Differential scanning calorimetry (DSC) is the most widely used thermal analytical technique in food research and it has a great utility in quality assurance of food. Proteins are the most studied food components by thermal analysis including studies on conformation changes of food proteins as affected by various environmental factors, thermal denaturation of tissue proteins, food enzymes and enzyme preparations for the food industry, as well as effects of various additives on their thermal properties. Freezing-induced denaturation of food proteins and the effect of cryoprotectants are also monitored by DSC. Polymer characterization based on DSC of polysaccharides, gelatinization behaviour of starches and interaction of starch with other food components can be determined, and phase transitions during baking processes can be studied by DSC. Studies on crystallization and melting behaviour of fats observed by DSC indicate changes in lipid composition or help characterizing products. Thermal oxidative decomposition of edible oils examined by DSC can be used for predicting oil stability. Using DSC in the freezing range has a great potential for measuring and modelling frozen food thermal properties, and to estimate the state of water in foods and food ingredients. Research in food microbiology utilizes DSC in better understanding thermoadaptive mechanisms or heat killing of food-borne microorganisms. Isothermic microcalorimetric techniques provide informative data regarding microbial growth and microbial metabolism.
Authors:Cs. Mohácsi-Farkas, J. Farkas, L. Mészáros, O. Reichart and É. Andrássy
Thermal stability of vegetative cells of Listeria monocytogenes, Escherichia coli and Lactobacillus plantarum was studied by counting viable fractions and determining DSC curves of their suspensions. DSC curves in the 5–99°C range showed a series of endothermic transitions between 50 and 60°C, where the heat destruction of cells occurred. Heat denaturation of DNA required a higher temperature than cell killing. Thermal death was strongly influenced by the pH, composition and NaCl content of the suspending buffer. A mathematical model developed by us enabled comparison of DSC peak temperatures and temperatures required for loss of viability.
Authors:Franciska Könczöl, Nelli Farkas, Tímea Dergez, J. Belágyi and D. Lőrinczy
DSC and EPR experiments were performed on human erythrocyte membranes and DPPC vesicles in order to study the effect of the
anaesthetic drug tetracaine on structure and dynamics of the lipid region. Experiments using spin label technique showed that
tetracaine induced fluidity changes of the lipid region in the environment of the fatty acid probe molecules incorporated
into the membranes in the vicinity of the lipid-water interface. Similarly to EPR observations, DSC measurements reported
decrease of the main melting and the pretransition temperature in comparison to control DPPC vesicles, which is the sign of
destabilisation of the structure in the head group region of the lipids. Similar effect was observed in the case of erythrocytes
where the protein conformation was also controlled in the presence of drug. A separated membrane melting with well distinguished
membrane protein phase transition was found that was affected significantly by tetracaine. These results suggest that tetracaine
is able to modify not only the internal dynamics of erythrocyte membranes and produce destabilisation of the lipid structure,
but the protein system as well. These might lead to further damage of the biological functions.
Authors:T. Dergez, F. Könczöl, N. Farkas, J. Belágyi and D. Lőrinczy
Summary The heat capacity of contractile proteins actin and myosin was studied in psoas muscle of rabbit in strongly and weakly binding state of myosin to actin as a function of temperature by DSC. Deconvolution of the unfolding scans makes possible to characterize the structural domains of the macromolecules. We tried to approach the unfolding process in different intermediate state of ATP hydrolysis. The thermal transitions were calorimetrically irreversible, therefore the two-state irreversible model that describes fairly well the denaturation of different proteins was used for evaluation of the denaturation processes in muscle fibers in strongly (rigor, ADP) and weakly binding states (ATP·Vi, ADP·AlF4) of myosin to actin. Deconvolution resulted in four transitions, the first three transition temperatures were almost independent of the intermediate states of muscle, the last transition temperature was shifted to higher temperature, when the buffer solution was manipulated. The mean values in strongly binding states were Tm1=52.9±0.7°C, Tm2=57.9±0.7°C, Tm3=63.7±1.0°C and Tm4=67.8±0.7°C, but the last transition increased to higher temperature depending on the Pi analogue.
Authors:D. Lőrinczy, N. Hartvig, N. Farkas and J. Belagyi
Differential scanning calorimetry and electron paramagnetic resonance experiments were performed on glycerinated muscle fibres
to study the effect of the binding of nucleotides (ADP and AMP⋅PNP) to myosin. The thermal unfolding of muscle fibres showed
three discrete domain regions with thermal stabilities of 52.2, 58.8 and 67.8°C. AMP⋅PNP markedly affected the transitions,
implying the strong interaction between AMP⋅PNP and catalytic domain, and partial dissociation of heads from actin. ADP produced
only small changes in transition temperatures.
Spectrum deconvolution performed on isothiocyanate-labelled fibres in AMP⋅PNP-state resulted in two populations; 50% of labels
was highly ordered with respect to fibre axis, whereas the other 50% of labels was randomly oriented. The conformation of
the myosin heads which showed high degree of order were in the strongly binding ADP-state, the heads being attached to actin
differ from those of heads in rigor. The results support the suggestion that the attached heads in strongly binding state
to actin might have different local conformations.
Authors:D. Lőrinczy, F. Könczöl, L. Farkas, J. Belagyi and C. Schick
Electron paramagnetic resonance (EPR, ST-EPR) and differential scanning calorimetry(DSC) were used in conventional and temperature
modulated mode to study internal motions and energetics of myosin in skeletal muscle fibres in different states of the actomyosin
ATPase cycle. Psoas muscle fibres from rabbit were spin-labelled with an isothiocyanate-based probe molecule at the reactive
sulfhydryl site (Cys-707) of the catalytic domain of myosin. In the presence of nucleotides (ATP, ADP, AMP⋅PNP) and ATP or
ADP plus orthovanadate, the conventional EPR spectra showed changes in the ordering of the probe molecules in fibres. In MgADP
state a new distribution appeared; ATP plus orthovanadate increased the orientational disorder of myosin heads, a random population
of spin labels was superimposed on the ADP-like spectrum.
In the complex DSC pattern, higher transition referred to the head region of myosin. The enthalpy of the thermal unfolding
depended on the nucleotides, the conversion from a strongly attached state of myosin to actin to a weakly binding state was
accompanied with an increase of the transition temperature which was due to the change of the affinity of nucleotide binding
to myosin. This was more pronounced in TMDSC mode, indicating that the strong-binding state and rigor state differ energetically
from each other. The different transition temperatures indicated alterations in the internal microstructure of myosin head
region The monoton decreasing TMDSC heat capacities show that Cp of biological samples should not be temperature independent.
Authors:M. Kiss, F. Könczöl, N. Farkas, D. Lőrinczy and J. Belagyi
The effect of free radicals obtained in hydroxyl and cerium(IV)-nitrilotriacetic acid free radical generating systems on contractile
proteins (actin, myosin and their complexes in glycerinated muscle fibres) was studied using differential scanning calorimetry
and spin trapping electron paramagnetic resonance technique. The analysis of spectra showed that selective attack of thiol
groups – Cys-257 and Cys-374 residues of actin, and among others Cys-707 residue of myosin – and random attack of sidechains
of the main proteins of muscle tissue produced structural and functional changes, which affected the ATP hydrolysis cycle
and very likely the dynamics of actin. The melting curves obtained on protein systems support the view that global conformational
changes accompany the local damage of free radicals.
Authors:J. Németh, G. Oroszi, B. Jakab, M.i Magyarlak, Z. Szilvassy, E. Rőth and B. Farkas
The iodination and separation of various diagnostically and/or experimentally important peptides including (Tyr1)-somatostatin-14, rat Tyr-a-calcitonin gene-related peptide (23-37), motilin and vasoactive intestinal peptide, furthermore bovine serum albumin are described. All species were iodinated by the iodogen method. The 125I-labeled peptide products were separated by reversed-phase HPLC, the specific activities of mono-iodinated forms are near identical with the theoretical value. The labeled bovine serum albumin was separated by Sephadex G-100 gel filtration.