Degree Of Renaturation Research Articles

Binding of platinum-diaminonitroxyl complexes to animal DNA

Reaction of Pt II(DAPO)X 2 complexes (where DAPO is trans-3,4-diamino-2,2,6,6-tetramethylpiperidine-1-oxyl, X 2=(NO 3) 2, oxalato (Ox) or 1,1-cyclobutanedicarboxylato (Cbdca)) with a bovine spleen DNA in 0.01 M NaHCO 3 at 37°C for 24 h gives rise to formation of platinated DNA. The [bound Pt II(DAPO)]/[nucleotide] ratio ( r) depends on the initial ratio of the reagents and on the nature of leaving ligands X. Nitroxyl–nitroxyl distances in platinated DNA determined by the ESR suggest that at r⩾0.1 Pt II(DAPO) fragments are uniformly attached to DNA. But at lower r, the thermal characteristics of modified DNA (melting temperature T m, melting range width Δ T) and the guanine-to-adenine platination degree ratios G Pt/A Pt imply that the nature of leaving ligands X affect the selectivity of DNA platination. At r⩾0.1, nitroxyl groups can approach each other so close that, in an acidic medium, the electron transfer from one nitroxyl group to another becomes possible, and the nitroxyls readily disproportionate to diamagnetic products. Correlation time of nitroxyl rotation in Pt II(DAPO)–DNA adducts is ∼10 −8 s, which is related to predominantly bifunctional bonding of Pt II(DAPO) with DNA. Platination-induced distortion of DNA was evidenced by changes in T m, Δ T and degree of hyperchromicity H. The major part of adducts form the intrastrand cross-links which destabilize the structure of DNA duplex. The interstrand Pt II(DAPO) cross-linking (∼1% of the adducts) facilitates renaturation of despiralized DNA molecules upon cooling. Two types of Pt II(DAPO)–DNA adducts are revealed, which differ substantially in their rates of deplatination with NaCN. ESR, electron spin resonance; r, degree of modification; cisplatin, cis-diamminedichloroplatinum(II); T m, melting temperature; Δ T, melting range width; H, degree of hyperchromicity; R, degree of renaturation; AAS, atomic absorption spectroscopy; HPLC, high performance liquid chromatography.

A calorimetric study of the thermal denaturation of whey proteins in simulated milk ultrafiltrate

SummaryDifferential scanning calorimetry (DSC) was used to study thermal transitions of the following whey proteins and enzymes in milk ultrafiltrate solution: β-lactoglobulin, α-lactalbumin, serum albumin, γ-globulin, apo- and Fe-lactoferrin, lysozyme, ribonuclease, α-chymotrypsin and xanthine oxidase. Denaturation enthalpies (ΔHD), denaturation temperatures (TD) and the half width of the denaturation peaks in DSC thermograms (ΔT½D) were determined and the degree of renaturation was estimated by rescanning previously denatured samples. A fair correlation between the results obtained by DSC and other more classical methods was found in general. However, for some proteins (α-lactalbumin, lysozyme, ribonuclease and xanthine oxidase), which have so far been considered relatively thermostable, calorimetry reveals conformational changes starting at temperatures as low as about 45 °C. In these cases thermostability observed after heat treatment of milk should be interpreted in terms of renaturation and not of high temperatures of denaturation.

Relationships among mycobacteria and nocardiae based upon deoxyribonucleic acid reassociation.

The degree of renaturation between Nocardia farcinica 330 deoxyribonucleic acid (DNA) and DNA from 17 nocardial strains and 11 mycobacterial strains, and between Mycobacterium smegmatis 405 DNA and DNA from 11 mycobacterial strains and 14 nocardial strains was determined by using the nitrocellulose membrane filter technique. These results indicated that some cultures designated N. farcinica were identical to some cultures designated N. asteroides but that other strains called N. asteroides were distinctly different. The species M. smegmatis was homogeneous and distinct from the other species examined. Data comparing the extent of nucleotide sequences shared by M. smegmatis 405 DNA and DNA from nine other mycobacterial strains, as determined by the membrane filter technique and by monitoring DNA reassociation optically, were similar. The extent of reassociation between M. tuberculosis H37Ra DNA and DNA from 14 mycobacterial strains was determined optically. DNA from M. bovis BCG was 86% homologous with M. tuberculosis H37Ra DNA. The other mycobacterial DNA preparations examined were less than 40% homologous with M. tuberculosis H37Ra DNA. The genome sizes of the mycobacteria examined ranged between 2.5 x 10(9) daltons and 4.5 x 10(9) daltons.

Reaction of dihydropyrrolizines with deoxyribonucleic acids in vitro.

1. Thermal denaturation profiles of Escherichia coli DNA pretreated with monocrotaline pyrrole in vitro showed no difference from control DNA samples during heating. A substantial increase in the degree of renaturation during cooling of the pretreated DNA samples was observed. The degree of renaturation was dependent on the concentration of pyrrole used. 2. When rat liver DNA was pretreated with monocrotaline pyrrole there was a greater degree of renaturation after heat treatment than was found with E. coli DNA. 3. Equilibrium-density-gradient centrifugation in alkaline caesium chloride showed that DNA pretreated with monocrotaline pyrrole and heat denatured, renatured to a greater extent on quenching in ice than did untreated control DNA. The degree of renaturation was similar whether the initial treatment of the DNA with pyrrole was for 1 or for 15min. The reaction also appeared to be independent of pH between 5.5 and 9.0. 4. Retrorsine pyrrole was as effective as monocrotaline pyrrole in cross-linking the DNA, but monocrotaline pyrrole exposed to water for 5min before the addition of the DNA was ineffective. Pretreatment of E. coli DNA with synthetic bis-hydroxymethylpyrrole esters also caused renaturation after heat treatment. Monoesters were ineffective. 5. After treatment of rats with retrorsine, no cross-linking of the liver DNA could be demonstrated.

On the reversibility of thermal denaturation of Delphinus delphis ferrimyoglobin derivatives

The effects of protein concentration, alkaline pH and specific bound ligands on the reversibility of thermal denaturation of Delphinus delphis myoglobin have been studied. Denaturation has been followed by absorption changes in the Soret band. It was established experimentally that by lowering the protein concentration below 1 · 10 −5 M at pH 9.0 a decrease in the degree of renaturation was obtained for the three ferrimyoglobin derivatives studied. It is shown that reversibility depended strongly on the ionization of the amino acid residues lying on the surface of the protein molecule and probably associated in “cluster groups”. These probably are: (a) 6 lysyl groups titrated in the region pH 9.5–10.2; (b) the distal histidine (E 7), depending on the nature of the sixth haem ligand; (c) the abnormally titrated tyrosine (H23). Reversibility was definitely influenced by the nature of the ligands in two ways, namely by their effect on the conformational stability of the molecule and on the stability of the hydrogen bonding between the ligand and the distal histidine. The specific effect of tyrosyl groups on the mechanism of reversibility is discussed. Total renaturation of cyanide ferrimyoglobin at pH 9.5 was shown and conditions for it were selected on the basis of these studies.