Nunu Metreveli

UV-vis and FT-IR spectra of ultraviolet irradiated collagen in the presence of antioxidant ascorbic acid.

The influence of deleterious UV radiation on collagen molecules in the absence and presence of ascorbic acid using UV-vis and FT-IR spectroscopy has been studied. Intensity of UV-vis absorption spectrum of collagen with a maximum at 275 m due to the aromatic residues (tyrosine and phenylalanine) increases with the increasing dose of UV radiation. This effect is significantly hindered in the presence of antioxidant ascorbic acid. Intensities of FT-IR bands (amide A, B, I and II) of collagen decrease with the increase of the UV radiation dosage. Intensities of bands are also decreased in the presence of ascorbic acid. Results suggest that increasing the concentration of ascorbic acid increases the photo-stability of collagen, and the collagen becomes less sensitive to UV radiation. It is possible that hydrogen bonds form between the groups N-H of collagen and C=O of ascorbic acid. It is believed that under UV radiation free radicals appear in acid soluble collagen and resulting in photodegradation of the macromolecule restore due to the ability of ascorbic acid donating one or two electrons. Increasing the dose of radiation causes more molecules of ascorbic acid to slow down, and their antioxidant effect is diminished accordingly.

UV Damage of Collagen: Insights from Model Collagen Peptides

Fibrils of Type I collagen in the skin are exposed to ultraviolet (UV) light and there have been claims that collagen photo-degradation leads to wrinkles and may contribute to skin cancers. To understand the effects of UV radiation on collagen, Type I collagen solutions were exposed to the UV-C wavelength of 254 nm for defined lengths of time at 4°C. Circular dichroism (CD) experiments show that irradiation of collagen leads to high loss of triple helical content with a new lower thermal stability peak and SDS-gel electrophoresis indicates breakdown of collagen chains. To better define the effects of UV radiation on the collagen triple-helix, the studies were extended to peptides which model the collagen sequence and conformation. CD studies showed irradiation for days led to lower magnitudes of the triple-helix maximum at 225 nm and lower thermal stabilities for two peptides containing multiple Gly-Pro-Hyp triplets. In contrast, the highest radiation exposure led to little change in the T(m) values of (Gly-Pro-Pro)(10) and (Ala-Hyp-Gly)(10) , although (Gly-Pro-Pro)(10) did show a significant decrease in triple helix intensity. Mass spectroscopy indicated preferential cleavage sites within the peptides, and identification of some of the most susceptible sites of cleavage. The effect of radiation on these well defined peptides gives insight into the sequence and conformational specificity of photo-degradation of collagen.

Mechanisms of the Influence of UV Irradiation on Collagen and Collagen-Ascorbic Acid Solutions

The study of the influence of UV irradiation on collagen solutions has shown the destabilization of the collagen molecule by calorimetric method. It is reflected both in changes of thermodynamic parameters of transition (Tm, ΔH, Cp=f(t)) and in the appearance of a low temperature peak, that is practically irreversible against rescanning. All these indicate that the important defects in the molecule occur. The ESR measurements have shown that the above-mentioned thermal changes are connected with the occurrence of free radicals in solution under UV irradiation. They interact with proline (Pro) residues of the protein with the appearance of secondary free radicals, with following migration to glycine (Gly) residues. The emergence of the free radicals at the Pro and then at the Gly residues may cause the dramatic structural defect resulting from the UV irradiation, which significantly alters the network of hydrogen bonds in the triple helix of the collagen molecule. All this is connected with destabilization of the collagen molecule, because the defects in amino acid residues probably lead to cleavage of covalent bonds near the damaged sites maintaining the triple helical structure. The presence of ascorbic acid in collagen solution protects the collagen molecule from occurring of secondary free radicals.

Partial heat capacity change — Fundamental characteristic of the process of thermal denaturation of biological macromolecules (proteins and nucleic acids)☆

Abstract We prove experimentally that for any biopolymers (proteins and nucleic acids), the process of thermal denaturation in aqueous medium is characterized by change of the heat capacity regardless a unique spatial structures of different conformations. Absolute values and sign of ΔCP=Cp, denatured−Cp, native, depends primarily on conformational peculiarities of macromolecules and concrete mechanisms of interaction of water molecules with biopolymers chains — interaction is very clearly found to be of critical importance for the stability of the spatial structure of any biopolymers in aqueous medium.

Calorimetric investigation of DNA in the native and denatured states

Abstract The results of determination of apparent heat capacities of naturally occurring DNA in native (helix) and denatured (coils) states in dilute aqueous solutions have been reported. These results, supplemented by direct heat capacity measurements as a function of temperature, using a capillary scanning calorimeter, clearly show that the apparent heat capacity of polynucleotide chains of DNA in aqueous solution is strongly dependent upon the conformational state of the macromolecule. Comparison of data obtained for DNA in the “solid state”, at different levels of hydration and for DNA in H 2 O and D 2 O solutions reveals the possibility that the heat capacity increment ( Δc p = 0.36 ± 0.04 J g −1 K −1 ) is determined by an increase in the number of vibrational degrees of freedom, by hydrophobic effects and as a result of hydrogen bond destruction in water (including the ordered water clusters in the hydration shells of double helix of DNA).

Identification of free radicals induced by UV irradiation in collagen water solutions

Electron paramagnetic resonance (EPR) method has shown that hydrogen atoms and acetic acid free radicals appear in surrounding acetic acid-water solution of collagen under ultraviolet (UV) irradiation. These free radicals interact with the collagen molecule; consequently, seven superfine components of EPR spectrum with the split of aH = 11.3G and g-factor 2.001 appear. It is assumed that this spectrum is related to the free radical occurred on the proline residue in collagen molecule. In order to discover .OH hydroxyl radicals even in minor concentration, spin trap 5.5-dimethyl-1-pyrroline N-oxide (DMPO) has been applied. During the irradiation of collagen water solution in the presence of spin trap, EPR spectrum of the DMPO/.OH adduct has not been identified, while the above mentioned spectrum has been observed once the hydrogen peroxide H2O2 and FeSO4 were added to the sample. That means that water photolysis does not take place in collagen water-solution due to UV irradiation. It was suggested that occurrence of hydrogen radical is connected with the electron transmission to the hydrogen ion. The possible source of free electrons can be aromatic residues, photo ionization of which takes place in collagen molecule due to UV irradiation.

A microcalorimetric and electron spin resonance study of the influence of UV radiation on collagen

It was demonstrated microcalorimetrically that UV radiation cardinally changed the behavioral pattern of the temperature dependence of heat capacity of a collagen solution and decreased the enthalpy of collagen denaturation. It was discovered by electron spin resonance (ESR) that the primary products of UV-irradiated acid-soluble collagen were atomic hydrogen and the anion radical of acetic acid. The latter, when exposed to long-wavelength UV light, converted into the methyl radical, which interacted with acetic acid to produce the acetic acid radical. These free radicals interacted with the collagen molecule, leading to the appearance of seven superfine components with the split ΔH = 1.13 mT in the ESR spectrum. It was assumed that this spectrum is related to the free radical that occurred in the proline residue of the collagen molecule, which, in this particular case, was the major defect in the triple helix of collagen that caused instability of the macromolecule.

Stable Domain Assembly of a Monomolecular DNA Quadruplex: Implications for DNA-Based Nanoswitches

In the presence of K+ ions, the 5′-GGGTGGGTGGGTGGG-3′ (G3T) sequence folds into a monomolecular quadruplex with unusually high thermal stability and unique optical properties. In this study we report that although single G3T molecules unfold and fold rapidly with overlapping melting and refolding curves, G3T multimers (G3T units covalently attached to each other) demonstrate highly reproducible hysteretic behavior. We demonstrate that this behavior necessitates full-length tandem G3T monomers directly conjugated to each other. Any modification of the tandem sequences eliminates the hysteresis. The experimentally measured kinetic parameters and equilibrium transition profiles suggest a highly specific two-state transition in which the folding and unfolding of the first G3T monomer is rate-limiting for both annealing and melting processes. The highly reproducible hysteretic behavior of G3T multimers has the potential to be used in the design of heat-stimulated DNA switches or transistors.

Sr(2+) induces unusually stable d(GGGTGGGTGGGTGGG) quadruplex dimers.

Guanine‐rich sequences are able to form quadruplexes consisting of G‐quartet structural units. Quadruplexes play an important role in the regulation of gene expression and have therapeutic and biotechnological potential. The HIV‐1 integrase inhibitor, (GGGT)4, and its variants demonstrate unusually high thermal stability. This property has been exploited in the use of quadruplex formation to drive various endergonic reactions of nucleic acids such as isothermal DNA amplification. Quadruplex stability is mainly determined by cations, which specifically bind into the inner core of the structure. In the present work, we report a systematic study of a variant of the HIV‐1 integrase inhibitor, GGGTGGGTGGGTGGG (G3T), in the presence of alkali and alkaline‐earth cations. We show that Sr2+‐G3T is characterized by the highest thermal stability and that quadruplex formation requires only one Sr2+ ion that binds with low micromolar affinity. These concentrations are sufficient to drive robust isothermal quadruplex priming DNA amplification reaction. The Sr2+‐quadruplexes are also able to form unusually stable dimers through end‐to‐end stacking. The multimerization can be induced by a combination of quadruplex forming cations (i.e., K+ or Sr2+) and non‐specific Mg2+.