ALBERT

Notes: Based on equilibrium binding studies, as well as on kinetic investigations, two types of interactions of Cu2+ ions with native DNA at low ionic strength could be characterized, namely, a nondenaturing and a denaturing complex formation. During a fast nondenaturing complex formation at low relative ligand concentrations and at low temperatures, different binding sites at the DNA bases become occupied by the metal ions. This type of interaction includes chelate formation of Cu2+ ions with atoms N(7) of purine bases and the oxygens of the corresponding phosphate groups, chelation between atoms N(7) and O of C(6) of the guanine bases, as well as the formation of specific intestrand crosslink complexes at adjacent G°C pairs of the sequence dGpC. CD spectra of the resulting nondenatured complex (DNA-Cu2+)nat may be interpreted in terms of a conformational change of DNA from the B-form to a C-like form on ligand binding. A slow cooperative denaturing complex formation occurs at increased copper concentrations and/or at increased temperatures. The uv absorption and CD spectra of the resulting complex, (DNA-Cu2+)denat, indicate DNA denaturation during this type of interaction. Such a conclusion is confirmed by microcalorimetric measurements, which show that the reaction consumes nearly the same amount of heat as acid denaturation of DNA.From these and the kinetic results, the following mechanism for the denaturing action of the ligands is suggested: binding of Cu2+ ions to atoms N(3) of the cytosine bases takes place when the cytosines come to the outside of the double helix as a result of statistical fluctuations. After the completion of the binding process, the bases cannot return to their initial positions, and thus local denaturation at the G·C pairs is brought about. The probability of the necessary fluctuations occurring is increased by chelation of Cu2+ ions between atoms N(7) and O of C(6) of the guanine bases during nondenaturing complex formation, which loosens one of the hydrogen bonds within the G·C pairs, as well as by raising the temperature. The implications of the new binding model, which comprises both the sequence-specific interstand crosslinks and the described mechanism of denaturing complex formation, are discussed and some predictions are made. The model is also used to explain the different renaturation properties of the denatured complexes of Cu2+, Cd2+, and Zn2+ ions with DNA.In temperature-jump experiments with the nondenatured complex (DNA-Cu2+)nat, a specific kinetic effect is observed, namely, the appearance of a lag in the response to the perturbation. The resulting sigmoidal shape of the kinetic curves is considered to be a consequence of the necessity of disrupting a certain number of the crosslinks existing in the nondenatured complex before the local unwinding of the binding regions (a main step of denaturing complex formation) may proceed.

Notes: Photo-ionization (PI) mass spectrometry performed with a monochromatic photon beam was applied to a series of peptide derivatives. PI mass spectra of ten N-acylpeptide methyl esters containing two to four residues of glycine, alanine, valine, leucine, proline, tryptophan, tyrosine, phenylalanine, methionine, carboxymethylcysteine, lysine and ornithine were studied. Comparative analysis of PI (10.2 eV) and electron-impact ionization (EI) (70 eV) mass spectra shows the total number of peaks on PI to be much less than that obtained with EI, especially in the low m/e region (〈 250 to 300). At the same time the relative abundance of ‘heavy’ ions, including molecular ions, is much higher in PI. The amino acid fragmentation pattern followed by N-acylpeptide esters in PI was found to be the same as for EI.

Notes: Photoionisation mass spectra of N-methylamides of N-acetyl derivatives of the following amino acids have been studied: alanine, valine, leucine, serine, threonine, phenylalanine, tyrosine, tryptophan and proline. The study also included methyl esters of N-acetyl tyrosine, N-acetyl phenyl alanine and N-trideuteroacetyl phenylalanine. The photon energies were within the range of 7.5 to 13.0 eV. The appearance potentials of the main ions were also determined. The compounds under study contain CH—CO, CO—NH and N—Cα bonds as well as sidechains and may therefore be regarded as models of short peptides.Under photoionisation conditions, decomposition of compounds containing aliphatic sidechains involves mainly CH—CO bonds; the appearance potentials of the corresponding fragments decrease with an increase in the size of sidechain. The presence of hydroxyl in the sidechain results in an increase in the appearance potential of a fragment formed due to CH—CO bond rupture, whereas the presence of an aromatic moiety favours an even greater increase in the appearance potential and results in predominant rupture of the N—Cα bond.Analysis of the mass spectra obtained confirms the conclusion made previously that application of the photoionisation technique enables one to increase considerably the relative intensity of characteristic peaks and to suppress secondary process taking place upon ionisation by the electrons with energy values of the order of 70 eV.

Notes: Mass spectra of the volatile derivatives of short peptides were studied by the photoionisation method with the use of monochromatic photons. The dependence of the intensity of the main peaks on the photon energy was analysed from 7·5 to 13·0 eV. The data obtain reveal the influence of the chemical structure of amino acid residues on the relative probability of the decomposition of peptide molecular ions at the CH—CO and CO—NH bonds, resulting in the formation of positively charged aldimine and amino acid N-terminal fragments, respectively. These data may be used as a basis for the application of the photoionisation technique to mass spectrometric sequential studies in peptides.In peptides containing residues of aliphatic amino acid the decomposition results mainly in the formation of aldimine ions, the stability of which increase with the increase of the alkyl chain size. In peptides containing residues of aromatic amino acids the decomposition is usually observed leading to formation of the amino acid ions.Ionisation potentials, as well as photoionisation efficiency curves and appearance potentials were determined for characteristic ions. Comparison was made of the values of the appearance potentials of different fragments formed upon decomposition of molecular ions. It has been shown that for peptides containing aliphatic amino acid moieties the appearance potentials of aldimine fragments are always lower than those inherent to peptides containing aromatic amino acid residues. The larger the size of an aliphatic chain, the lower is the energy of formation of these fragments. For all the compounds studied, including the peptides containing aromatic amino acid residues, the appearance potentials of the aldimine ions did not exceed those of the amino acid ions. These data indicate that, contrary to the experiments with electron-impact with energy of about 70 eV, upon ionisation with photons with energy from 7·5 to 13·0 eV, the aldimine fragments appear directly due to primary decomposition of molecular ions, independent of the formation of the amino acid fragments. The photoionisation efficiency curves for peptides containing different types of amino acid residues facilitate the choice of an optimal photon energy providing unequivocal information on the amino acid sequence in the peptide under study.