas in East Asia by randomly amplified polymorphic DNA. Ten unique DNA bands for a commercial Hm1-1 strain and the Hm3-10 strain were extracted and their sequences were determined. Primer sets were designed based on the determined sequences. PCR reactions with the primer sets revealed that four primer sets successfully discriminated the new strains from other commercial strains and are thus suitable for commercial purposes.     

Synthesis and characterization of six sequential polypeptides containing tyrosine, glutamic acid, alanine, and glycine by circular dichroism and difference spectroscopy

Six sequential polytetrapeptides containing equimolar amounts of tyrosine, glutamic acid, alanine, and glycine were characterized by CD and difference spectroscopy over a wide range of pH. As the pH was lowered from physiological values, each of the polymers underwent pH-sensitive transitions. The CD spectra indicated that two polymers, poly(Tyr-Glu-Ala-Gly) and poly(Tyr-Ala-Glu-Gly), had some α-helical conformation at pH 7.0 and approached maximum helicity around pH 6.0; two others, poly(Ala-Tyr-Glu-Gly) and poly(Glu-Ala-Tyr-Gly), had no α-helical conformation at pH 7.0 and about one-third of the ellipticities of the above two polymers at pH 5.5; and the remaining two, poly(Ala-Glu-Tyr-Gly) and poly(Glu-Tyr-Ala-Gly) had little or no α-helix, even at pH 5.5. Difference spectroscopy at 286 nm yielded results quite different. The molar extinction coefficients for poly(Tyr-Glu-Ala-Gly) and poly(Tyr-Ala-Glu-Gly) continued to change, even below pH 5.5, and the total changes in absorbance between pH 8.0 and 4.5 were of intermediate magnitudes among the six polymers. Poly(Ala-Tyr-Glu-Gly) and poly(Glu-Ala-Tyr-Gly), which had similar CD spectra, had the lowest and highest pH-related changes in the molar extinction coefficients. It thus appears that amino acid composition alone cannot account for the apparent differences in conformation among the polytetrapeptides. Other factors, such as amno acid sequence, must play a major role in the determination of conformation. The intrinsic viscosity of poly(Tyr-Glu-Ala-Gly) increased markedly between pH 6.0 and 5.5, which was below the pH of the CD transition but above the pH at which the largest absorption perturbation change, at 286 nm, took place. The model that can best account for the relatively low pH at which the absorption transition of tyrosine occurred is a progressive immobilization of side chains in the α-helix as the pH decreases.     

Electron paramagnetic resonance studies on Pseudomonas nitrosyl nitrite reductase. Evidence for multiple species in the electron paramagnetic resonance spectra of nitrosyl haemoproteins.

The e.p.r. spectra of reduced 14NO- and 15NO-bound Pseudomonas nitrite reductase have been investigated at pH 5.8 and 8.0 in four buffer systems. At pH 8.0, absorption spectra indicated that only the haem d1 was NO-bound, but, although quantification of the e.p.r. signals in all cases accounted for NO bound the the haem d1 in both subunits of the enzyme, the precise form of the signals varied with buffer and temperature. A rhombic species, with gx = 2.07, gz = 2.01 and gy = 1.96, represented in the low-temperature spectra seen in all the buffers was converted at high temperatures (approx. 200K) into a form showing a reduced anisotropy. Hyperfine splitting on the gz component of this rhombic signal indicated a nitrogen atom trans to NO and it is proposed that histidine provides the endogenous axial ligand for haem d1. At pH 5.8, absorption spectra indicated NO binding to both haems c and d1 and e.p.r. quantifications accounted for NO-bound haems c and d1 in both enzyme subunits. The e.p.r. spectra at pH 5.8 were generally similar to those at pH 8.0 with respect to g-values and hyperfine coupling constants, but were broader with less well defined hyperfine splittings. As at pH 8, rhombic signals present in spectra at low temperatures were converted to less anisotropic forms at high temperatures. The results are discussed in relation to work on model nitrosyl-protohaem complexes [Yoshimura, Ozaki, Shintani & Watanabe (1979) Arch. Biochem, Biophys. 193, 301-313]. No. e.p.r. signal was observed from oxidized NO-bound Pseudomonas nitrite reductase at pH 6.0, over the temperature range 6-100K.     

Study of the structure of the histidine decarboxylase of Micrococcus sp. n. by the method of circular dichroism]

Ring dichroism spectra (RD) of histidine decarboxylase (HDC) from Micrococcus sp. n. at the regions of peptide bonds (200-240 nm) and aromatic amino acids (250-300 nm) absorption are studied. The treatment of RD spectra according to methods of Greenfield-Fasman, Saksena-Vetlaufer and Mayer permits to conclude that at the pH range within 4-8 the content of ordered structures of alpha-helix type comprises 20%, that of beta-structure type-40%, while the rest 40% are represented with polypeptide chain in a disordered globular state. When pH is varied from 1 to 12, the content of alpha-helices decreases from 17 to 5%. There are two distinct dichroic bands in the spectrum of aromatic chromophores absorption (at 270 and 290 nm), the former containing tirosine, tryptophane and phenylalanine residues and the latter being induced with triptophane residues. The study of HDC RD spectra at the regions of peptide bonds and aromatic acids absorption at different temperatures has shown that a part of triptophane, tyrosine and phenylalanine residues is in an ordered structure of the alpha-helix type. The HDC undergoes irreversible changes under heating to 70 degrees and in 8 M urea. 5 M guanidine chloride eliminates the ordered HDC structure, while sodium dodecylsulphate at concentrations up to 1% does not affect the enzyme structure.     

Calorimetric study of the complexes between polyuridylic acid and adenylic nucleotides.

Soluble complexes of poly (U) and adenylic nucleotides in NaCl solutions were studied by scanning microcalorimetry. The melting enthalpies, delta Hm, of poly (U) complexes with adenosine, 2',3' -cAMP, 2'(3')-AMP, 5-AMP, ADP, ATP in 1 M NaCl are 50.5; 45.0; 42.9; 28.6; 26.1 and 25.6 kJ/mole triplets, respectively. Delta Hm is independent of the complex melting temperature, Tm. The calorimetric enthalpies are considerably lower than the apparent delta Hv.H. obtained from Tm dependence on free monomer concentration. The enthalpy of complex formation in 1 M NaCl depends neither ob the number nor on the degree of ionization of the phosphate groups but is essentially determined by their 5' - or 2'(3')-position. In contrast to 2'(3')- AMP. 2 poly (U), delta Hm of 5'AMP. 2 poly (U) increases considerably at lowering Na+ concentration. The enthalpy of poly (U) double helix melting in 1 M NaCl is 8.8 kJ/mole pairs which is 2.5 times lower than that in MgCl2 solutions.