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We have investigated in detail radiation and thermal stabilities and transformations of adenosine mono- and triphosphates in liquid and frozen solid aqueous solutions within a wide range of absorbed radiation dose (up to 75 kGy) and temperature (up to 160 degrees C). Dephosphorylation is the main pathway of high temperature hydrolysis of adenine nucleotides. Basic thermodynamic and kinetic parameters of this process have been determined. Radiolysis of investigated compounds at room temperature results in scission of N-glycosidic bond with a radiation yield about of 1 mol/100 eV. Solution freezing significantly enhances radiation stability of nucleotides as well as other biomolecules. This circumstance is essential in the discussion of panspermia concepts.

Reversed-phase high-performance liquid chromatography (RP-HPLC) was used to detect differences in the secondary structures of two self-complementary oligodeoxyribonucleotides. The [d(CCTTTAAAGG)] 2 duplex assumes an ordinary B-conformation in aqueous solution, while [d(GGAAATTTCC)] 2 is known to contain in its central part a stretch of a more rigid B′-form conformation with significantly lowered fluctuational mobility of base pairs. The latter factor causes a marked difference in the amounts of thymine cyclobutane photodimers formed under UV irradiation of corresponding duplexes as revealed by chromatography of two single-stranded oligonucleotides. Increasing the temperature below the duplex melting temperature (stimulation of the B′-B structural transition) results in an increase in photodimer formation that was inhibited in the B′-form. Thus, we demonstrate the usefulness of RP-HPLC for duplex DNA structural studies.

Degradation of the behaviorally active peptide ACTH/MSH(4–10) and its synthetic analog semax was studied in serum in the presence of several specific peptidase inhibitors. Bestatin and puromycin were used to inhibit aminopeptidase activity, lisinopril for angiotensin-converting enzyme, phosphoramidon for neutral endopeptidase 24.11, and Z-Pro-prolinal for prolyl endopeptidase. Bestatin inhibited up to 66%, puromycin about 33%, and lisinopril about 15% of total degrading activity against both ACTH/MSH(4–10) and semax. Involvement of neutral endopeptidase and prolyl endopeptidase in hydrolysis of the two peptides was less definitive. These studies showed that aminopeptidases and angiotensin-converting enzyme are responsible for the major part of the hydrolysis of ACTH/MSH(4–10) and semax in rat serum.

Tetrabutylammonium (TBA) salts of amino acids and peptides have increased solubility, as compared with that of alkali metals salts, in organic solvents. We have compared the reaction rates for tripeptide formation in methylene chloride from Boc-Gly-Phe activated with various phenols, N-oxysuccinimide and azide, and TBA-salt of tryptophan, as well as Trp-OCH3. H-Trp-O− TBA+ as an amino component significantly accelerates the rate of reaction. Although a significant degree of racemization has been found, the use of TBA-salt of amino acids and peptides is justified in many cases due to high conversion rates.

Tetrabutylammonium (TBA) salts of amino acids and peptides have increased solubility, as compared with that of alkali metals salts, in organic solvents. We have compared the reaction rates for tripeptide formation in methylene chloride from Boc-Gly-Phe activated with various phenols, N-oxysuccinimide and azide, and TBA-salt of tryptophan, as well as Trp-OCH3. H-Trp-O− TBA+ as an amino component significantly accelerates the rate of reaction. Although a significant degree of racemization has been found, the use of TBA-salt of amino acids and peptides is justified in many cases due to high conversion rates.

In view of known central effects of N-terminal ACTH fragments, a possibility of their entry into the brain was studied. Rat blood and brain extracts after intravenous injection of the tritiated synthetic ACTH(4–10) analogue, Met-Glu-His-Phe-Pro-Gly-Pro, were subjected to a high-performance liquid chromatographic analysis. At two time points the labelled peptide was detected in brain extracts. The brain to blood ratios of peptide content in brain and blood were found to be significantly higher than those calculated for a distribution of labelled bovine serum albumin in rat brain capillaries and blood. This strongly suggests that this peptide penetrates into the brain tissues, its quantity not exceeding 0.01% of dose injected. Peptide diffusion through the vascular epithelium of brain capillaries could account for the data obtained.

Summary Degradation of a regulatory peptide ACTH(4–10) and its synthetic analog semax in rat blood and serum was studied using high-performance liquid chromatography. About one third to one half of the serum degrading activity could be ascribed to bestatin-sensitive aminopeptidase which cleaved first and second N-terminal residues Met and Glu producing relatively stable intermediates. Comparable areas under the degradation/accumulation curves for intact peptides and intermediates implied that the latter can contribute to effects of intact peptides. Semax turned out to be more stable than ACTH(4–10) against the action of other enzymes that took part in degradation.

Reversed-phase high-performance liquid chromatography in a gradient of acetate or phosphate buffer and methanol was used for separating short (n  2–4) oligodeoxyribonucleotides and the products of their photomodification which contained thymine dimers. The modified oligomers have shorter retention times and are readily separated from the original ones. The modified oligonucleotides differing in the type (cyclobutane and non-cyclobutane) of the thymine dimer and its position in the molecule are effectively resolved. To establish the type of thymine photodimers in the modified oligomers, the use of their spectral properties, viz., detection at several wavelengths, is sufficient.

The potential of reversed-phase high-performance liquid chromatography for the separation of various ribo and deoxyribo components of nucleic acids containing photohydrates of pyrimidines has been investigated. The chromatographic properties of the modified nucleobases, nucleosides and nucleotides of uracil and cytosine as well as of the dinucleoside monophosphate, ApU, have been studied. Monomers were separated isocratically in pure water and various water-buffer eluents containing methanol at different pH values. Modified and unmodified dinucleoside monophosphates were separated in acetate buffer with a methanol concentration gradient. The modified compounds have shorter retention times and are readily separated from the original ones. Satisfactory resolution has also been obtained for most diastereoisomeric derivatives of the photohydrates.

DNA films with psi +/- CD spectra have been investigated. X-ray analysis has shown the sign of the psi spectra to be independent of the secondary structure of DNA. The appearance of the psi spectra is attended by the formation of a characteristic polygonal texture of the cholesteric type in the DNA film.

Sedimentation of DNA ethanol-water solutions has been studied over the range of ethanol concentrations corresponding to the B to A transition (65-80% ethanol, v/v). High ethanol concentrations (more than 75%) have been found to promote aggregate formation in solution. The molecular weight of DNA under fixed ionic conditions in solution (5x10(-4)M NaCl) has been shown to influence the value of ethanol concentration at which aggregates appear. On the other hand, the fact that DNA molecular weight has not been found to exert any influence on B to A transition curves obtained from CD measurements suggests that the changes observed in DNA CD spectra on adding ethanol to the solution are independent of aggregate formation. The date obtained show that, first, aggregation is not a necessary condition for the DNA transition from the B to the A-conformation and, second, changes in CD spectra of DNA under the influence of ethanol are not related to the process of aggregation.