Dénes Lörinczy

Differential scanning calorimetric examination of the human intervertebral disc: a preliminary study

Abstract Intervertebral disc degeneration is a common orthopaedic disorder with significant social and economic impact. The major pathological changes occur in the structure of anulus fibrosus and nucleus pulposus. Differential scanning calorimetry (DSC) is a well-established method for the demonstration of thermal consequences of local and global conformational changes in biological systems. It has never been applied for the investigation of intervertebral disc. According to the present study, the thermograms may prove and follow the changes in the structure of degenerated intervertebral discs. Differences were clearly demonstrated between the two major parts of the intervertebral discs as well as healthy and degenerated samples with the changes in total enthalpy and heat capacity.

Nucleotide-induced changes in muscle fibres studied by DSC and TMDSC

Abstract Differential scanning calorimetry (DSC) was used in conventional and temperature-modulated mode to study the energetics of myosin in skeletal muscle fibres in different states of the actomyosin ATPase cycle. Psoas muscle fibres from rabbit were used in the experiments with and without the presence of nucleotides (ATP, ADP, AMP–PNP) and ATP or ADP+orthovanadate. In the complex DSC pattern, the 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 the C p of biological samples should not be temperature independent.

Calorimetric and spectroscopic properties of small globular proteins (bovine serum albumin, hemoglobin) after free radical generation

Mild oxidation of –SH-containing proteins (serum albumin, hemoglobin) by Ce(IV)-ions in the presence of the spin trap phenyl-tert-butylnitrone (PBN) resulted in the appearance of strongly immobilized nitroxide free radicals which evidences the formation of thiyl radicals on the thiol site of the proteins. In hydroxyl free radical generating system a fraction of strongly immobilized nitroxide radicals was also detected in these proteins, which implies that the oxidation of a fraction of the thiol groups was also involved in the free radical reaction. According to the differential scanning calorimetry (DSC) experiments the melting processes of the proteins were calorimetrically irreversible, therefore the two-state kinetic model was used to evaluate the experiments. The results support the view that site-specific interaction of SH-containing proteins with hydroxyl and thiyl free radicals is able to modify the internal dynamics of proteins and affect the conformation of large molecules.

Effect of polycyclic aromatic hydrocarbons on erythrocyte membranes by DSC and EPR

Differential scanning calorimetry (DSC) and electron paramagnetic resonance spectroscopy (EPR) experiments were performed on human erythrocyte membranes and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) model systems in order to study the effect the polycyclic aromatic hydrocarbons on lipid structure and dynamics. Eight different compounds among others naphthalene and pyrene were compared, which occur in significant concentrations in dust collected from the air in large cities. Experiments using spin label technique showed that the compounds induced mobility changes in the lipid region in the environment of the fatty acid probe molecules incorporated into the membranes. The effects depended on the structure and concentration of the different compounds. Similarly to EPR observations, DSC measurements reported decrease of transition temperature in comparison to control DPPC vesicles. These results suggest that polycyclic aromatic hydrocarbons were able to modify the internal dynamics of erythrocyte membranes which might lead to damage of the biological functions.

Interaction between lipopolysaccharide and detergents detected by differential scanning calorimetry

The Gram-negative bacteria can cause very serious diseases. One of their pathogenetic factors is the endotoxic lipopolysaccharide (LPS). This molecule is a component of outer membrane of Gram-negative bacteria. The LPS is released from the living bacteria in a modest measure, but after killing off bacteria by antibiotics a larger quantity of endotoxin is released into the surrounding. This molecule can induce different biological effects including pyogenicity and toxic shock. The LPS is an amphiphilic molecule: it has a hydrophobic and a hydrophilic part of molecule. In water, the LPS can aggregate. This aggregation process has been followed in our experiment by calorimetry. When we mixed the LPS extracted from Shigella sonnei Re 4350 with different detergents (polymyxin B (PmB), deoxicholat (DOC)) in different concentration, we could diminish the measure of aggregation. The heat treatment process was reversible within the temperature range and detergent concentration used by us.

Effect of nucleotides and their analogues on essential light chains in myosin head

The domain movement in myosin head plays a decisive role in the energy transduction process of the muscle contraction. During hydrolysis of ATP, the specific formation of strong binding of myosin head for actin causes conformational changes. As a consequence, the light chain-binding motif generates the powerstroke. In our work maleimide spin labels were covalently attached to Cys-177 residue of ELC in subfragment-1 (S1). Our goal was to study the orientation dependence and the motion of S1, which were incorporated into glycerinated skeletal muscle fibres. The electron paramagnetic resonance spectroscopy (EPR) spectra of the probes depended strongly on the orientation of the fibre axis relative to the magnetic field, indicating that the essential light chain (ELC) and the neck were ordered. The probes were undergoing rapid motion within a cone. The half-width of the cone was estimated to be 65+/-5 degrees (SD, n=8). Addition of ADP affected little the hyperfine splitting and the angular spread of the probe distribution. In the presence of ADP and orthovanadate the intensity of the spectra decreased, which showed the dissociation of S1 and this was accompanied with the disappearance of the orientation dependence.