Denaturation Point Research Articles

Characterization of recombinant amino-terminal NC4 domain of human collagen IX: interaction with glycosaminoglycans and cartilage oligomeric matrix protein.

The N-terminal NC4 domain of collagen IX is a globular structure projecting away from the surface of the cartilage collagen fibril. Several interactions have been suggested for this domain, reflecting its location and its characteristic high isoelectric point. In an attempt to characterize the NC4 domain in more detail, we set up a prokaryotic expression system to produce the domain. The purified 27.5-kDa product was analyzed for its glycosaminoglycan-binding potential by surface plasmon resonance and solid-state assays. The results show that the NC4 domain of collagen IX specifically binds heparin with a K(d) of 0.6 microm, and the full-length recombinant collagen IX has an even stronger interaction with heparin, with an apparent K(d) of 3.6 nm. The heparin-binding site of the NC4 domain was located in the extreme N terminus, containing a heparin-binding consensus sequence, whereas electron microscopy suggested the presence of at least three additional heparin-binding sites on full-length collagen IX. The NC4 domain was also shown to bind cartilage oligomeric matrix protein. This interaction and the association of cartilage oligomeric matrix protein with other regions of collagen IX were found to be heparin-competitive. Circular dichroism analyses of the NC4 domain indicated the presence of stabilizing disulfide bonds and a thermal denaturation point of about 80 degrees C. The pattern of disulfide bond formation within the NC4 domain was identified by tryptic peptide mass mapping of the NC4 in native and reduced states. A similar pattern was demonstrated for the NC4 domain of full-length recombinant collagen IX.

Temperature-dependent Raman spectra of collagen and DNA

Raman spectra of collagen and DNA were discussed at different temperatures. The temperature-dependence of Raman intensity was obtained in the region from −150 to 200 °C. Four denaturation points at 0, 40, 68 and 90 °C of collagen and two peaks at 38 and 82 °C for DNA were obtained. The wavenumbers of many vibrational modes were found to increase for lower temperature, but the peak at 1302 cm −1 of collagen and the peak at 1101 cm −1 of DNA showed the opposite trend. In all of the vibrational modes of DNA, the bases showed the most sensitive to different temperatures and there is a pronounced shift of bands at 70 °C, the starting point of denaturation.

Contribution of secondary structure to the heat capacity and enthalpy distribution of the unfolded state in proteins.

We have recently shown that one can construct the enthalpy distribution for protein molecules from experimental knowledge of the temperature dependence of the heat capacity. For many proteins the enthalpy distribution evaluated at the midpoint of the denaturation transition (corresponding to the maximum in the heat capacity vs temperature curve) is broad and biphasic, indicating two different populations of molecules (native and unfolded) with distinctly different enthalpies. At temperatures above the denaturation point, the heat capacity for the unfolded state in many proteins is quite large and using the analysis just mentioned, we obtain a gaussian-like enthalpy distribution that is very broad. A large value of the heat capacity indicates that there are structural changes going on in the unfolded state above the transition temperature. In the present paper we investigate the origin of this large heat capacity by considering the presence of changing amounts of secondary structure (specifically, alpha-helix) in the unfolded state. For this purpose we use the empirical estimates of the Zimm-Bragg sigma and s factors for all of the native amino acids in water as determined by Scheraga and co-workers. Using myoglobin as an example, we calculate probability profiles and distribution functions for the total number of helix states in the specific-sequence molecule. Given the partition function for the specific-sequence molecule, we can then calculate a set of enthalpy moments for the molecule from which we obtain a good estimate of the enthalpy distribution in the unfolded state. This distribution turns out to be quite narrow when compared with the distribution obtained from the raw heat capacity data. We conclude that there must be other major structural changes (backbone and solvent) that are not accounted for by the inclusion of alpha-helix in the unfolded state.

The interaction of new 4,9-diazapyrenium compounds with double stranded nucleic acids

Interactions of double stranded nucleic acids were studied with 4,9-diazapyrenium compounds, including monofunctional monocationic derivatives [4-methyl- (1) and 4-benzyl- (2)], monofunctional dicationic derivatives [4,9-dimethyl- with substituents H- (3) or Ph- (4) in the 5,10 positions and Me- (5) in the 2,7-positions], and bifunctional derivatives [4,4′-p- (6) and m- (7) xylylene bridged], which were described in the preceding paper. NMR spectra indicate intercalation for all ligands, with line width increases of up to 70 Hz. Thermal melting experiments and UV or fluorescence titrations were used to characterize affinities; these are essentially independent of the number of charges present in the ring systems, in line with negligible electrostatic binding contributions and with the corresponding affinities towards nucleotides (reported in the preceding paper). Substituents at the pyrenium rings have relatively little influence on the binding, with the exception of two phenyl groups, which lower the affinity, probably due to steric hindrance. Several melting curves are biphasic; in particular with the RNA-type polyA-polyU one observes transition points above and below the original denaturation point. Ligands containing two diazapyrenium rings bridged either by a m- or by a p-xylylene unit show distinctly higher affinities for the latter, and in Scatchard analyses a ligand to nucleotide ratio of n = 0.08, suggesting bisintercalation. Viscometry, however, shows a rather uniform length increase of the calf thymus DNA double helix with slopes of α = 1.1, similar to the known monointercalator ethidium bromide (α = 1.0). The monofunctional compounds exhibit some noteworthy RNA selectivity.

Physico-chemical properties of lupin seed proteins ( Lupinus albus, ssp. Graecus)

Some general physico-chemical properties of lupin seed ( Lupinus albus, ssp. Graecus) protein isolate solutions were studied. These included: molecular weight, isoelectric point, amino acids, temperatures and enthalpies of denaturation.

The effect of temperature on enzymes used in diagnostics

A number of enzymes that are used in clinical analysis have been studied in relation to the effect of temperature on their activity. Both V max and K m were determined over a temperature range from 13 to 55°C. Whereas V max values increased steadily until denaturation point with all enzymes, the effect of temperature on K m was more variable. With most enzymes there was a gradual increase in K m, often with a sharp rise close to the denaturation temperature. In most cases, K m did not increase as fast as V max consequently the enzyme efficiency, V max K m , also increased slightly with temperature. However this was not the case with rabbit muscle lactate dehydrogenase, for which the K m for pyruvate increased faster than V max. As a consequence, it was predicted and confirmed that enzymatic analysis of pyruvate using lactate dehydrogenase is more rapid at 20°C than at 35°C or 50°C.

Relationship between the Thermal Denaturation and Gelling Properties of Legumin from Broad Beans.

The effects of NaCl and heating temperature on the gel-forming ability of legumin were studied. The addition of NaCl progressively increased the denaturation temperature of legumin. Heating to around the denaturation point, i.e., below the onset temperature (zone 1), between the onset and maximal temperatures (zone 2), between the maximal and final temperatures (zone 3), and above the final temperature (zone 4), affected both the gel-forming ability and gel properties. No gel was formed in zone 1, while the gel was harder in zone 3 than in zones 2 and 4. The gel hardness gradually decreased with increasing temperature in zone 4. Differences in the viscoelastic and microstructural properties between gels heated at various temperatures around the denaturation point were observed.

Thermodynamic analysis of the folding of the streptococcal protein G IgG-binding domains B1 and B2: why small proteins tend to have high denaturation temperatures.

We have cloned, expressed, and characterized two naturally occurring variations of the IgG-binding domain of streptococcal protein G. The domain is a stable cooperative folding unit of 56 amino acids, which maintains a unique folded structure without disulfide cross-links or tight ligand binding. We have studied the thermodynamics of the unfolding reaction for the two versions of this domain, designated B1 and B2, which differ by six amino acids. They have denaturation temperatures of 87.5 degrees C and 79.4 degrees C, respectively at pH 5.4, as determined by differential scanning calorimetry. Thermodynamic state functions for the unfolding reaction (delta G, delta H, delta S, and delta Cp) have been determined and reveal several interesting insights into the behavior of very small proteins. First, though the B1 domain has a heat denaturation point close to 90 degrees C, it is not unusually stable at physiologically relevant temperatures (delta G = 25 kJ/mol at 37 degrees C). This behavior occurs because the stability profile (delta G vs temperature) is flat and shallow due to the small delta S and delta Cp for unfolding. Related to this point is the second observation that small changes in the free energy of unfolding of the B-domain due to mutation or change in solvent conditions lead to large shifts in the heat denaturation temperature. Third, the magnitude and relative contributions of hydrophobic vs nonhydrophobic forces (per amino acid residue) to the total free energy of folding of the B-domain are remarkably typical of other globular proteins of much larger size.

Assignment of aromatic spin systems in the 1H nuclear magnetic resonance spectrum of adenylate kinase

A combination of selective spin decoupling, two-dimensional double quantum spectroscopy, correlated spectroscopy (COSY), and pH titration experiments brought about the assignment of all tyrosyl spin systems and completed the assignment of the histidyl spin systems in porcine adenylate kinase. In the detection of the tyrosyl spin systems it proved to be advantageous to resort to the COSY method rather than to two-dimensional double quantum spectroscopy. In the titration experiments, His189 revealed a second apparent p K value at pH 8.3, which is explained by deprotonation of the adjacent residue Cysl87. None of the seven tyrosyl side-chains shows any evidence for deprotonation up to the point of denaturation of the protein, which took place around pH 10.

Thermal injury to bone: A vital-microscopic description of heat effects

A titanium implant, the thermal chamber, allowing vital microscopic observations of bone tissue during heating and bone temperature measurements to be made simultaneously is described. The tissue reactions to a defined heat trauma may be followed and registered on slides and ciné film. The thermal chamber technique allows repeated observations of the same bone tissue compartment for indefinite follow-up periods. In the present experiment, the chamber was inserted in the rabbit tibia and heating to 53°C was applied for 1 min. During heating to the assigned temperature, the blood-flow velocity initially increased. At 53°C the blood flow stopped in some vessels and became sluggish in others. 2 days after heating the blood flow in the pre-existing vessels had stopped. Gradually thereafter, all original vessels became replaced with ingrowing newly-developed vascular pathways. Fat cells seen before burning became resorbed and bone remodelling started 3–5 weeks after the heat injury. A temperature of 53°C, i.e. below the denaturation point of alkaline phosphatase, caused an irreversible bone injury, after which healing occurred from the surrounding tissues.

Zur Systematik von Actinobacillus, Haemophilus und Pasteurella: Basenzusammensetzung der DNS, Atmungschinone und kulturellbiochemische Eigenschaften repräsentativer Sammlungsstämme

In a comparative study, 63 collection cultures representing 38 nomenspecies of, or assigned to, the genera Actinobacillus, Haemophilus, or Pasteurella were characterized by phenotypical features and deoxyribonucleic acid base composition. The latter was calculated from the thermal denaturation point. Biochemical reactions were tested in differential media commonly used for Enterobacteriaceae, and two test procedures were compared: (i) pure cultures with haematin and nicotine adenine dinucleotide added, where necessary, and (ii) xenocultures with an asaccharolytic Acinetobacter strain (ST 661/60). Furthermore, the respiratory quinones, and the effect of fumarate on oxygen-limited growth were considered. On the basis of these and some additional physiological and morphological criteria, a definition of the Actinobacillus-Haemophilus-Pasteurella group as a whole was established which appears to rank as a family. Several misclassified species, i.e. the so-called Actinobacillus actinoides, Haemophilus piscium, Haemophilus vaginalis, Pasteurella anatipestifer, and the organisms of the Bovine Lymphangitis group were eliminated, and the position of so-called Pasteurella piscicida was questioned. Some principles of subdivision of the group, and some of the practical identification procedures were discussed.

The bone-cement interface temperature during total joint replacement

Sets of thermoprobe measurements of the temperature of the bone-cement interface were made during twenty total joint-replacement procedures. Although the range of rise in temperature was wide, from 3 to 17 degrees centigrade, the highest temperature obtained was 48 degrees centigrade, which is well below the denaturation point (56 degrees centigrade) of proteins. Several factors, such as the presence of blood and moisture at the interface and the large surface area and poor heat conductivity of methylmethacrylate, prevent the interface from experiencing the high rise in temperature that occurs at the center of the polymerizing cement mass.

DNA conformation in N,N‐dimethyl formamide–H2O solutions

AbstractOptical density, viscosity, and light scattering measurements for calf thymus DNA in water–N,N dimethyl formamide (DMF) solutions are presented. DMF content varied between 0 and 60% (v/v) and DNA molecular weight varied between 15 × 106 and 0.5 × 106. Complementary measurements of the solubility of adenine, thymine, guanine, and cytosine in H2O–DMF mixtures are presented. The denaturation temperature of DNA, manifested by about a 35% increase of optical density, is gradually depressed by increasing DMF content. However, a significant increase of OD occurs even before (and even after) the denaturation point, when DMF content is increased isothermally. The intrinsic viscosity also exhibits a large decrease when DMF content is increased both before and after the denaturation point. Light scattering data for high‐molecular‐weight DNA in the predenaturation range indicate a decrease of the mean‐square radius and a constant molecular weight on increasing DMF content. The results, interpreted in terms of the wormlike chain of Kratky and Porod, indicate a large decrease of the persistence length of DNA. For low‐molecular‐weight DNA, radius and molecular weight increase with DMF content, indicating intermolecular aggregation. The formation of compact structures of native DNA is discussed in terms of an increased solubility of uncharged bases, and a decreased solubility of phosphate and deoxyribose groups, when a less polar environment is provided by the addition of DMF.

Quasi-Elastic Light Scattering by Diffusional Fluctuations in RNase Solutions

Using measurements of quasi-elastic light scattering spectra, we have investigated diffusional fluctuations of RNase. The diffusion coefficient for individual protein molecules, together with the corresponding calculated effective molecular radius R(eff), were determined. Between room temperature and the point of irreversible denaturation at 63.5 degrees C, R(eff) increased from 20-250 A. This is comparable to the plateau in R(eff) of 300 A reached after about 200 min following chemical denaturation in 10 M urea. The measurements indicated the presence of a large size component even in the freshly prepared and chromatographically purified solutions. From the diffusion constants deduced for this large component we obtained effective sizes from 1000-5000 A. Concentration and temperature dependent measurements exclude the possibility that these large particles are impurities and indicate that they are the result of aggregations of RNase molecules.

Interim report of the Committee on Histologic Mounting Media; resinous media.

The Fisher “Permount” naphthalene polymer, the Hartman Leddon “H.S.R.” terpene polymer resin, a Monsanto polystyrene P-1, the Will Corporation “Diaphane” and “Green Diaphane”, and the du Pont “Lucite” methyl methacrylate polymer were examined, and the possibility of use of some other plastics was also explored. The first 5 mentioned were tested for color preservation of a variety of stains in comparison with Canada balsam and Clarite X. From this point of view polystyrene, the Hartman Leddon “H.S.R.” and the Fisher “Permount” resins were the most satisfactory, then the “Diaphanes”. Both “Permount” and “H.S.R.” show some yellowing. The H.S.R. with a melting point of 115°C, the Permount with 150°C. melting point, and the Polystyrene with a thermal denaturation point above 220°C. all excell Canada balsam in heat resistance. Trimethylbenzene, cymene and monoamylbenzene appear to be the best solvents for polystyrene. Mounts made in a solution of 20 g. polystyrene in 100 ml. trimethylbenzene can be packed flat ...