On the various types of circular dichroism induced on acridine orange bound to poly(S-carboxymethyl-L-cysteine).

Circular dichroism and absorption spectra are measured on mixed solutions of acridine orange and poly(S-carboxymethyl-L -cysteine) at different pH and P/D mixing ratios. The observed circular dichroism spectra are classified into several types, mainly based on the number and sign of circular dichroic bands in the visible region. Three of them are associated with the absorption spectra characteristic of dimeric dye or higher aggregates of dye. Type I is observed with solutions, of which the pH is acid and P/D is higher than 4, and it has an unsymmetrical pair of positive and negative dichroic bands at 470 and 430 nm. This type is induced on the dye bound to the polymer in the β-conformation. Types II and III are considered to be characteristic of randomly coiled polymers. Type II is exhibited by solutions of P/D higher than 1 at pH 5–7 and has two dichroic bands around the same wavelengths as Type I but with opposite signs and an additional positive band at 560 nm. Type III, shown by solutions of P/D 2–0.6 at pH 6–10.5, has three dichroic bands around the same wavelengths as Type II but with signs opposite to it. The other two types of circular dichroism, induced for the solutions of P/D less than 1 at slightly acid pH, are associated with the absorption spectra of monomeric dye and are observed with disordered or randomly coiled polymer. They have a pair of dichroic bands at 540 and 425 nm, and the signs of these bands are opposite to each other in these two types.     

Differentiation between the α-helical and coillike conformations of poly(L-glutamic acid) in aqueous solution through binding of pinacyanol

Pinacyanol in the presence of an excess of poly(L -glutamic acid) [polymer/dye ratio (P/D) > 10] exhibits different absorption spectra in the visible region when bound to the slightly charged polypeptide in the α-helical conformation or to the nearly completely dissociated polypeptide in the coillike conformation. These spectra reveal aggregation of the dye bound to the macromolecular chain in both conformations, although in the coillike one different kinds of aggregates may be present. Dye binding is accompanied by the appearance of CD bands in the visible region which are also different for the α-helical and the coillike conformations. The aggregates formed in the presence of the latter change slowly in time and seem to induce some conformational changes in the polypeptide chain. Furthermore, they have been found to be, at least partially, stable with respect to a subsequent reversal to the α-helical conformation. All results could be qualitatively interpreted assuming that in the coillike conformation, ordered regions exist along the chain as proposed by Krimm and Tiffany.     

Induced optical activity of acridine orange bound to poly-S-carboxymethyl-L-cysteine

The absorption and rotatory properties of acridine orange-poly-S-carboxymethyl-L -cysteine system in water and in 0.2 M NaCl have been measured at different pH and polymer-to-dye mixing ratios. The absorption spectra indicate that the dyes are bound to the polymer in dimeric or highly aggregated forms. At neutral pH where the polymer is randomly coiled, no optical activity is induced on the absorption bands of bound acridine orange. At acid pH where the polymer has the β-conformation, a pair of positive and negative circular dichroic bands occur at each of the absorption bands, centered around 458 and 261 mμ. The signs of those bands are opposite to those found for α-helical poly-L -glutamic acid. A model for the binding of dye to the β-form polymer is presented, in which dimeric dyes are attached to ionized carboxyl groups and slack one another to form linear arrays on both sides of an extended polypeptide chain. The observed circular dichroism spectra can be explained by the Tinoco's exciton mechanism, based on this model. Low molecular weight poly-S-carboxymethyl-L -cysteine induces quite a different circular dichroism on bound acridine orange.     

Induced circular dichroism of acridine orange bound to double-stranded RNA and transfer RNA

The induced circular dichroism (CD) in the visible region of acridine orange bound to the double-stranded RNA from cytoplasmic polyhedrosis virus and to yeast tRNA has been measured as a function of RNA phosphate-to-dye ratio (P/D), under the conditions of 0.01 M Na⁺ at pH 7.0. The shape of the CD spectrum of acridine orange bound to the double-stranded RNA was quite different from the spectrum of the dye bound to DNA. The CD spectral features of acridine orange bound to the double-stranded regions in tRNA closely resembled those of the double-stranded RNA-dye complex, suggesting that the dyes bind similarly to the two RNA's. It was further found that the CD spectrum of the tRNA-dye complex at sufficiently high P/D ratios, which is assignable to monomeric, intercalated dye to the base-paired parts in tRNA, is also distinct from the corresponding spectrum of the DNA-dye complex.     

Unsaturated amino-acid residues as probes for the conformation of polypeptides in solution

Acetyl-(dehydro-Phe) and acetyl-bis(dehydro-Phe) groups have been attached to the ε-amino group of the lysine residues of the copolymer poly(Glu⁹²Lys⁸) by reacting this last with acetyl-(dehydro-Phe)-azlactone and acetyl-bis(dehydro-Phe)-azlactone, respectively. In the latter case induced CD is observed between 250 and 330 nm, due to the relative dissymmetric disposition of the two dehydro-Phe groups under the chiral field of the polypeptide chain. pH dependence of the induced CD, observed for the copolymer and lacking in the lowmolecular-weight structural model, is related to the α-helical and random coiled conformation of the polypeptide chain.     

Molecular conformation of poly-S-carboxymethyl-L-cysteine in aqueous solutions

Poly-S-carboxymethyl-L -cysteine has been prepared by debenzylation of poly-S-carbobenzoxymethyl-L -cysteine with hydrogen bromide in acetic acid. By the infrared spectroscopic method the polymer is found to be in the extended β-conformation with an antiparallel arrangement of polypeptide chains in solid film, if it has been regenerated from dimethyl sulfoxide solution. Aqueous solutions of the polymer have been investigated by measurements of optical rotatory dispersion and viscosity. Various properties sharply change around pH 5 at different ionic strengths. By combining these with infrared studies in D₂O solutions, it has been shown that the polymer exists in the random coil conformation at higher ionization but associates into the intermolecular β-conformation at lower ionization. At the lowest pH attainable in solution, the β-form is partly converted into the random coil as the temperature is raised. The rotatory dispersion of the polymer is described by the Moffitt equation. While the random coil form has a large negative a₀ value and a zero b₀ value, the β-form is characterized by a positive a₀ value and a negative b₀ value, ?130°.     

Circular dichroism of the complex between N-N′-diethylpseudoisocyanine and sodium poly(L-glutamate) in aqueous solutions

In the presence of sodium poly(L -glutamate) at pH = 7.5 the dye pseudoisocyanine in dilute aqueous solution (C_d = 1.10 × 10^?5 M) and low P/D values exhibits an absorption spectrum with a very sharp red-shifted J-band. Under the same conditions circular dichroism (CD) in the visible region is observed with an extremely sharp peak at the position of the J-band. At pH = 4.6, where the polypeptide is in the α-helix conformation, no such J-band is observed and no CD spectrum can be detected at the same P/D values. CD spectra in the uv range demonstrate that the occurrence of the dye–polypeptide complex which gives rise to the J-band at slightly alkaline pH is not accompanied by a conformational transition of the polypeptide towards the α-helical form.     

Conformation of poly-S-carboxyethyl-L-cysteine in aqueous solutions

Poly-S-carboxyethyl-L -cysteine, a higher side-chain homolog of poly-S-carboxymethyl-L -cysteine, has been prepared from poly-S-carbobenzoxyethyl-L -cysteine with hydrogen bromide in chloroform or acetic acid. The polymer is found to be in the β-conformation of an antiparallel arrangement of polypeptide chains in solid films, both in acid and salt forms, when examined by infrared spectra. Aqueous solutions of t he polymer have been investigated by measurements of rotatory dispersion and circular dichroism as well as by infrared spectra in D₂O. These properties show sharp changes around pH 5.5, as the pH of solution is varied. At higher ionization the polymer is randomly coiled, but at lower ionization it is in the β-conformation. Dependence of the rotatory properties upon polymer concentration as well as on ionic strength has been observed even at the lowest degree of ionization attained, and this has been attributed to the formation of intermolecular β-conformation in solutions. The β-structure is characterized by a negative circular dichroic band at 223 mμ and a positive dichroic band at a wavelength lower than 200 mμ, and furt her by a negative b_o value, ?140°. The pH-induced coil-β transition of the polymer is compared with that of poly-S-carboxymethl-L -cysteine.     

Circular dichroism and the pH-induced β-coil transition of poly(S-carboxymethyl-L-cysteine) and its side-chain homolog

The CD of aqueous solutions of poly(S-carboxymethyl-L -cysteine) and poly(S-carboxyethyl-L -cysteine) has been measured at different pH, and the pH-induced β-coil transition is observed by changes in residue ellipticity of dichroic bands around 200 and 225 nm. The residue ellipticity at 200 nm of the former polypeptide is twice as large as that of the latter, when the β-conformation is formed in solution. However, the β-conformation of the latter polypeptide is more stable against electrostatic repulsion than that of the former. The transition curve of poly(S-carboxymethyl-L -cysteine) has also been determined for different molecular weights. The curves were found to be completely coincident with one another if the degree of polymerization were higher than about 100. Such a transition curve is generally divided into three steps: initiation, cooperative formation, and rearrangement of hydrogen bonds. The cooperative step is very sharp, occurring at a constant pH. These steps become agglomerated into two or one when the polypeptide concentration or added salt concentration is increased.     

Interaction between acridine orange and polyriboadenylic acid

The absorption spectra and circular dichroism of aqueous solutions of acridine orange mixed with polY(riboadenylic acid) [poly(rA)] have been measured for different mixing ratios at acid and neutral pH. The binding ratio of dye to poly(rA) has been determined by equilibrium dialysis. At acid pH where poly(rA) is in a double-stranded helix, monomeric dye molecules are intercalated between base pairs, first sparsely and then at neighbouring sites with mutual coupling, as the nucleotide-to-dye mixing ratio decreases. In the presence of excess dye, dimeric dye molecules of antiparallel type are bound to phosphate groups electrostatically and stack together to form linear sequences along a poly(rA) chain. At neutral pH where poly(rA) is single-stranded, isolated intercalation of monomeric dye molecules can occur in the helical parts. At intermediate mixing ratios, half-intercalated dimeric dye molecules are bound to adjacent sites and electronically coupled, inducing characteristic circular dichroism. In the presence of higher amounts of dye, external stacking of dimeric dye molecules of antiparallel type occurs along a poly(rA) chain. The binding of dye cations is suppressed to some degree at acid pH compared to that at neutral pH, owing to the repulsion exerted by protonated adenine bases.     

Structure and optical activity of the DNA-aminoacridine complex

The electronic absorption and circular dichroism spectra of the DNA-acridine orange complex have been measured over a range of ionic strength, pH, and DNA phosphate to dye (P/D) ratios. Three circular dichroism bands associated with the long wavelength absorption band of acridine orange are induced on complex formation with DNA. Two of the dichroism bands, due mainly to dimeric dye molecules, are favored by low ionic strength, low pH (3.2), and a low P/D ratio (～3), while the third, deriving primarily from monomeric dye, is optimum at high ionic strength, neutral pH, and a larger P/D ratio (9). The data suggest that monomeric acridine orange binds to DNA in the form of a left-handed helical array with four dye molecules per turn, while the bound dimer has a skewed sandwich conformation which is itself dissymmetric. The stereochemical relations between the bound monomer dye and the DNA are consistent with a modified intercalation model for the DNA-acridine complex.     

Circular dichroism studies of copper(II)- hyaluronic acid complexes in relation to conformation of the polymer

The study of the Cu(II)-hyaluronate complexes by absorption and CD spectra, as well as by acid–base titration and viscosity, provides information about the nature of ligands and the conformation of the polymer. Three different complexes have been identified. The first (complex I), which is formed between pH 3 and 6, involves mainly the carboxyl groups of the polymer as ligands and is characterized by a strong absorption band at 238 nm. In this complex formation, the CD properties of hyaluronate do not charge appreciably. The second (complex II) forms between pH 6 and 8 bad shows a major change in CD properties. The changes include (1) a new positive CD band at 250 nm and a strong negative on in the π → π* amide transition region and (2) the disappearance of the negative n → π* amide CD band near 210 nm. A sharp increase in absorbance at 238 nm from complex I to II has been attributed to a conformational transition which is also manifested in the CD features of hyaluronate. Complex II involves, in addition to the carboxyl group, the nitrogen atom of the deprotonated acetamido group coordinated to Cu(II). The absorption at 230–280 nm is associated with the optically active charge-transfer transitions involving ligands to metal ion. At higher concentrations of the polymer or at higher pH, complex II aggregates to a gel, complex III. Chondroitin, differing from hyaluronic acid in the C-4 hydroxyl group configuration of the glucosamine moiety, does not show any CD change in the presence of Cu(II).The results provide further support to our fourfold helical structure of Cu(II)–hyaluronate complex at pH between 6 and 8. Intrinsic viscosities of hyaluronate in the presence of the cupric ion is lower than in the presence of other monovalent or bivalent cations, indicating a compact conformation of the polymer when it is complexed with Cu(II).     

Binding of methylene blue to alginate: Effect of acid treatment on metachromasy

The spectral properties of methylene blue (MB) in solutions of Na alginate depend on the severity of prior acid treatment of the polysaccharide. The spectral properties affected are the fraction of MB in monomeric form, the relative amounts of metachromatic dye absorbing near 570 and near 595 nm, and the intensity and sign of circular dichroism (CD) activity associated with the 570 nm bands, at various ratios of polymer equivalents to dye (P/D) from 1300 to 4. Acid treatment consisted of reaction of dry, alcohol-precipitated and presumably native alginate with 0·3m HCl at room temperature for 5 min to 8 h. Acid-induced changes showed immediate (5 min) and slow (4–8 h) stages. In both stages the fractions of MB in monomeric form and in the 595 nm metachromatic form increased. CD activity was little affected by brief acid treatment (except in range P/D=165 to 55), but diminished at all P/D values on prolonged acid treatment. Minor changes were observed in the infrared spectra of alginate films. Fresh alcohol-precipitated alginate, untreated with acid, did not precipitate when dye was in excess, nor did it form gel beads in CaCl₂ solution. It is concluded that dilute acid treatment alters the stereospecific properties of native alginate, perhaps by inducing conformational changes in the constituent copolymer segments.     

Effect of counterions on the conformation of hyaluronic acid.

To study the effect of metal ions on the conformation of hyaluronic acid, circular dichroism (CD) and optical rotatory dispersion (ORD), along with viscosity measurements of the Na, Li, Ca, and Mg salts of the polymer, were carried out. With divalent cations, the results show a decrease in CD minima at 210 nm and an increase in ORD troughs at 220 nm, as compared to monovalent ions. To account for this behavior, the ORD in the visible range corresponding to the observed CD bands was directly calculated from the Moscowitz equation using Kronig-Kramer's transform. The background rotation was found to be more levorotatory in bivalent than in monovalent cations. The ORD spectra of various metal hyaluronates differ significantly from each other in the far ultraviolet region, especially at lower pH values. The values of intrinsic viscosities of these hyaluronates, on the other hand, are almost the same in the pH range of 1–3. These results indicate a local conformation variation rather than any appreciable change in the chain conformation of the molecule in the presence of different counterions.     

Circular dichroism study of modified nucleosides

CD study of four modified nucleosides, constituents of tRNA molecules, revealed that 2-thio-5-methyluridine and 5-methyluridine in aqueous solution, 0.1N HCl, and organic solvents essentially occur in an anti-conformation. 5-Methylcytidine also occurs in an anti-conformation similar to cytidine in aqueous solution and organic solvents, while 2-thiocytidine dihydrate appears to occur in an anti-conformation. It is stressed that the CD data of thionucleosides might be applied to the successfully conformational analysis of tRNA molecules.     

Kinetic and circular dichroism studies of enzymes adsorbed on ultrafine silica particles

Negatively charged ultrafine silica particles (average diameter 20 nm) were used as support materials for adsorption immobilization of porcine trypsin, horseradish peroxidase, and bovine catalase under various conditions, and the changes in the enzyme activities and the circular dichroism (CD) spectra of these enzymes upon adsorption were measured. Since the light scattering intensity of the ultrafine particles was very low, the activities and the CD spectra of the enzymes adsorbed on the particle surfaces could be measured. The enzymes adsorbed at pH around and above their isoelectric points (pI) showed high activities. On the other hand, the enzymes adsorbed at pHs below their pI had significantly diminished activities and showed large CD spectral changes upon adsorption. The extent of CD spectral changes in the enzymes upon adsorption correlated very closely with that of the activity reduction. Therefore, the conformational changes in enzymes upon adsorption are one of the important factors that reduce the activities of adsorbed enzymes. These results demonstrate that the ultrafine particles are not only a novel support for enzyme immobilization but also are helpful for the molecular understanding of the immobilized enzymes. Correspondence to: A. Kondo     

Specificity in the interaction between poly(L-glutamate) and iron (III) complex ions in aqueous solution

The binding process between sodium poly(L -glutamate) and trans-2,2′,2″,2?-tetrapyridyl-Fe(III) complex ions in aqueous solution at pH around 7 has been studied by means of equilibrium dialysis and optical measurements. The binding isotherm indicates the occurrence of a cooperative process, whereby bound molecules facilitate the association of additional molecules. According to circular dichroism (CD) data, this effect is coupled with that which sees a conformational change in the charged polypeptide upon progessive binding of complex counterions. All these features are discussed in the light of the structural characteristics of the interacting species. A stereochemical model of the association “complex” is proposed.     

Soluble and Membrane-bound Quinoprotein D-Glucose Dehydrogenases of the Acinetobacter calcoaceticus: The Binding Process of PQQ to the Apoenzymes

Acinetohacter calcoaceticus LMD 79.41 is a unique bacterium containing a soluble quinoprotein D-glucose dehydrogenase (sGDH) in addition to the membrane-bound quinoprotein D-glucose dehydrogenase (mGDH) which is distributed extensively in Gram-negative bacteria. sGDH has been shown to be a distinct enzyme from mGDH, though both enzymes contain a tightly bound pyrroloquinoline quinone (PQQ) as their prosthetic group. In this study, sGDH was detectable in all strains tested of A. calcoaceticus but not in other Gram-negative bacteria tested, indicating that sGDH can be useful as a taxonomic marker for A. calcoaceticus.

The binding process of PQQ with both enzymes was examined by using the apoenzymes purified from a PQQ-deficient mutant strain of A. calcoaceticus. sGDH was able to bind two moles of PQQ in one mole of the homodimer with a fairly high affinity. The binding reaction was much faster at alkaline pH than at acidic pH, and required the presence of some divalent cations such as Cd²⁺, Ca²⁺, Sr²⁺, or Mn²⁺. On the other hand, mGDH bound one mol of PQQ in the monomeric enzyme with a relatively slow reacting process, which was optimum at acidic pH and in the presence of different types of divalent cations such as Mg²⁺, Ca²⁺, Zn²⁺, or Sr²⁺. Thus, it is suggested that sGDH and mGDH have distinct structures around their PQQ binding site. Furthermore, binding of PQQ affects the conformation of both enzymes, which can be shown from the diminishing intrinsic fluorescence of the enzymes and the increase in resistance against proteolysis upon PQQ binding. Data also suggest that the conformational changes caused by PQQ-binding are more dramatic in sGDH than in mGDH. Based on the results obtained, the differences in PQQ-binding mode between the enzymes and the physiological meanings of sGDH are discussed.     

Characterization of the isolated calcium-binding vesicles from the green alga Mougeotia scalaris,and their relevance to chloroplast movement

The calcium-binding vesicles from the green alga Mougeotia scalaris were isolated and characterized after staining in vivo by neutral red or rhodamine B. They were found to possess, a protonated group with a pK_a-9.9, typifying phenolic hydroxyl groups; upon titration, both, phenolic compound(s) and vital dye were concomitantly released from the vesicular matrix. A shift in peak absorbance from 450 nm to 540 nm of the vitally stained vesicles indicated that the neutral form of neutral red was bound to the vesicular, matrix as an intermediate form, stabilized via intermolecular hydrogen bonds to the phenolic compound(s). Up to 8.5.10⁹ dye molecules were calculated to be adsorbed to a mean-size vesicle. Analysis of Langmuir adsorption isotherms, indicated that there were two binding sites each for both neutral red and rhodamine B. The isolated vesicles were devoid of calcium, probably because vesicular calcium, bound to the vesicle matrix, was displaced upon dye binding. Dye adsorption to the vesicles in vivo results in substantial inhibition of the reorientational movement of the Mougeotia chloroplast and is explained by dye-mediated disorder of the cellular calcium homoeostasis.Abbreviations NR neutral red - RB rhodamine B - SDS sodium dodecyl sulfate This paper is part of the Ph.D. thesis of F. Grolig at Justus-Liebig-Universität Giessen, FRG     

Interaction of sodium decyl sulfate with poly(L-ornithine) and poly(L-lysine) in aqueous solution

The binding isotherms of sodium decyl sulfate to poly(L -ornithine), poly(D ,L -ornithine), and poly(L -lysine) at neutral pH were determined potentiometrically. The nature of a highly cooperative binding in all three cases suggests a micelle-like clustering of the surfactant ions onto the polypeptide side groups. The hydrophobic interaction between the nonpolar groups overshadows the coulombic interaction between the charged groups. The titration curves can be interpreted well by the Zimm–Bragg theory. The average cluster size of bound surfactant ions is sufficiently large to promote the β-structure of (L -Lys)_n even at a very low binding ratio of surfactant to polypeptide residue, whereas the onset of the helical structure for (L -Orn)_n begins after about 7 surfactant ions are bound to two turns of the helix. The CD results are consistent with this explanation.