A circular dichroism study uncovers a two-step interaction of antitumor azolato-bridged dinuclear platinum(II) complexes with calf thymus DNA

The interactions of four antitumor azolato-bridged dinuclear platinum(II) complexes, [{cis-Pt(NH(3))(2)}(2)(μ-OH)(μ-azolato)](2+), with calf thymus DNA were monitored dose- and time-dependently, by using circular dichroism. Complexes 1-4 reacted with DNA via a two-step interaction that comprised a prompt diffusion-controlled reaction, which induced a B- to C-form transition, and a relatively slow temperature-dependent reaction.     

Magnetic circular dichroism of myoglobin-thiolate complexes.

Various complexes of myoglobin (Mb) with thiolate were studied by use of magnetic circular dichroism (MCD) spectroscopy. 1. MetMb-ethyl, n-propyl and isopropylmercaptan complexes offered MCD spectra similar to that of cytochrome P-450 (P-450) with respect to shape and intensity ratio of Soret MCD to Q0-0 MCD. The MCD spectra did not show any pH dependence. The complexes reduced by sodium dithionite exhibited the MCD spectrum of deoxyMb, indicative of release of thiolate anion from the heme iron. 2. Cysteine and cysteine methyl ester coordinated to the heme iron at pH 9.18 but not at pH 6.86 and 11.45. The complex formed at pH 9.18 gave an MCD spectrum similar to that of P-450, and an MCD spectrum of deoxy Mb on reduction with sodium dithionite. 3. The 2-mercaptoethanol complex exhibited three A terms associated with the Q0-0-1, and Soret transitions at pH 6.86 similar to those of Fe(II) cytochrome c, which indicates that Mb was reduced by this reagent at pH 6.86. At pH 9.18 2-mercaptoethanol gave an MCD spectrum similar to that of alkyl mercaptan just after the addition. With the time changed into deoxy Mb through some intermediate of reduced Mb-thiolate complex. At pH 11.45 2-mercaptoethanol formed complex which exhibited an MCD spectrum similar to those of other alkylmercaptans. 4. Sodium sulfide gave an MCD spectrum which resembled that of the normal thiol Mb complex just after addition at pH 6.86. The complex was gradually reduced to give 610 nm trough in addition to the MCD of deoxy Mb. The Mb-sulfur complex formed at pH 9.18 was gradually reduced to give an MCD spectrum which was fairly different from that of deoxy Mb. A similar MCD spectrum was observed at pH 11.45 just after the addition of Na2S. These results were considered to suggest the saturation of one of the conjugated double bonds of the porphyrin by sulfur.     

Conformation and circular dichroism of DNA.

CD spectra of calf thymus, C. perfringens, E. coli, and M. luteus DNA have been measured in the vacuum-uv region to about 168 nm for the A-, B-, and C-forms. The positive band at about 187 nm and the negative band at about 170 nm found for each type and form of DNA are sensitive to the source of the DNA and the base–base interactions of the double-stranded helix. The A-form spectra confirm that these bands are indeed sensitive to secondary structure. In the near-uv, the CD of B-form DNA is well analyzed as a linear combination of 27% A-form and 78% C-form. However, an analysis of the extended spectrum demonstrates that the near-uv analysis is not correct. The extended analysis shows that the base–base interactions are similar for B- and C-forms in solution, which implies that these two forms have nearly the same number of base pairs per turn. Various types of CD difference spectra are also discussed.     

Molecular biology of copper. A circular dichroism study on copper complexes of thionein and penicillamine.

Chicken liver Cd, Zn-thionein (metallothionein) was isolated from Cd-pretreated chickens weighing 1 500 g. The native Cd, Zn-thionein contained 9 g-atoms of metals per 12 000 g of protein. Upon the addition of Cu(CH3CN)4ClO4, all Cd2 and Zn2 were successfully replaced. 15 g-atoms of Cu from the acetonitrile perchlorate complex were bound to the protein. Due to the absence of aromatic amino acid residues, thionein has unique ultraviolet and circular dichroism properties. The shoulder of the ultraviolet spectrum at 250 nm (A250 X A280(-1) = 23.9) was shifted to 275 nm (A250 X A280(-1) = 1.6). No significant absorption was detected in the visible region. Th conformational changes of the protein moiety were much more visible in the circular dichroism spectra. The titration with Cu(CH3CH)2 caused the appearence of three new Cotton effects: 257.5 nm (+), 350 nm (+) and 301 nm (-). The negative Cotton effect at 239 nm of the original metallothionein was completely levelled off. The binding strength of copper with thionein is extraordinarily high: it survives proton treatment up to pH 1.9. Displacement of the Cd2 by Cu employing Cd-thionein which was formed at pH 2.2 resulted in the same circular dichroism properties as observed for Cu-thionein. D-Penicillamine proved a suitable model for the metal-free thionein, since redox reactions and polymerization of the sterically hindered thiol residue are known to be slow. The correlation of the circular dichroism properties of either copper complex using thionein or D-penicillamine was surprisingly high. Circular dichroism measurements of Cu(I)-D-penicillamine revealed Cotton effects at 255 nm (+), 280 nm (+) and 355 nm (-). Upon examining the red-violet mixed Cu(-i)-cu(II)-D-penicillamine complex, Cotton bands in the visible region at 425 nm (-) and 495 nm (+) were seen. In many blue copper enzymes, the copper is assumed to be in the neighborhood of both cysteine and aromatic amino acid residues, which are known to play an important role in the electron transfer. This is not the case in the Cu-thionein, which would explain many different properties of this copper protein. It is very attractive to conclude that the sterically hindered SH-group of D-penicillamine reacts with excess copper in a specific way, similar to the Cu-thionein. This phenomenon could explain the considerable success of D-penicillamine in the treatment of Wilson's disease.     

Ceruloplasmin-anion interaction. A circular dichroism spectroscopic study.

The effect of anion binding to ceruloplasmin has been studied using absorption and cirbular dichroism spectral data. At anion to ceruloplasmin molar ratios approaching infinite, OCN-, N3- and SCN- bind to ceruloplasmin giving rise to similar alterations in circular dichroism and absorption spectra. The positive bands at 610 and 520 nm in circular dichroism spectra disappear, a negative one apperars at 600 nm and the peak at 450 nm is only slightly modified. There is a new negative band at 410 nm well-defined in OCN- ceruloplasmin spectra. The decrease in absorption at 610 nm is ascribed to the disruption of one type I Cu-S(cysteine) bond owing presumably to the changes induced by anions in the protein secondary structure. The new band at 410 nm is assigned to a charge transfer transition from the ligand replacing cysteine at its binding site. Both absorption and circular dichroism spectra show isobestic points indicating that anion binding to the enzyme, disruption of one of the two type I Cu-S bonds and coordination of this Cu to another protein residue take place simultaneously.     

A circular dichroism and fluorescence quenching study of the interactions between rhodium(II) complexes and human serum albumin.

Various divalent rhodium complexes Rh2(L)4 (L = acetate, propionate, butyrate, trifluoroacetate and trifluoroacetamidate) have been found to bind to non-defatted human serum albumin (HSA) at molar ratios about 8:1. The circular dichroism measurements showed that the more liposoluble carboxylates, butyrate and trifluoroacetate, caused the major alterations of the secondary structure of HSA. Stern-Volmer constants for the fluorescence quenching of the buried Trp214 residue by these complexes were also higher for the lipophilic metal compounds. In the case of the rhodium carboxylates it was observed that their denaturating and quenching properties could be explained in terms of their liposolubilities: the higher their lipophilic characters, the higher their abilities to penetrate inside the protein framework leading to structural alterations, and the closer they could get to the Trp residue causing fluorescence quenching. The liposoluble amidate complex, Rh2 (tfc)4, presented an intermediate quenching and did not cause structural alterations in the protein, presumably not penetrating inside the peptidic backbone. This study shows that it is possible to design new antitumor metal complexes which bind, to a large extent, to a transport protein causing little structural damage.     

Absorption, circular dichroism, magnetic circular dichroism and emission study of rat kidney Cd,Cu-metallothionein

Absorption, circular dichroism (CD), magnetic circular dichroism (MCD) and emission spectra of rat liver and rat kidney cadmium-, zinc- and copper-containing metallothioneins (MT) are reported. The absorption, CD and MCD data of native rat kidney Cd,Cu-MT protein closely resemble data recorded for the rat liver Cd,Zn-MT. This suggests that the major features in all three spectra of the native Cd,Cu-MT are dominated by cadmium-related bands. The CD spectrum of the Cd,Cu-MT recorded at pH 2.7 has the same band envelope that is observed for a Cd,Cu-MT formed in vitro by titration of Cd,Zn-MT with Cu(I), suggesting that the copper occupies the zinc sites in Cd,Cu-MT formed both in vivo and, at low molar ratios, in vitro. Remetallalion of the metallothionein from low pH in the presence of both copper and cadmium results in considerably less cadmium bound to the protein than was present in the native sample. It is suggested that this is due to the effect of the distribution of the copper amongst all available binding sites, thus inhibiting cluster formation by the cadmium. Emission spectra are reported for the first time for a cadmium- and copper-containing metallothionein. An emission band at 610 nm is shown to be a sensitive indicator of Cu(I) binding to metallothionein. Both the native Cd,Cu-MT and a Cd,Cu-MT formed in vitro exhibit an excitation spectrum with a band in the copper-thiolate charge-transfer region.     

Effects of monofunctional adducts of platinum(II) complexes on thermodynamic stability and energetics of DNA duplexes

Effects of adducts of [PtCl(NH3)3]Cl or chlorodiethylenetriamineplatinum(II) on DNA stability were studied with emphasis on thermodynamic origins of that stability. Oligodeoxyribonucleotide duplexes (15-bp) containing the single, site-specific monofunctional adduct at G-residues of the central sequences TGT/ACA or 5'-AGT/5'-ACT were prepared and analyzed by differential scanning calorimetry, temperature-dependent ultraviolet absorption and circular dichroism. The unfolding of the platinated duplexes was accompanied by relatively small unfavorable free energy terms. This destabilization was enthalpic in origin. On the other hand, a relatively large reduction of melting temperature (T(m)) was observed as a consequence of the monofunctional adduct in the TGT sequence, whereas T(m) due to the adduct in the AGT sequence was reduced only slightly. We also examined the efficiency of the mammalian nucleotide excision repair system to remove from DNA the monofunctional adducts and found that these lesions were not recognized by this repair system. Thus, rather thermodynamic than thermal characterization of DNA adducts of monofunctional platinum compounds is a property implicated in the modulation of downstream effects such as protein recognition and repair.     

A circular dichroism study of modified nucleosides

The conformation of 5-methoxycarbonylmethyluridine and 5-methoxycarbonylmethyl-2-thiouridine was studied by means of circular dichroism in various solvents. In order to calculate the accurate spectral parameters of the Cotton effects, the circular dichroism spectra were resolved into component Gaussian functions which simultaneously fit the adsorption spectra. On the basis of circular dichroism and proton magnetic resonance spectra, these nucleosides were found to occur in the β-configuration with the ³E-gg-anti conformation preferred. Due to the fact that the long-wavelength Cotton effect of mcm⁵s²U is not masked by the Cotton effects of the other nucleic acid monomers, the molecular parameters of this band may be useful for the conformational analysis of tRNA segments.     

Specific DNA binding of the cyclic AMP receptor protein to a synthetic oligodeoxyribonucleotide. A circular dichroism study

The interaction of the cAMP receptor protein (CRP) of Escherichia coli with a synthetic DNA undecamer (11 mer) comprising a portion of the specific target site in the gal operon and containing 8 basepairs out of the 10 basepair concensus making up specific CRP sites, has been studied by circular dichroism spectroscopy. The binding constants for the interaction of CRP with the 11 mer in the presence and absence of cAMP have been determined, and it is shown that CRP, both in the presence and absence of cAMP, induces a B-C transition in the conformation of the 11 mer.     

Immunological and circular dichroism studies of maleylated f-1 (A) histone and complexes with DNA

Complexes of calf thymus f-1 (A) histone and homologous DNA were examined by circular dichroism. The maleylation of f-1 (A) produces a polypeptide with decreased ability to modify the circular dichroism spectrum of f-1 (A)-DNA complexes. By the introduction of two to three maleyl groups per f-1 (A) molecule, the alteration of the DNA CD spectrum is reduced by nearly half compared to that induced by the native nonmaleylated f-1 (A). Similarly maleylation reduces the serological reactivity of the histone, i.e., the reaction of the maleylated f-1 (A) with specific complement fixing f-1 (A) antibodies. On the other hand, moderate maleylation of f-1 (A) improves the cross-inhibition of the f-2b-anti-f-2b reaction by native f-1 (A) while extensively maleylated f-1 (A) is inert with respect to the same reaction. These results are interpreted in terms of possible conformational changes induced in f-1 (A) by maleylation, partially due to decreasing the histone net charge and perhaps as well as removal of specific site charges necessary for correct binding and interaction. Such an interpretation is consistent with the altered CD spectrum of maleylated f-1 (A) (i.e., a decreased and slightly red-shifted [θ]₁₉₈) and moreover explains why maleylation of two to three lysines per f-1 (A) molecule hinders simultaneously the very different DNA-histone and histone-complement fixing antibody interactions.     

Change in conformation of various DNAs on melting as followed by circular dichroism

The intensities of the CD bands at about 275 and 190 nm were monitored for DNAs with different G + C contents as a function of temperature. The 190-nm bands showed a nearly complete and cooperative collapse on melting of the DNA, demonstrating that the CD arises from base–base interactions. The small cooperative change on melting shown by the 275-nm bands indicates that base–base interactions do not contribute much CD intensity here. No significant difference in melting temperature was found between the two wavelengths, but the lack of premelting in the 190-nm bands contrasted with the significant premelting in the 275 nm bands. Since the 190-nm bands are particularly sensitive to base–base interactions, the relative positions of the bases must not change much during premelting. Still, changes in such interactions would be noticeable on top of the low intensity of the 275-nm bands. Premelting is discussed in the light of recent studies on DNA conformation.     

Double-helical DNA and RNA circular dichroism. Calculations on base-sugar-phosphateehlix interactions

Theoretical calculations of the near ultraviolet (uv) circular dichroism of double-helical DNA and RNA models were performed in order to evaluate the effects, on the calculated circular dichroism, of including the interactions of near uv quantum transitions of the nucleic acid bases with classical polarizable bonds of the sugar-phosphate backbone. Double-helical models (A-form, B-form, and C-form DNA and RNA-11) from X-ray diffraction data were used in the calculations. The results indicate that the contributions to the circular dichroism in the near uv region, of these types of interactions, provide calculated spectra that are slightly altered from calculated spectra when only base–base transition interactions were considered.     

Conformational characteristics of deoxyribonucleic acid-butylamine complexes with C-type circular dichroism spectra. 2. A Raman spectroscopic study

The derivatives of calf thymus DNA in which n-butylamine is covalently attached as described in the preceding paper in this series [Chen, C. Y., Pheiffer, B. H., Zimmerman, S. B., & Hanlon, S. (1983) Biochemistry (preceding paper in this issue)] were examined by Raman spectroscopy. As previously mentioned, these complexes exhibit profoundly decreased rotational strengths of the positive band of the circular dichroism (CD) spectrum above 260 nm, with the most heavily substituted (ca. 0.12 mol of amine/mol of nucleotide) resembling that of DNA in 11 m LiCl. Raman spectra of all complexes and their controls in the form of either fibers at 98% relative humidity or gels at 40 mg/mL in 20 mM NaCl, pH 7, show typical B-type spectra with no evidence of significant amounts of C, A, Z, or disordered forms. We have thus concluded that the assignment of the nonconservative CD spectrum of DNA typically observed in concentrated electrolyte solutions to a C form is in error. Both these Raman data and the X-ray results reported in the previous paper indicate that the structure giving rise to the C CD spectrum has B-form backbone geometry.     

Condensation of DNA by the C-terminal domain of histone H1. A circular dichroism study

The condensation of DNA by the C-terminal domain of histone H1 has been studied by circular dichroism in physiological salt concentration (0.14 M NaF). As the intact H1 molecule, its C-terminal domain induces the so-called psi state of DNA that is characterized by a nonconservative circular dichroism spectrum which is currently attributed to ordered aggregation of the DNA molecules. On a molar basis, intact H1 and its C-terminal domain give spectra of similar intensity. Neither the globular domain of H1 nor an N-terminal fragment, that includes both the globular and N-terminal domains, has any effect on the conservative circular dichroism of DNA. From these results it is concluded that the condensation of DNA mediated by histone H1 is mainly due to its C-terminal domain. The effect of the salt concentration and the size of DNA molecules on the circular dichroism of the complexes are also examined.     

Vibrational circular dichroism and IR absorption of DNA complexes with Cu2+ ions

Vibrational circular dichroism (VCD) spectroscopy and simultaneous IR absorption measurements are applied to study the interaction of natural calf thymus DNA with Cu2+ ions at room temperature in a Cu2+ concentration range of 0-0.4M (a Cu2+/phosphate molar ratio [Cu]/[P] of 0-0.7). In some important instances, VCD provides more detailed insights than previous IR investigations whereas in several others it leads to the same interpretations. The Cu2+ ions bind to phosphate groups at a low metal concentration. Upon increasing the ion concentration, chelates are formed in which Cu2+ binds to the N7 of guanine (G) and a phosphate group. Detectable only by VCD, significant distortion of most guanine-cytosine (GC) base pairs occurs at a [Cu]/[P] ratio of 0.5 with only a minor affect on adenine-thymine (AT) base pairs, which favors a "sandwich" complex in which a Cu2+ ion is inserted between two adjacent guanines in a GpG sequence. The AT base pairs become significantly distorted when the metal concentration is increased to 0.7 [Cu]/[P]. A number of GC base pairs, which are possibly involved in sandwich complexes, remain stacked and paired even at 0.7 [Cu]/[P], preventing complete strand separation. The DNA secondary structure changes considerably from the standard B-form geometry at a [Cu]/[P] ratio of 0.4 and higher. A further transition to some intermediate conformation that is inconsistent with either the A- or Z-form or a completely denatured state is suggested in agreement with other works. In general, VCD proves to be a reliable indicator of the 3-dimensional structure of the DNA-metal ion complexes, which reveals structural details that cannot be deduced from the IR absorption spectra alone.