The induced circular dichroism of bilirubin complexed with the alpha-helix form of poly(L-lysine)

Binding of bilirubin by the alpha-helix conformation of poly(L-lysine) in water induces optical activity. The bisignate circular dichroism spectrum exhibits exciton bands centred at 444 nm, negative, and at 525 nm, positive. The magnitude of the induced circular dichroism depends on the concentration of total bilirubin and total lysine residues, the molar ratio of total lysine residues-to-total bilirubin molecules, the pH and the degree of polymerization of poly(L-lysine). Although bilirubin binds to the random coil conformation of poly(L-lysine), as evidence by the absorption spectrum, the complex is optically inactive. The results suggest that bilirubin binds to the poly(L-lysine) in the form of dimers and oligomers.     

Synthesis of poly(L-Tyr-L-Glu-L-Ala-Gly) and poly(L-Glu-L-Lys-L-Ala) and their circular dichroism spectra in aqueous solution

Two sequential polypeptides, poly(O-benzyl-L -Tyr-γ-benzyl-L -Glu-L -Ala-Gly) and poly(ε-benzyloxycarbonyl-L -Lys-L -Glu-L -Ala), were synthesized, the former by the pentachlorophenyl ester of the tetrapeptide monomer and the latter by the azide of the tripeptide monomer. After deprotection and dialysis, poly(L -Tyr-L -Glu-L -Ala-Gly) was obtained in 71% yield and had a molecular weight of 53,000. The circular dichroism spectra (CD) of the polymer at pH's 7.2, 10.5, and 11.8 and of oligomers and of the monomer at pH 7.2 indicated that the polymer exists in an α-helical conformation. After deprotection, poly(L -Lys-L -Glu-L -Ala) was obtained in 37% yield and had a molecular weight of 3000. The CD spectra of the polymer at pH 7.2 and 2.8, and of the monomer at pH 7.2, indicated that the polymer is in a randomly coiled configuration.     

Induced circular dichroism and mode of binding of acridine orange adsorbed on β-form poly(S-carboxyethyl-L-cysteine) in aqueous solutions

The absorption spectra and circular dichroism (CD) have been measured for aqueous solutions of acridine orange of a constant concentration, [D] = 5 × 10^?5M, mixed with poly(S-carboxyethyl-L -cysteine) in various mixing ratios, [P]/[D], ranging from 330 to 11, at different pH. The absorption spectra of the dye–polymer solutions are hypochromic, and the main band is located at 470 nm, accompanying a shoulder at 500 nm. At alkaline pH, no CD is induced in the visible region. At neutral and acidic pH, where the polymer is in the β-conformation, CD is induced in the visible and near-uv regions. A pair of CD bands is located at the region around 450 nm, when the pH is around the neutrality, while it appears at the region around 500 nm at acidic pH. Thus, the optically active species of bound dye changes from dimer to monomer on lowering the pH. These species form dissymmetric arrays along a polypeptide chain. The fraction of bound dye forming dissymmetric sequences is not high, but most of bound dye is adsorbed randomly on the ionized carboxyl groups of polypeptide chain and gives rise to hypochromism only. A dissymmetric structure of dye–polymer complexes is presented, in which the polymer has the β-conformation and the dye cations, either dimeric or monomeric, bind to its side chains, in such a way that the longer axes of molecular planes of bound dye form a two-fold, right-handed helix along the extended polypeptide chain. A zeroth-order calculation of CD based on the coupled oscillator model leads to the result that each dissymmetric array of dye consists, on the average, of two dimeric or monomeric cations. This low number of bound cations in a dissymmetric array and the large fraction of randomly adsorbed dye suggest that the hydrophobic interaction of dye with the polymer is strong, so that dye cations are adsorbed sparsely on both sides of the extended polypeptide chain.     

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.     

Isodichroic point and the β–random coil transition of poly(S-carboxymethyl-L-cysteine) and poly(S-carboxyethyl-L-cysteine) in the absence of added salt

The β-coil transition of poly(S-carboxymethyl-L -cysteine) (poly[Cys(CH₂CO₂H)]) and poly(S-carboxyethyl-L -cysteine) (poly[Cys((CH₂)₂CO₂H)]) was followed by CD, potentiometric titration, and viscosity in the absence of added salt. These different properties give consistent results for poly[Cys((CH₂)₂CO₂H)]. The CD spectra of poly[Cys(CH₂CO₂H)] change considerably with the degree of neutralization α even for a low-molecular-weight sample incapable of forming the β-structure. Because of the superposition of this additional effect, the dependence of CD on α is inconsistent with titration data for the case of poly[Cys(CH₂CO₂H)], particularly when the nπ transition is used to follow the β-coil transition. The change of CD inherent to the β-coil transition is characterized by an isodichroic point: 215 nm for poly[Cys((CH₂)₂CO₂H)] and 218 nm for poly[Cys(CH₂CO₂H)]. A criterion supporting the stacking of the pleated sheet is suggested based on the isodichroic point.     

Absorption and circular dichroism spectra of methylene blue bound to alginate

The addition of methylene blue to certain samples of Na alginate produces a complex succession of spectrally distinguishable aggregated (metachromatic) dye species. Three of these species are active in CD; they are interpreted as aggregates of dye, but probably dimers, bound in orientations characteristic of the constituent copolymer blocks of alginate to which they are tentatively assigned. The aggregates compete with divalent metal ions and hydrogen ion for the binding sites of the polymer. Other samples of alginate give a modified succession of aggregated spectral species, which are almost devoid of CD activity. Mild treatment with acid, insufficient for hydrolysis, converts forms of alginate with CD activity into modified forms without it, the absorption spectra of which resemble those of samples originally devoid of activity. It is implied, subject to confirmation, that the chiral properties of the binding sites of the native polysaccharide are diminished or lost by acid treatment during commercial preparation.     

Absorption and circular dichroism spectra of different forms of mushroom tyrosinase.

The circular dichroism spectrum of resting mushroom tyrosinase between 800 and 400 nm showed two bands at 755, and 653 nm. The CD spectrum of resting tyrosinase between 400 and 250 nm showed oxygen-sensitive changes at 350 nm upon treatment of tyrosinase with hydroxylamine or hydrogen peroxide. These were similar to changes observed on regeneration of aged hemocyanin by similar procedures. A structural relationship between the active sites of hydroxylamine- or hydrogen peroxide-treated tyrosinase and hemocyanin is suggested by these observations, confirming inferences based upon other studies (Jolly, Jr., R.L., Evans, L.H., Makino, N. and Mason, H.S. (1974) J. Biol. Chem. 249, 335-345 and Schoot Uiterkamp, A.J.M. and Mason, H.S. (1973) Proc. Natl. Acad, Sci. U.S. 70, 993-996).     

Absorption flattening in the circular dichroism spectra of small membrane fragments

The inhomogeneous distribution of chromophore occurring in a particulate suspension can result in a reduction in the apparent molar ellipticity recorded in circular dichroism (CD) spectra. The possibility of such a systematic error has often been a matter of concern when CD spectra of cell membrane proteins are recorded. The recent publication of CD spectra for bacteriorhodopsin in native and sonicated membranes, in detergent-solubilized form, and reconstituted into small unilamellar vesicles [Mao, D., & Wallace, B. A. (1984) Biochemistry 23, 2667-2673] gives a unique opportunity to apply the theoretical analysis of Gordon and Holzwarth [Gordon, D. J., & Holzwarth, G. (1971) Arch. Biochem. Biophys. 142, 481-488] so as to provide a definitive answer to the question of whether absorption flattening is significant for membrane particles. We show here that the data of Mao and Wallace can be combined with the theoretical analysis of Gordon and Holzwarth to rule out significant absorption flattening effects over the range 200-240 nm for submicrometer-sized membranes. In addition, the results show that absorption flattening can be disregarded even at 190 nm for membranous material in the size range below 100 nm. The demonstration that there are no major flattening effects in the CD spectra of bacteriorhodopsin, particularly in the region of 200-240 nm, means that the experimental spectra are incompatible with the proposal that this transmembrane protein contains seven transmembrane helices.     

Vacuum-ultraviolet circular dichroism of chondroitins and their complexes with poly(L-arginine)

The vacuum-ultraviolet circular dichroism (VUCD) of chondroitin and chontroitin-6-sulfate has been measured to 160 nm for films and to 170 nm for D₂O solutions. The pD-dependent dichroic behavior of these glycosaminoglycans in D₂O is similar above 200 nm and is in agreement with previous studies. Near 190 nm, the CD band sign is also dependent on pD. VUCD spectra were recorded for films and solutions of poly(L -arginine). In trifluoroethanol the polypeptide is α-helical, while in D₂O it exists as a random coil. The well-characterized coil–helix transition of poly(L -arginine) during complexation with chondroitin-6-sulfate was observed by VUCD, including the previously inaccessible entire π → π* band. By construction of difference spectra it was also possible to monitor the VUCD of the polysaccharide component during complexation.     

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.     

Circular dichroism of N-phenylnaphthylamine derivatives complexed with the β-form of poly(L-lysine)

Complex formation of 1-anilinonaphthalene-8-sulfonate (ANS) and 2-p-toluidinonaphthalene-6-sulfonate (TNS) with the β-form of poly(L -lysine) [(β-Lys)_n] has been studied by circular dichroism (CD) and absorption spectra measurements. Not only hydrophobic interactions but also hydrogen-bonding and electrostatic interactions contribute to complex formation. The relative importance of these stabilizing factors depends on the relative position of the arylamino group and the sulfonate. For example, ionic interactions play a significant role in the binding of 1,8-ANS and 1,8-TNS, but not in the case of 2,6-TNS. The induced CD of the complexes of (β-Lys)_n with 1,8-ANS and 1,8-TNS is consistent with theoretical calculations for nonplanar conformations of these dyes, twisted in a left-handed sense. As expected for steric reasons, the dominant isomer is one in which the arylamino group is oriented away from the 8-sulfonate (α₁). The induced CD of complexes with 2,6-TNS can be accounted for by an equimolar mixture of left-handed isomers in which the arylamino group is oriented toward the 1-position (β₂) and toward the 3-position (β₁). Our results demonstrate that (β-Lys)_n is capable of chiral discrimination and suggest its general utility for CD studies of racemic anionic dyes.     

Fe(III) and Cu(II) conalbumin visible circular dichroism spectra.

The single polypeptide chain of conalbumin strongly binds two Fe(III) or two Cu(II) ions to yield intense absorption in the visible region similar to that shown by the related protein transferrin. Comparison of the metal-ion-binding sites in the two proteins is made by exploiting the sensitivity to ligand geometry of circular dichroism (CD). For the Fe(III) proteins strong similarities of the CD spectra outweigh marginal differences. For Cu(II) conalbumin an additional negative extremum near 506 nm appears between two positive ones at 634 and 410 nm suggesting greater subtraction of oppositely signed CD components leading to lesser magnitudes for the two positive peaks than are found in Cu(II)-transferrin. The two Fe(III)-binding sites within conalbumin are compared by noting the strong similarities of the CD and MCD of proteins with Fe(III) in one site and Ga(III) in the other site, and vice versa, with the protein containing Fe(III) in both sites. Due to features of the amino acid sequences of the single protein chains, the four strong metal ion binding sites in conalbumin and transferrin cannot be identical in all particulars, yet CD spectra of their metal ion complexes are closely similar. From a study of model phenolate complexes and the wavelength maxima of visible absorption in the Fe(III), Cu(II), and Co(III) proteins near 465, 440, and 405 nm, respectively, these strong absorption bands are identified as ligand to metal ion electron-transfer transitions. It is suggested that tyrosyl residues are the donors in the electron transfer transitions and that they lock in the metal ions after being keyed into position by binding of bicarbonate or other anions.     

Circular dichroism and magnetic circular dichroism spectra of chlorophylls a and b in nematic liquid crystals. II. Magnetic circular dichroism spectra

Absorption and magnetic circular dichroism (MCD) spectra are reported for chlorophyll (Chl) a and Chl b dissolved in nematic liquid crystal solvents. The spectra were measured with the dye molecules oriented uniaxially along the direction of. the magnetic field and measuring light beam. It is significant that under such conditions the MCD spectra recorded in the wavelength region of the Q and Soret bands of the chlorophyll are essentially unchanged with respect to rotation of the sample cell around this axis, even though there is almost complete orientation of the chlorophyll molecules by the liquid crystals. The MCD spectra of Chl a and b in the nematic liquid crystal solvents used in this study are surprisingly similar to the spectra obtained under isotropic conditions. These results illustrate an important technique with which to examine the optical spectra of dyes oriented in liquid crystal matrices in which the anisotropic effects can be reduced the negligible proportions by the application of a strong magnetic field parallel to the direction of the measuring light beam. The first deconvolution calculations are reported that describe the deconvolution of pairs of absorption and MCD spectra, in the Q and B band regions, for both Chl a and b. The spectral analysis to obtain quantitative estimates of transition energies was accomplished by carrying out detailed deconvolution calculations in which the both the absorption and MCD spectral envelopes were fitted with the same number of components; each pair of components had the same hand centres and bandwidth values. This procedure resulted in an assignment of each of the main transitions in the absorption spectra of both Chl a and b. Chl a is clearly monomeric, with Qy, Qx, By and Bx located at 671, 582, 439 and 431 nm, respectively. Analysis of the spectral data for Chl b located Qy, By and Bx, at 662, 476 and 464 nm, respectively.     

Vibrational circular dichroism spectra of three conformationally distinct states and an unordered state of poly(L-lysine) in deuterated aqueous solution

Fourier transform ir vibrational circular dichroism (VCD) spectra in the amide I′ region of poly(L-lysine) in D₂O solutions have confirmed the existence of three distinct conformational states and an unordered conformational state in this homopolypeptide. Characteristic VCD spectra are presented for the right-handed α-helix, the antiparallel β-sheet, an extended helix conformation previously referred to as the so-called “random coil,” and a completely unordered conformation characterized by the absence of any amide I′ VCD. VCD for the antiparallel β-sheet in solution and the unordered chain conformation are presented for the first time. Each of the four different VCD spectra is unique in appearance and lends weight to the view that VCD has the potential to become a sensitive new probe of the secondary structure of proteins in solution.     

Absorption flattening as one cause of distortion of circular dichroism spectra of Delta-RuPhen3 . H2TPPS complex

To extend the model that explains why and how much absorption flattening (AF) influences circular dichroism (CD) signals, we have investigated the interesting case of exciton CD in the Soret region of a noncovalent complex formed by (Delta-RuPhen(3))(2+) and the tetraanionic porphyrin H(2)TPPS. Different concentrations have been studied by using an AF emulator and spectra simulation. The CD spectra of this compound occasionally show distortions in the solution sampling mode with the increase of concentration; the inhomogeneous distribution in the cell volume is due to aggregation and is the source of the AF effect. On the basis of these results, we conclude that AF is an important cause of distortions in CD spectra for Delta-RuPhen(3) . H(2)TPPS complexes and might affect the CD bands of other aggregated systems as well.