Deconvolution of complex differential scanning calorimetry profiles for protein transitions under kinetic control

A frequent outcome in differential scanning calorimetry (DSC) experiments carried out with large proteins is the irreversibility of the observed endothermic effects. In these cases, DSC profiles are analyzed according to methods developed for temperature-induced denaturation transitions occurring under kinetic control. In the one-step irreversible model (native → denatured) the characteristics of the observed single-peaked endotherm depend on the denaturation enthalpy and the temperature dependence of the reaction rate constant, k. Several procedures have been devised to obtain the parameters that determine the variation of k with temperature. Here, we have elaborated on one of these procedures in order to analyze more complex DSC profiles. Synthetic data for a heat capacity curve were generated according to a model with two sequential reactions; the temperature dependence of each of the two rate constants involved was determined, according to the Eyring's equation, by two fixed parameters. It was then shown that our deconvolution procedure, by making use of heat capacity data alone, permits to extract the parameter values that were initially used. Finally, experimental DSC traces showing two and three maxima were analyzed and reproduced with relative success according to two- and four-step sequential models.     

Melatonin strongly interacts with zwitterionic model membranes—evidence from Fourier transform infrared spectroscopy and differential scanning calorimetry

Interactions of melatonin with zwitterionic dipalmitoyl phosphatidylcholine (DPPC) multilamellar liposomes (MLVs) were investigated as a function of temperature and melatonin concentration (1-30 mol%) by using two noninvasive techniques, namely Fourier transform infrared (FTIR) spectroscopy and differential scanning calorimetry (DSC). The investigation of the C-H, CO, and PO₂⁻ antisymmetric double stretching modes in FTIR spectra and DSC studies reveal that melatonin changes the physical properties of the DPPC bilayers by decreasing the main phase transition temperature, abolishing the pretransition, ordering the system in the gel phase, and increasing the dynamics of the system both in the gel and liquid crystalline phases. It also causes significant decrease in the wavenumber for the CO stretching and PO₂⁻ antisymmetric double bond stretching bands, which indicates strong hydrogen bonding The results imply that melatonin locates in the interfacial region of the membrane. Furthermore, in the DSC curve, more than one signal is observed at high melatonin concentrations (24 and 30 mol%), which indicates melatonin-induced phase separation in DPPC membranes.     

Small angle X-ray scattering (SAXS) and differential scanning calorimetry (DSC) studies of amide phospholipids

Varying chemically the structure of phospholipids in the region between hydrophobic and hydrophilic segments is expected to have a strong influence on the interaction with water and the phase behavior. This is studied in this work with the motivation to investigate these lipids as potential inhibitors of phospholipase A2. Thus the amide phospholipids L-ether-amide-PC (1-O-hexadecyl-2-N-palmitoyl-2-amino-2-deoxy-sn-glycero-3-phosphocholine), L-ester-amide-PC (1-palmitoyl-2-N-palmitoyl-2-amino-2-deoxy-sn-glycero-3-phosphocholine) and L-ether-amide-PE (1-O-hexadecyl-2-N-palmitoyl-2-deoxy-sn-glycero-3-phosphoethanolamine) have been synthesized and characterized. The phase behavior and thermal transitions in buffer dispersions are examined by a combination of high-sensitivity differential scanning calorimetry (DSC) and small angle X-ray scattering (SAXS) experiments between 10 and 80 degrees C at pH 8.9. The onset temperatures determined from DSC measurements agree well with the starting temperatures of changes in the repeat distance obtained by SAXS measurements. The phases observed are lamellar both below and above the main phase transition. The phase transition temperatures and enthalpies depend strongly on the substitutions in sn-1 position and head group structure. The lamellar repeat distance in gel and liquid-crystalline phases increases with increasing temperature for L-ester-amide-PC and L-ether-amide-PC, whereas the temperature dependence is opposite for the L-ether-amide-PE. The observed behavior is discussed and compared with that of DPPC and DPPE, indicating the strong dependence of hydration and phase behavior on head group structure.     

Successive phase-transition phenomena and phase diagram of the phosphatidylcholine-water system as revealed by differential scanning calorimetry

A completely dehydrated dipalmitoylphosphatidylcholine (DPPC) was prepared with dehydration under high vacuum and at a temperature above its main transition temperature. Thermal analyses on about forty different samples of the DPPC-water system indicated that the main transition temperature decreased stepwise with an increase in the water content to the limiting temperature at 42.6°C, reflecting the thermal behaviors of a total of five endothermic peaks. The pretransition appeared at a water content above 17 g%, and the predominant role of ‘newly incorporated water’ between the bilayers of DPPC molecules at the pretransition was made evident.     

The circular dichroism and differential scanning calorimetry study of the properties of DNA aptamer dimers

We have applied circular dichroism (CD), temperature-gradient gel electrophoresis (TGGE) and differential scanning calorimetry (DSC) to study the properties of novel bioengineered DNA aptamer dimers sensitive to fibrinogen (F) and heparin (H) binding sites of thrombin and compared them with canonical single stranded aptamer sensitive to fibrinogen binding site of thrombin (Fibri). The homodimer (FF) and heterodimer (FH) aptamers were constructed based on hybridization of their supported parts. CD results showed that both FF and FH dimers form stable guanine quadruplexes in the presence of potassium ions like those in Fibri. The thermal stability of aptamer dimers was slightly lower compared to those of canonical aptamers, but sufficient for practical applications. Both FF and FH aptamer dimers exhibited a potassium-dependent inhibitory effect on thrombin-mediated fibrin gel formation, which was on average two-fold higher than those of canonical single stranded Fibri aptamers.     

Phase separations induced by melittin in negatively-charged phospholipid bilayers as detected by fluorescence polarization and differential scanning calorimetry

Interactions between melittin and a variety of negatively-charged lipid bilayers have been investigated by intrinsic fluorescence, fluorescence polarization of 1,6-diphenylhexatriene and differential scanning calorimetry. (1) Intrinsic fluorescence of the single tryptophan residue of melittin shows that binding of this peptide to negatively-charged phospholipids is directly related to the surface charge density, but is unaffected by the physical state of lipids, fluid or gel, single-shell vesicles or unsonicated dispersions. (2) Changes in the thermotropic properties of negatively-charged lipids upon melittin binding allow to differentiate two groups of lipids: (i) A progressive disappearance of the transition, without any shift in temperature, is observed with monoacid C₁₄ lipids such as dimyristoylphosphatidylglycerol and -serine (group 1). (ii) With a second group of lipids (group 2), a transition occurs even at melittin saturation, and two transitions are detected at intermediate melittin content, one corresponding to remaining unperturbed lipids, the other shifted downward by 10–20°C. This second group of lipids is constituted by monoacid C₁₆ lipids, dipalmitoylphosphatidylglycerol and -serine. Phosphatidic acids also enter this classification, but it is the net charge of the phosphate group which allows to discriminate: singly charged phosphatidic acids belong to group 2, whereas totally ionized ones behave like group 1 lipids, whatever the chain length. (3) It is concluded that melittin induces phase separations between unperturbed lipid regions which give a transition at the same temperature as pure lipid, and peptide rich domains in which the stoichiometry is 1 toxin per 8 phospholipids. The properties of such domains depend on the bilayer stability: in the case of C₁₆ aliphatic chains and singly charged polar heads, the lipid-peptide domains have a transition at a lower temperature than the pure lipid. With shorter C₁₄ chains or with two net charges by polar group, the bilayer structure is probably totally disrupted, and the new resulting phase can no longer lead to a cooperative transition.     

Monitoring the kinetics and thermodynamics of interfacial enzymatic catalysis by differential scanning calorimetry

Using phase transition profile as an indicator of thermodynamic property and phase transition heat as the second indicator of the percentage of substrates unhydrolyzed, differential scanning calorimetry has been used to observe in detail the kinetics and thermodynamics of phospholipase A(2)-catalyzed 1,2-dipalmitoyl-sn-glycero-3-phosphocholine large unilamellar vesicle (LUV) hydrolysis. Phase transition profiles show that the original LUV almost completely changes into a novel aggregate at the end of the latency, followed by an abrupt activation of the reaction. The phase transition profiles are asymmetric between the heating and cooling curves, indicating a thermodynamic mesostatic property of the system. The reaction in activated phase follows a single first-order kinetics and all of the substrates in vesicles can be hydrolyzed. All these evidences indicate that the products and substrates can freely exchange between the outer and the inner layers of the vesicles and the membrane of the vesicle in the activated phase is permeable. This permeability favors the exchange of the substrates and products, thus, resulting in the activation of the fast reaction.     

Interaction between dry starch and plasticisers glycerol or ethylene glycol, measured by differential scanning calorimetry and solid state NMR spectroscopy

The interaction of crystalline amylose and of crystalline and amorphous amylopectin with the plasticisers glycerol or ethylene glycol in the absence of water was studied, by using differential scanning calorimetry (DSC) and solid state nuclear magnetic resonance (NMR) spectroscopy. Upon heating starch freshly mixed with plasticisers, a strong exothermal interaction enthalpy of ΔH−35 J/g was detected by DSC. At room temperature glycerol interacts mainly with the amorphous starch regions, the interaction taking 8 days to reach equilibrium. For ethylene glycol the interaction is faster, taking four days to reach equilibrium, and the rate is not affected by crystallinity. Ethylene glycol interacts in a more ordered manner with amorphous than with crystalline material, resulting in a narrower ethylene glycol cross-polarisation magic angle spinning (CP/MAS) signal when equilibrium is reached at room temperature. Upon heating, more glycerol or ethylene glycol is immobilised, but in a less ordered manner than upon storage at room temperature. This results in a more intense, but broader plasticiser CP/MAS signal upon heating. Interaction in a more ordered manner probably implies interaction with more of the hydroxy groups of the plasticiser. The polysaccharide mobility is increased more when the plasticiser interacts in a more ordered manner, as observed by small starch signals in HP/DEC spectra.     

Quantitative measurement of indomethacin crystallinity in indomethacin-silica gel binary system using differential scanning calorimetry and X-ray powder diffractometry

Differential scanning calorimetry (DSC) and X-ray powder diffractometry (XRPD) methods were developed for the quantitative analysis of the crystallinity of indomethacin (IMC) in IMC and silica gel (SG) binary system. The DSC calibration curve exhibited better linearity than that of XRPD. No phase transformation occurred in the IMC-SG mixtures during DSC measurement. The major sources of error in DSC measurements were inhomogeneous mixing and sampling. Analyzing the amount of IMC in the mixtures using high-performance liquid chromatography (HPLC) could reduce the sampling error. DSC demonstrated greater sensitivity and had less variation in measurement than XRPD in quantifying crystalline IMC in the IMC-SG binary system. Published: February, 10, 2006     

Interactions of glycerol monooleate and dimethylsulphoxide with phospholipids A differential scanning calorimetry and 31P-NMR study

1. A comparative study has been made of the effects of the fusogens glycerol monooleate and dimethylsulphoxide on the polymorphic phase behaviour of dipalmitoyl phosphatidylcholine and dipalmitoyl phosphatidylethanolamine by differential scanning calorimetry and ³¹P-NMR techniques. 2. Glycerol monooleate induces a reduction in the temperature, cooperativity and enthalpy of the gel to liquid-crystal transitions of dipalmitoyl phosphatidylcholine and dipalmitoyl phosphatidylethanolamine, whereas dimethylsulphoxide induces an increase in the temperature and enthalpy and a reduction in the cooperativity of the gel to liquid-crystal transitions for those same phospholipids. 3. Glycerol monooleate induces the formation of isotropic and hexagonal (H_II) phases when mixed with either dipalmitoyl phosphatidylcholine or dipalmitoyl phosphatidylethanolamine. By contrast, in the presence of dimethylsulphoxide, those same phospholipids retain the lamellar configuration observed in the absence of fusogen. 4. These results are discussed in terms of the mechanisms of chemically induced cell fusion.     

A structure for the yeast prohibitin complex: Structure prediction and evidence from chemical crosslinking and mass spectrometry

The mitochondrial prohibitin complex consists of two subunits (PHB1 of 32 kD and PHB2 of 34 kD), assembled into a membrane-associated supercomplex of approximately 1 MD. A chaperone-like function in holding and assembling newly synthesized mitochondrial polypeptide chains has been proposed. To further elucidate the function of this complex, structural information is necessary. In this study we use chemical crosslinking, connecting lysine side chains, which are well scattered along the sequence. Crosslinked peptides from protease digested prohibitin complexes were identified with mass spectrometry. From these results, spatial restraints for possible protein conformation were obtained. Many interaction sites between PHB1 and PHB2 were found, whereas no homodimeric interactions were observed. Secondary and tertiary structural predictions were made using several algorithms and the models best fitting the spatial restraints were selected for further evaluation. From the structure predictions and the crosslink data we derived a structural building block of one PHB1 and one PHB2 subunit, strongly intertwined along most of their length. The size of the complex implies that approximately 14 of these building blocks are present. Each unit contains a putative transmembrane helix in PHB2. Taken together with the unit building block we postulate a circular palisade-like arrangement of the building blocks projecting into the intermembrane space.     

Influence of collagen denaturation on the chemorheological properties of skin,assessed by differential scanning calorimetry and hydrothermal isometric tension measurement

The curves obtained for skin samples of different ages and species by hydrothermal isometric tension (‘HIT’) measurement are compared to those obtained by differential scanning calorimetry (DSC) under the same thermal conditions (for a rise in temperature at a rate of 1.0°C/min). Collagen denaturation, observed by DSC, directly affects the kinetics of the tension variations in the first part of the ‘HIT’ curves, including the early peak due to the presence and destruction of the heat-labile cross-links in the collagen network. The presence of cross-links is in term shown to delay collagen denaturation to an extent which depends in part on their heat-stability. The final part of the ‘HIT’ curves reflecting the effects of heat in the stable polymeric collagen network is no longer affected by collagen denaturation. Thus, both ‘HIT’ and DSC are useful methods to evaluate collagen reticulation in connective tissues.     

On the assessment of the stability of vitrified cryo-media by differential scanning calorimetry: A new tool for biobanks to derive standard operating procedures for storage,access and transport

When a vitrified sample is heated over the glass transition temperature it may start to devitrify endangering the sample. The ability to estimate the stability of the vitrified state can help in the development of new vitrification media as well as handling procedures. By employing differential scanning calorimetry, we can measure the ice crystallization rate in a vitrified sample and thus study the devitrification kinetics. Using this technique, we have studied samples comprised of PBS with cryoprotective additives (CPA) as dimethylsulfoxide (Me₂SO), ethylene glycol (EG) and mixtures thereof, regarding the dependence of the devitrification kinetics on the CPA concentration. We found that already small concentration changes lead to significant changes in the devitrification times. Changing the CPA concentration by 4 wt% changed the devitrification time with a factor of 342 and 271 for Me₂SO and EG, respectively. Concentration changes in EG/Me₂SO mixtures was found to have a smaller impact on the devitrification kinetics compared to the pure CPA samples. Our data suggest that these significant increases in the devitrification times are primarily due to a relation between nucleation rates and the CPA concentration. Finally, we investigated an established vitrification medium used to preserve human embryonic stem cells. This medium was found to have the poorest glass stability in this study and reflects the tradeoff between stability and biocompatibility. The present work finally provides a tool to evaluate handling and storage procedures when employing vitrification as a cryopreservation method and underlines the importance of these.     

Thermotropic phase behaviour of the pseudobinary mixtures of DPPC/C12E5 and DMPC/C12E5 determined by differential scanning calorimetry and ultrasonic velocimetry

The present paper reports on the phase behaviour of the pseudobinary aqueous mixtures of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)/pentaethylene glycol monododecyl ether (C12E5) and 1,2-dimyristoyl-sn-glycero-3-phosphocholine monohydrate (DMPC)/C12E5. Both systems exhibit a variety of mesophases, such as lamellar gel, liquid crystalline and micellar phases. The phase diagrams show peritectic and eutectic behaviours. The existence of a compound complex is established. From the phase diagrams, the temperature dependence of the solubilisation parameters is obtained. The phase diagrams, especially with respect to the solubilisation process were qualitatively explained assuming that the packing of the constituents plays a dominating role. Finally, differential scanning calorimetry and ultrasonic velocimetry are compared concerning their potentials to determine characteristics of phase transitions in pseudobinary phospholipid/surfactant mixtures.     

Quantitative electrospray ionization mass spectrometry of zinc finger oxidation: the reaction of XPA zinc finger with H(2)O(2)

Oxidation plays an important role in the functioning of zinc fingers (ZFs). Electrospray ionization mass spectrometry (ESI-MS) is a very useful technique to study products of ZF oxidation, but its application has been limited largely to qualitative analysis of reaction products. On the other hand, ESI-MS has been applied successfully on several occasions to determine binding constants in metalloproteins. We used a synthetic 37-residue peptide acetyl-DYVICEECGKEFMDSYLMNHFDLPTCDNCRDADDKHK-amide (XPAzf), which corresponds to the Cys4 ZF sequence of human nucleotide excision repair protein XPA, to find out whether ESI-MS might be used quantitatively to study ZF reaction kinetics. For this purpose, we studied oxidation of the Zn(II) complex of XPAzf (ZnXPAzf) by H(2)O(2) using three techniques in parallel: high-performance liquid chromatography (HPLC) of covalent reaction products, 4-(2-pyridylazo)-resorcinol monosodium salt (PAR)-based spectrophotometric zinc release assay, and ESI-MS. Single and double intrapeptide disulfides were detected by ESI-MS to be the sole reaction products. All three techniques yielded independently the same reaction rate, thereby demonstrating that ESI-MS may indeed be used in quantitative kinetic studies of ZF reactions. The comparison of experimental information demonstrated that the formation of the Cys5-Cys8 single disulfide was responsible for zinc release.     

Interaction of cAMP receptor protein from Escherichia coli with cAMP and DNA studied by differential scanning calorimetry

The cyclic AMP receptor protein (CRP) regulates the expression of many genes in Escherichia coli. The protein is a homodimer, and each monomer is folded into two distinct structural domains. In this study, we have used differential scanning calorimetry (DSC) and circular dichroism (CD) to measure the enthalpy change and melting temperature of the apo-CRP and CRP complexes with cAMP or DNA sequences lac, gal, and palindromic ICAP. DSC and CD measurements showed irreversible thermal denaturation process of CRP. Enthalpy of dissociation of the protein–DNA complex, as measured by DSC, depends on the DNA sequence. The thermal transition of the protein in CRP-DNA complexes, measured by CD, indicates that the protein stability in the complex is also DNA sequence-dependent.     

The thermotropic phase behaviour and phase structure of a homologous series of racemic beta-D-galactosyl dialkylglycerols studied by differential scanning calorimetry and X-ray diffraction

The thermotropic phase behaviour of aqueous dispersions of some synthetic 1,2-di-O-alkyl-3-O-(beta-D-galactosyl)-rac-glycerols (rac-beta-D-GalDAGs) with both odd and even hydrocarbon chain lengths was studied by differential scanning calorimetry (DSC), small-angle (SAXS) and wide-angle (WAXS) X-ray diffraction. DSC heating curves show a complex pattern of lamellar (L) and nonlamellar (NL) phase polymorphism dependent on the sample's thermal history. On cooling from 95 degrees C and immediate reheating, rac-beta-D-GalDAGs typically show a single, strongly energetic phase transition, corresponding to either a lamellar gel/liquid-crystalline (L(beta)/L(alpha)) phase transition (N< or =15 carbon atoms) or a lamellar gel/inverted hexagonal (L(beta)/H(II)) phase transition (N> or =16). At higher temperatures, some shorter chain compounds (N=10-13) exhibit additional endothermic phase transitions, identified as L/NL phase transitions using SAXS/WAXS. The NL morphology and the number of associated intermediate transitions vary with hydrocarbon chain length. Typically, at temperatures just above the L(alpha) phase boundary, a region of phase coexistence consisting of two inverted cubic (Q(II)) phases are observed. The space group of the cubic phase seen on initial heating has not been determined; however, on further heating, this Q(II) phase disappears, enabling the identification of the second Q(II) phase as Pn3 m (space group Q(224)). Only the Pn3 m phase is seen on cooling. Under suitable annealing conditions, rac-beta-D-GalDAGs rapidly form highly ordered lamellar-crystalline (L(c)) phases at temperatures above (N< or =15) or below (N=16-18) the L(beta)/L(alpha) phase transition temperature (T(m)). In the N< or =15 chain length lipids, DSC heating curves show two overlapping, highly energetic, endothermic peaks on heating above T(m); corresponding changes in the first-order spacings are observed by SAXS, accompanied by two different, complex patterns of reflections in the WAXS region. The WAXS data show that there is a difference in hydrocarbon chain packing, but no difference in bilayer dimensions or hydrocarbon chain tilt for these two L(c) phases (termed L(c1) and L(c2), respectively). Continued heating of suitably annealed, shorter chain rac-beta-D-GalDAGs from the L(c2) phase results in a phase transition to an L(alpha) phase and, on further heating, to the same Q(II) or H(II) phases observed on first heating. On reheating annealed samples with longer chain lengths, a subgel phase is formed. This is characterized by a single, poorly energetic endotherm visible below the T(m). SAXS/WAXS identifies this event as an L(c)/L(beta) phase transition. However, the WAXS reflections in the di-16:0 lipid do not entirely correspond to the reflections seen for either the L(c1) or L(c2) phases present in the shorter chain rac-beta-D-GalDAGs; rather these consist of a combination of L(c1), L(c2) and L(beta) reflections, consistent with DSC data where all three phase transitions occur within a span of 5 degrees C. At very long chain lengths (N> or =19), the L(beta)/L(c) conversion process is so slow that no L(c) phases are formed over the time scale of our experiments. The L(beta)/L(c) phase conversion process is significantly faster than that seen in the corresponding rac-beta-D-GlcDAGs, but is slower than in the 1,2-sn-beta-D-GalDAGs already studied. The L(alpha)/NL phase transition temperatures are also higher in the rac-beta-D-GalDAGs than in the corresponding rac-beta-D-GlcDAGs, suggesting that the orientation of the hydroxyl at position 4 and the chirality of the glycerol molecule in the lipid/water interface influence both the L(c) and NL phase properties of these lipids, probably by controlling the relative positions of hydrogen bond donors and acceptors in the polar region of the membrane.     

Electrospray ionization mass spectrometry of zinc, cadmium, and copper metallothioneins: evidence for metal-binding cooperativity

Electrospray ionization (ESI) mass spectra of both well-characterized and novel metallothioneins (MTs) from various species were recorded to explore their metal-ion-binding modes and stoichiometries. The ESI mass spectra of the zinc- and cadmium-binding MTs showed a single main peak corresponding to metal-to-protein ratios of 4, 6, or 7. These findings combined with data obtained by other methods suggest that these MTs bind zinc or cadmium in a single predominant form and are consistent with the presence of three- and four-metal clusters. An unstable copper-specific MT isoform from Roman snails (Helix pomatia) could be isolated intact and was shown to preferentially bind 12 copper ions. To obtain additional information on the formation and relative stability of metal-thiolate clusters in MTs, a mass spectrometric titration study was conducted. One to seven molar equivalents of zinc or of cadmium were added to metal-free human MT-2 at neutral pH, and the resulting complexes were measured by ESI mass spectrometry. These experiments revealed that the formation of the four-metal cluster and of the thermodynamically less stable three-metal cluster is sequential and largely cooperative for both zinc and cadmium. Minor intermediate forms between metal-free MT, Me4MT, and fully reconstituted Me7MT were also observed. The addition of increasing amounts of cadmium to metal-free blue crab MT-I resulted in prominent peaks whose masses were consistent with apoMT, Cd3MT, and Cd6MT, reflecting the known structure of this MT with two Me3Cys9 centers. In a similar reconstitution experiment performed with Caenorhabditis elegans MT-II, a series of signals corresponding to apoMT and Cd3MT to Cd6MT species were observed.     

Label‐free determination of protein–ligand binding constants using mass spectrometry and validation using surface plasmon resonance and isothermal titration calorimetry

We performed a systematic comparison of three label‐free methods for quantitative assessment of binding strengths of proteins interacting with small molecule ligands. The performance of (1) nanoelectrospray ionization mass spectrometry (nESI‐MS), (2) surface plasmon resonance (SPR), and (3) isothermal titration calorimetry (ITC) was compared for the determination of dissociation constants (K_D). The model system studied for this purpose was the human carbonic anhydrase I (hCAI) with eight known and well characterized sulfonamide inhibitors (Krishnamurthy et al., Chem. Rev. 2008, 108: 946–1051). The binding affinities of the inhibitors chosen vary by more than four orders of magnitude e.g., the K_D value determined for ethoxzolamide by nESI‐MS was 5 ± 1 nM and the K_D value for sulfanilamide was 145.7 ± 10.0 µM. The agreement of the determined K_D values by the three methods investigated was excellent for ethoxzolamide and benzenesulfonamide (variation with experimental error), good for acetazolamide and 4‐carboxybenzenesulfonamide (variation by ～ one order of magnitude), but poor for others e.g., sulpiride. The accuracies of the K_D values are determined, and advantages and drawbacks of the individual methods are discussed. Moreover, we critically evaluate the three examined methods in terms of ease of the measurement, sample consumption, time requirement, and discuss their limitations. Copyright © 2009 John Wiley & Sons, Ltd.     

Effect of thermal denaturation on water–collagen interactions: NMR relaxation and differential scanning calorimetry analysis

The dependence of the proton spin–lattice relaxation rate, and of the enthalpy and temperature of denaturation on water content, were studied by nmr and differential scanning calorimetry (DSC) in native and denatured collagen. Collagen was first heated at four different temperatures ranging from 40 to 70°C. The percentage of denatured collagen induced by these preheating treatments was determined from DSC measurements. The DSC results are discussed in terms of heat‐induced structural changes. A two‐exponential behavior for the spin–lattice relaxation was observed with the appearance of denatured collagen. This was attributed to the presence of a noncollagen protein fraction. The variations in the different longitudinal relaxation rates as a function of the moisture content and of the denatured collagen percentage are described within the multiphase water proton exchange model. This study highlights the complementarity of the information obtained from the two analytical tools used. © 1999 John Wiley & Sons, Inc. Biopoly 50: 690–696, 1999