What properties of non-electrolyte solutions does a DNA molecule feel?

As early as in 1953 it was reported that at low relative humidity of the atmosphere (about 70 %) DNA films were in A-form, while at higher humidity (more than 80%) they were in B-form. Relative humidity of the atmosphere corresponds to the water activity in the system. It was found in the beginning of the 70th that DNA transfers to the A-form when non-electrolytes, such as monoatomic alcohols, dioxane, tetrahydrofurane were added to water. It was reasonable to suppose that B to A transition of DNA occurs in the non-electrolyte solutions at the same values af water activity as in the moist atmosphere. This prediction was borne out. But in water solutions of very polar non-electrolytes such as methanol and ethylene glycol B to A DNA does not occur even at very low water activity values. It was supposed that A form of DNA can arise only in the medium with sufficiently low polarity. We used hyperfine splitting constant (A) of a nitroxide spin label determined from the EPR spectra as a measure of the liquid solvent polarity. It was found that DNA transition into B form occurs when A reached a certain value. Polarity of methanol and ethylene glycol is much higher than that of the solutions in which B to A DNA transition takes place. This transition also occurs in water solutions of trifluoroethanol. But in these solutions the transition takes place at hyperfine splitting values, which are much greater than in other non-electrolyte solutions. Water activity in the zone of B-A DNA transition in trifluoroethanol solutions does not correspond water activity which was calculated for other nob-electrolyte solutions in which B-A transition is observed.

First Synthesis of C-Phenyl N-tert-Butyl [15N]nitrone (PBN-15N) for the EPR Spin Trapping Methodology

As compared to normal PBN, about fifty percent increase of electron paramagnetic resonance (EPR) spin trapping sensitivity has been gained by using a new 100% 15N-enriched spin trap, C-phenyl N-tert-butyl[15N]nitrone (PBN-15N). PBN-15N has been prepared by a convenient four-step route using ammonium-15N chloride as the starting material. This synthetic method produces 2-methyl-2-[15N]nitropropane which is useful for the synthesis of many other PBN-15N type spin traps for the purpose of increasing spin trapping sensitivity. EPR spin trapping with PBN-15N in benzene and in phosphate buffer has been investigated. The 15N hyperfine splitting constant (15N-hfsc) is larger than 14N-hfsc by 40%. The larger 15N-hfsc gives more opportunity to identify different radical addends within the same system.

An EPR and MS Investigation of Hexa-Substituted Pyrrolidine-l-oxyl Aminoxyl Stable Radicals. Unexpectedly Large y-Hydrogen Splittings

The y-hydrogen hyperfine splitting constant (γ-H hfsc) for stable aminoxyl (nitroxide) spin labels such as 2,2,5,5-tetramethylpyrrolidine-1-oxyl and their derivatives is usually very small (<1.0 G) and not distinguished with EPR spectrometry. Surprisingly, large γ-H hfsc’s (≥2 .0 G) have been detected with EPR for the first time from ten 2-alkyl-2-phenyl-3,3,5,5- tetramethylpyrrolidine-1-oxyl stable aminoxyl radicals. It is discovered that γ-H hfsc’s are very sensitive to the size and the substitution pattern of 2-alkyl groups. When the 2-alkyl group is CH3 or CD3, γ-H hfsc’s are not resolved in the EPR spectra. But if the 2-alkyl group is C2H5, one γ-H hfsc is very large, equal to 4.72 G in C6H6. If the substituent is longer than C2H5, such as n-C3H7, n-C4H9, n-C5H11, n-C6H13 and CH2=CHCH2- substituents, the γ-H hfsc is slightly smaller, equal to 4.59 G. For secondary substituents such as sec-C4H9 and cyclo-C6H11 , the γ-H hfsc decreases to 2.00 G. Intermediate γ-H hfsc’s correspond to C6H5CH2 (3.18 G) or a tertiary alkyl group such as t-C4H9 (3.47 G). Variation of γ-H hfsc’s is based on the change of the pyrrolidine ring conformation which is a result of the 2-alkyl group influence. The structures of these aminoxyl radicals are characterized also with mass spectrometry. Possible MS fragmentation mechanisms are discussed.

Hyperthermia stimulates nitric oxide formation: electron paramagnetic resonance detection of .NO-heme in blood

Previous experiments from our laboratory have demonstrated that severe hyperthermia results in a selective loss of splanchnic vasoconstriction. Using electron paramagnetic resonance spectroscopy to scan whole blood samples collected in vivo from the portal vein and femoral artery of conscious unrestrained rats, we observed an increase in the concentration of spectroscopy-detectable species in portal venous blood of all heat-stressed animals. These spectra consisted of at least three distinct species: one with a broad feature having an effective g factor for the unpaired electron (g) of 2.06 assigned to the copper-binding acute phase protein ceruloplasmin, and two with narrower features that evolved at core temperatures > 39 degrees C representing a semiquinone radical and .NO-heme. This heat-induced signal displays the classic nitrogen triplet hyperfine structure (nitrogen hyperfine splitting constant = 17.5 gauss, centered at g = 2.012) that is consistent with a five-coordinate heme complex and is characteristic of an unpaired electron coupled to nitrogen in the ferrous .NO-heme adduct [(alpha 2+NO) beta 3+]2. The intensity of this signal increased approximately twofold as core temperature rose to > 39 degrees C, peaking 1 h post-heat exposure at greater than threefold basal concentration. This species was not seen in corresponding arterial blood samples. This is the first demonstration that whole body hyperthermia produces increased concentrations of radicals and metal binding proteins in the venous blood of the rat and suggests that severe hyperthermia stimulates an enhanced local release of .NO within the splanchnic circulation.

Mechanism of inhibition of horseradish peroxidase-catalysed iodide oxidation by EDTA

EDTA inhibits horseradish peroxidase (HRP)-catalysed iodide oxidation in a concentration and pH-dependent manner. It is more effective at pH 6 than at lower pH values. A plot of log Kiapp. values as a function of pH yields a sigmoidal curve from which a pKa value of 5.4 can be calculated for an ionizable group on the catalytically active HRP for EDTA inhibition. Among the structural analogues of EDTA, tetramethylethylenediamine (TEMED) is 80% as effective as EDTA, whereas the EDTA-Zn2+ chelate and EGTA are ineffective. Kinetic studies indicate that EDTA competitively inhibits iodide oxidation. Spectral studies show that EDTA can quickly reduce compound I to compound II, but reduction of preformed compound II to the native enzyme is relatively slow, as demonstrated by the time-dependent spectral shift from 417 nm to 402 nm through an isosbestic point at 408 nm. Under steady-state conditions, in a reaction mixture containing HRP, EDTA and H2O2, the enzyme remains in the compound-II form, with absorption maxima at 417, 527 and 556 nm. Direct evidence for one-electron oxidation of EDTA by HRP intermediates is provided by the appearance of an e.s.r. signal of a 5,5-dimethyl-1-pyrroline N-oxide (spin trap)-EDTA radical adduct [aN (hyperfine splitting constant) = 1.5 mT] in e.s.r. studies. The signal intensity, however, decreases in the presence of iodide. The KD of the HRP-EDTA complex obtained from optical difference spectroscopy increases with an increase in iodide concentration, and the double-reciprocal plot for EDTA binding indicates that EDTA and iodide compete for the same binding site for oxidation. We suggest that EDTA inhibits iodide oxidation by acting as an electron donor.

N-14 ENDOR study of micellar solutions of sodium dodecylsulfate using small spin probes

ENDOR (electron nuclear double resonance) spectroscopy has been applied to the study of the state of solubilization of small nitroxide spin probes in micelles produced in sodium dodecylsulfate solution. The intensity ratio of the pair of N-14 ENDOR lines observed by fixing the field on the low-field ESR (electron spin resonance) line was monitored as a measure of the motion of spin probes. Neutral spin probes, when solubilized in micelles, show a discontinuous change of ENDOR spectral parameters such as hyperfine splitting constant and peak intensity ratio. Above the critical micelle concentration, the spectrum features change only gradually as the number of solubilized probes is increased. Spin probes in micelles appear to experience environments that are less polar than those met in aqueous solutions. However, the solubilized state is stable and is insensitive to changes in the concentration of the amphiphile. An anionic spin probe, Fremy's salt, shows no indication of interaction with the micelles. The applicability of the use of ENDOR peak intensities for the study of spin probe motion is discussed.

Azide-binding studies reveal type 3 copper heterogeneity in ascorbate oxidase from the green zucchini squash (Cucurbita pepo)

Titration of native ascorbate oxidase from green zucchini squash (Cucurbita pepo) with azide in 0.1 M-phosphate buffer, pH 6.8, exhibits a biphasic spectral behaviour. Binding of the anion with ‘high affinity’ (K greater than 5000 M-1) produces a broad increase of absorption in the 400-500 nm region (delta epsilon approximately 1000 M-1.cm-1) and c.d. activity in the 300-450 nm region, whereas azide binding with ‘low affinity’ (K approximately 100 M-1) is characterized by an intense absorption band at 420 nm (delta epsilon = 6000 M-1.cm-1), corresponding to negative c.d. activity and a decrease of absorption at 330 nm (delta epsilon = -2000 M-1.cm-1). The high-affinity binding involves a minor fraction of the protein containing Type 3 copper in the reduced state, and the spectral features of this azide adduct can be eliminated by treatment of the native enzyme with small amounts of H2O2, followed by dialysis before azide addition. As shown by e.s.r. spectroscopy, Type 2 copper is involved in both types of binding, its signal being converted into that of a species with small hyperfine splitting constant [12 mT (approximately 120 G)] in the case of the low-affinity azide adduct. The spectral similarities of the two types of azide adducts with the corresponding adducts formed by native laccase, which also exhibits Type 3 copper heterogeneity, are discussed.