Immunochemical studies on thermal denaturation of ovomucoid

The thermal denaturation of ovomucoid was investigated by immunochemical methods, namely immunoprecipitation analyses and antibody-Sepharose 4B column chromatography. In the immunoprecipitation analyses, heated ovomucoid (90 degrees C, 90 min, pH 7.2) required about twice the antigen addition of the native protein to approach maximal precipitation with specific antibody, and the maximal immunoprecipitation was decreased to 80% of that by native ovomucoid. However, heated protein inhibited the binding of antibody with native ovomucoid, and 100% inhibition was attained at about 4-times the antigen addition necessary for the native protein. Heated ovomucoid (100 degrees C, 120 min) showed little immunoprecipitation and inhibition. To ovomucoid antigenicity was diminished more slowly than the trypsin inhibitory activity by heating, e.g., heated ovomucoid (90 degrees C, 120 min) retained more than 30% of the antigenicity but little trypsin inhibitory activity. By passing through the immunoaffinity column, heated ovomucoid (90 degrees C, 90 min) was separated into two fractions, either with (fraction II) or without (fraction I) antigenicity. Fraction II contained smaller fractions of ordered secondary structure than native ovomucoid, and trypsin inhibitory activity of fraction II was only 24% of the native one. These results indicated that thermally denatured ovomucoid was heterogeneous regarding the conformational damage caused by heating, and the structure around some antigenic sites in an ovomucoid molecule was retained even after the backbone conformation was partially destroyed and trypsin inhibitory activity was lost.     

Kinetic studies on thermal denaturation of C-phycocyanin

The kinetics of thermal denaturation of a biliprotein, C-phycocyanin (C-PC) isolated from Spirulina platensis were studied at different pH values, ranging from 4.0 to 8.0. The denaturation of C-PC follows the first order kinetics and rate constant at pH 5.0 and temperature 55 degrees C is found to be 4.37 x 10(-5) s(-1), which increases to 5.46 x 10(-1) s(-1) at pH 7.0. The denaturation rate is much higher at 65 degrees C and pH 7.0 (7.96 x 10(-4)), as compared to at pH 5.0 (1.46 x 10(-4)). The thermal stability of C-PC is more at pH 5.0, as compared to other pH values. The observed differences in entropy values at pH 5.0, as compared to other pH values indicate a considerably close fit structure of the protein at pH 5.0, which increases the stability of native structure, even at higher temperature (65 degrees C).     

Thermal fluctuation spectroscopy of DNA thermal denaturation

We have developed the technique of thermal fluctuation spectroscopy to measure the thermal fluctuations in a system. This technique is particularly useful to study the denaturation dynamics of biomolecules like DNA. Here we present a study of the thermal fluctuations during the thermal denaturation (or melting) of double-stranded DNA. We find that the thermal denaturation of heteropolymeric DNA is accompanied by large, non-Gaussian thermal fluctuations. The thermal fluctuations show a two-peak structure as a function of temperature. Calculations of enthalpy exchanged show that the first peak comes from the denaturation of AT rich regions and the second peak from denaturation of GC rich regions. The large fluctuations are almost absent in homopolymeric DNA. We suggest that bubble formation and cooperative opening and closing dynamics of basepairs causes the additional fluctuation at the first peak and a large cooperative transition from a partially molten DNA to a completely denatured state causes the additional fluctuation at the second peak.     

High resolution thermal denaturation of DNA: thermalites of bacteriophage DNA.

High resolution thermal denaturation profiles are presented for the DNAs of bacteriophages lambda and T7. It is concluded that the temperature increment in data gathering and the method of calculating results meet the requirements for quantitative recording of the large amount of information found in the thermal transitions of both DNAs. The high resolution derivative denaturation profiles of these bacteriophage DNAs demonstrate that individual subtransitions (thermalites) of natural DNA are Gaussian in form and have narrow transition widths. Curve resolution performed on these profiles indicates that the mean thermalite width (2 sigma) is 0.33 degrees C and that this breadth is relatively invariant. Transition widths are not influenced by the position of thermalites in the profile or by cation concentration in the range from 5 to 30 mM Na+. However, the relative position of thermalites within a denaturation profile is a function of the solution ionic strength. The distribution of lengths of the DNA sequences which these thermalites represent is broad, with a number average length of 900 base pairs. Although we find an approximate similarity between the number of thermalites in the denaturation profile of T7 DNA and the number of looping regions in the electron microscopic partial denaturation map of Gomez and Lang ((1972), J. Mol. Biol. 70, 239-251) we conclude that free solution thermal denaturation experiments can be compared only superficially to the mapping results.     

Effect of diethylsulfoxide on the thermal denaturation of DNA

DNA thermal denaturation has been investigated in aqueous solutions of diethylsulfoxide (DESO) by means of UV-vis and densimetry methods. It is suggested that, on the one hand, the structural change of entire solutions and, on the other hand, a direct interaction of DESO with DNA are responsible for the observed peculiar behavior. The results obtained were compared with those of dimethylsulfoxide (DMSO), also known from literature.     

Thermal denaturation of DNA in concentrated salt-free solutions: comparison of microcalorimetric and spectrophotometric data

Scanning microcalorimetry and spectrophotometry were used to study the dependence of melting enthalpy (delta Hm) and temperature (Tm) on DNA concentration in salt free solutions and on NaCl concentration in solutions with constant DNA concentration. This data is used to calculate the Manning's charge density parameter which is found to be equal 1.8. The linear dependence of Tm on the logarithm of DNA concentration in salt free solution was obtained. An approximate evaluation of dissociation degree in native DNA at different concentrations was made by comparison of straight lines in the Tm = f(lg CNaCl) and Tm = f(lg Cp) coordinates.     

Circular dichroism and thermal denaturation studies of chromatin and DNA from BrdU-treated mouse fibroblasts

A simple procedure for the isolation and pruification of metallothionein from rat liver is described. This method involves only four steps and is especially useful for large scale isolation of this protein. The final isolated preparation was homogeneous both in Sephadex gel filteration and in polyacrylamide gel electrophoresis. Isoelectric focussing shows the presence of two cadmium binding proteins with isoelectric points of 4.2 and 4.7. Metallothionein is isolated from dog liver using this method.     

Calorimetric studies of the thermal denaturation of cytochrome c peroxidase

Two endotherms are observed by differential scanning calorimetry during the thermal denaturation of cytochrome c peroxidase at pH 7.0. The transition midpoint temperatures (tm) were 43.9 +/- 1.4 and 63.3 +/- 1.6 degrees C, independent of concentration. The two endotherms were observed at all pH values between 4 and 8, with the transition temperatures varying with pH. Precipitation was observed between pH 4 and 6, and only qualitative data are presented for this region. The thermal unfolding of cytochrome c peroxidase was sensitive to the presence and ligation state of the heme. Only a single endotherm was observed for the unfolding of the apoprotein, and this transition was similar to the high-temperature transition in the holoenzyme. Addition of KCN to the holoenzyme increases the midpoint of the high-temperature transition whereas the low-temperature transition was increased upon addition of KF. Binding of the natural substrate ferricytochrome c to the enzyme increases the low-temperature transition by 4.8 +/- 1.3 degrees C but has no effect on the high-temperature transition at pH 7. The presence of cytochrome c peroxidase decreases the stability of cytochrome c, and both proteins appear to unfold simultaneously. The results are discussed in terms of the two domains evident in the X-ray crystallographic structure of cytochrome c peroxidase.     

Nonhistone chromosomal protein HMG 1 interactions with DNA. Fluorescence and thermal denaturation studies

The interaction of high mobility group protein 1 (HMG 1) isolated from chicken erythrocytes with DNA has been characterized using the intrinsic tryptophan fluorescence of the protein as a probe. It was found that the fluorescence is quenched approximately 30% upon binding to either single- or double-stranded DNA. Fluorescent titrations indicate that the physical site size for HMG 1 binding on native DNA is approximately 14 base pairs (or 14 bases for binding to single-stranded DNA). Binding to single-stranded poly(dA) is only slightly dependent on ionic strength, although the affinity for double-stranded DNA is strongly ionic strength-dependent and has an optimum at approximately 100-120 mM Na+. Above this range, binding to native DNA is virtually all electrostatic in nature. Although the affinity of HMG 1 for single-stranded DNA is higher than that for double-stranded DNA at the extremes of the ionic range studied, no clear evidence for a helix-destabilizing activity was obtained. At low ionic strength, the protein actually stabilized DNA against thermal denaturation, while at high ionic strength, HMG 1 appears to undergo denaturation below the Tm of the DNA. Studies of the environment of the tryptophan fluorophores using collisional quenchers iodide, cesium, and acrylamide suggest that the predominant fluorophore is relatively exposed but constrained in a rigid, positively charged environment.     

Alteration in nucleosome structure induced by thermal denaturation.

Mononucleosomes prepared from goose erythrocyte nuclei exhibited limited heterogeneity with respect to number of electrophoretic components, histones and DNA composition. The components differ slightly in ionic strength induced self-association. Thermal denaturation of each component gave only two dominant, highly cooperative, melting transitions, T" and T"'. Urea and trypsin were used to establish the differential lability of these two transitions. Comparison of the morphologies of the mononucleosomes at various stages throughout the melting profile indicated that the 13.3 +/- 1.5 nm diameter mononucleosomes start to disrupt only in the latter half of transition T" and do not unfold until after reaching T"'. The resultant, open ended (17.4 +/- 2.2 nm diameter) toroids are still largely negatively staining and much more uniform in shape if fixed simultaneously with gluteraldehyde.     

Computer programs to assist in high resolution thermal denaturation and circular dichroism studies on nucleic acids

Computer programs are described that direct the collection, processing, and graphical display of numerical data obtained from high resolution thermal denaturation (1-3) and circular dichroism (4) studies. Besides these specific applications, the programs may also be useful, either directly or as programming models, in other types of spectrophotometric studies employing computers, programming languages, or instruments similar to those described here (see Materials and Methods).     

Calorimetric and circular dichroic studies of the thermal denaturation of beta-lactoglobulin

The thermal denaturation of beta-lactoglobulin in aqueous solutions at pH 5.5 and 2.0 was investigated by differential scanning calorimetry (DSC) and circular dichroic (CD) measurements. By calorimetry, the denaturation temperatures (Td), denaturation enthalpies, and specific heat capacity changes for thermal denaturation in the temperature range scanned, i.e., 20-100 degrees C. The unfolding process was found to be only partially reversible. Analysis of the far-ultraviolet CD spectra reveals that with increasing temperature the mean residue ellipticity [( theta]) becomes less negative, which reflects unfolding of the native protein. At the highest temperature of CD measurements, i.e., 80 degrees C, conformational changes are to a large extent reversible.