Mikrokalorymetryczne badania przemian konformacyjnych albuminy poddanej działaniu wybranych czynników fizykochemicznych

Thermal unfolding of albumin in aqueous solutions proceeding under different physico-chemical conditions has been investigated using differential scanning calorimetry (DSC). The dependence of the observed conformational restructuring on the kind of albumin (human, bovine), its form (fatty acid free and nondefatted), the properties of solvent (water, ethanol solutions), ionic strength, pH, protein concentration and experimental conditions has been discussed in this work. The endothermic unfolding transition has been shown to be modified by time changes and changes induced by such environmental factors as radio frequency radiation or UV radiation. Considering the process of albumin thermal unfolding within the equilibrium thermodynamics, a deconvolution of DSC traces have been performed using the appropriate mathematical models. Structurally independent subunits revealed during thermal denaturation of albumin has been found pH dependent. At pH range corresponding to the N form of albumin these subunits could be correlated with three albumin domains for human albumins and nondefatted bovine albumin. Under the same conditions two subunits have been revealed for fatty acid free bovine albumin: C-terminal fragment containing domain III and the greater part of domain II and the N-terminal fragment containing domain I and the smaller part of domain II. DSC study of albumin in ethanol solutions has revealed stronger binding of ethanol to defatted than to nondefatted albumin. The interaction of ethanol with fatty acid binding sites located in subdomain IIA has been confirmed. Ethanol has been observed to be a stabilizer of the folded state of albumin at a lower concentration contrary to the high denaturant concentration where its binding to the unfolded protein predominates. The obtained results indicate that the influence of radiofrequency radiation (from several to tens MHz) on albumin unfolding events could be detected using ultrasensitive microcalorimeter. That influence is not observed immediately, however, the differences between DSC profiles for irradiated and nonirradiated albumin solutions have appeared during their storage. The changes in irradiated samples outpace nonirradiated ones. Calorimetric and spectroscopic results have shown the conformational restructuring of albumin under UV irradiation. The differences in response to UV radiation between nondefatted and fatty acid free albumins have been found. Albumin devoid of endoge-nous fatty acids has been suggested to be more susceptible to aggregation caused by UV A—C as well as 254 nm UVC radiation. DSC curve deconvolution results allow to conclude that the C-terminal fragment of albumin macromolecule, containing domain III, is the most liable part to UV radiation. The studies presented by the author have revealed the fundamental role of the presence of fatty acids for the thermal stability, conformational rearrangement and binding properties of albumin macromolecule

Calorimetric monitoring of the effect of endurance training and testosterone treatment on rat serum denaturation transition

Differential scanning calorimetry (DSC) was applied to evaluate whether endurance training and/or testosterone treatment affect thermal behavior of rat serum. Thirty-six adolescent male Wistar rats were divided into six groups: three untrained and three trained groups, receiving weekly 0, 8, or 80 mg kgBM -1 of testosterone enanthate (TE) each. Total testosterone was significantly higher in both groups treated with the higher TE dose. The rats’ body mass and the levels of serum glucose, uric acid, and free fatty acids were additionally determined. The effect of TE treatment was clearly visible in DSC curves for sedentary rats and much less for trained rats. The differences between averaged endothermic denaturation transitions of serum from sedentary and physically active untreated groups were within an experimental error. A statistically significant increase of the width of peak at its half height and the ratio of excess heat capacity at 75 C and in the temperature of peak maximum (Cp75/Cpmax) were observed due to TE treatment. The same two parameters were significantly lower for physically active rats. Significantly lower values of the enthalpy of serum denaturation (DH) and the first moment of the transition with respect to the temperature axis (M1) were observed for endurance-trained than for sedentary rats

Modification of blood serum DSC profiles by sauna treatments in cross country skiers during the exercise cycle

The study has evaluated the effect of sauna treatments on the blood serum of professional cross-country skiers (3 women and 6 men) during two exercise sessions: the first without any treatments and the second after ten sauna treatments. Aqueous solutions of serum samples from the blood of athletes collected in four stages in both exercise sessions: at rest, after exercise, after 1 h and 24 h rest and additionally only at rest after the first sauna treatment, were measured by means of differential scanning calorimetry (DSC). DSC results allowed to register characteristic post-exercise changes in the profile of thermal denaturation transition of serum. A trend of these changes was similar regardless of the use of sauna, but their intensity was stronger in the exercise session after the sauna treatments. In particular, the differences relating to the use of sauna have revealed in the higher temperature range of serum denaturation transition. Regarding the recovery process after exercises, a large individual variability was observed in both sessions

Effects of low-dose ionizing radiation on α,β-globulins solutions studied by DSC

An attempt has been made to detect the effect of a small dose of ionizing radiation on the course of a,b-globulin thermal denaturation in aqueous solutions. Doses of 0.1 and 1.8 Gy have been delivered using c-rays emitted by 60Co isotope while doses of 10 and 100 Gy have been supplied by X-rays produced by linear accelerator. The highest dose has visibly changed DSC curve of protein solution while the changes due to lower doses are hardly detectable. Although very weak, the irradiation effect found has been dose dependent. The results suggest that the influence of ionizing radiation on globulins solution is bigger when the dose rate is lower at given dose. The opposite direction of differences between irradiated and control samples for fresh and stored protein solutions suggests various characters of changes in initial and later period of sample aging. This may be an important reason for difficulties in an investigation of the effect of ionizing radiation on protein solution, especially for low doses delivered very slowly

Blood serum calorimetry indicates the chemotherapeutic efficacy in lung cancer treatment

Chemotherapy is a primary treatment for the metastatic lung cancer patients. To select the most effective combination of drugs, we need an efficient way of assessing tumor response. Here, we showed that differential scanning calorimetry (DSC) analysis of blood serum proteins could reveal the patient response to the treatment. If chemotherapy is effective, serum proteins DSC curve of non-small cellular lung cancer (NSCLC) case is similar to the those of the healthy individuals. If treatment fails, notable changes occur in the DSC profile of NSCLC patient's blood serum. Our preliminary work illustrates how thermal analysis of changes in the heat capacity of blood serum proteins can provide an insight into patient response to chemotherapy - the essential information for any successive lung cancer treatment

Serum autofluorescence and biochemical markers in athlete’s response to strength effort in normobaric hypoxia: a preliminary study

The human organism has the ability to adapt to hypoxia conditions. Training in hypoxia is used in sport to improve the efficiency of athletes; however, type of training affects the direction and scope of this process. Therefore, in this study, the usefulness of serum fluorescence spectroscopy to study the assessment of athlete’s response to strength effort in hypoxia is considered in comparison with biochemical assay. Six resistance-trained male subjects took part in a research experiment. They performed barbell squats in simulated normobaric hypoxic conditions with deficiency of oxygen 11.3%, 13% 14.3% compared to 21% in normoxic conditions. Fluorescence intensity of tyrosine revealed high sensitivity on strength effort whereas tryptophan was more dependent on high altitude. Changes in emission in the visible region are associated with altering cell metabolism dependent on high altitude as well as strength training and endurance training. Significant changes in serum fluorescence intensity with relatively weak modifications in biochemical assay at 3000 m above sea level (ASL) were observed. Training at 5000 m ASL caused changes in fluorescence parameters towards the normobaric specific values, and pronounced decreases of lactate level and kinase creatine activity were observed. Such modifications of fluorescence and biochemical assay indicate increased adaptation of the organism to effort in oxygen-deficient conditions at 5000 m ASL, unlike 3000 m ASL. Fluorescence spectroscopy study of serum accompanied by biochemical assay can contribute to the understanding of metabolic regulation and the physiological response to hypoxia. The results of serum autofluorescence during various concepts of altitude training may be a useful method to analyze individual response to acute and chronic hypoxia. An endogenous tryptophan could be exploited as intrinsic biomarker in autofluorescence studies. However, these issues require further research

Diversity in athlete’s response to strength effort in normobaric hypoxia: Serum DSC study

The hypoxia may be used during exercise training sessions in humans with the aim of improving athletic performance. The effect of normobaric hypoxia strength training on thermal properties of blood serum has been evaluated in a group of 12 male and female athletes using differential scanning calorimetry (DSC). Each athlete was tested under normoxic and simulated hypoxic (4000 m, FiO2 = 13% and 5000 m, FiO2 = 11.3%) conditions during squats with a barbell (70% 1RM) exercise. A substantial inter-individual variation in the effects of hypoxia on serum DSC curves has been observed. The effect of exercising in normobaric hypoxia has been found greater for men than for the women. When the work intensity is high enough, the strength exercise in hypoxia can trigger an acute-phase response. Calorimetric and biochemical data have shown that men’s exercising in hypoxia could increase the concentration of acute-phase proteins: haptoglobin and/or C-reactive protein. Our results suggest that 24-h period of rest is sufficient to return to the pre-exercise state after normoxic as well as hypoxic training session for both men and women. The recovery seems to be faster after the training in normobaric hypoxia conditions than in normoxia in the male but not in the female group of athletes

Blood serum DSC analysis of well-trained men response to CrossFit training and green tea extract supplementation

Differential scanning calorimetry (DSC) has been used for the detection of post-exercise changes in blood serum resulting from participation in the CrossFit (CF) training combined with green tea extract (GTE) supplementation. Blood samples from 20 well-trained men were collected at rest, immediately post-exercise and after 1 h of recovery in two trials: first before and second after CF training combined with GTE or placebo administration in the supplemented (S) and control (C) groups, respectively. Selected muscle damage biomarkers have been compared in different phases of the experiment. A significant increase in blood lactate content has been observed post-exercise in both trials in both participants’ groups. The opposite trends have been noted for the C and S groups in creatine kinase (CK) activity changes recorded during the first to the second trial: an increase in CK for the control and a decrease for the supplemented group in all phases of the experiment: pre-exercise, post-exercise and after recovery. In the second trial, all CK values for the S group have been found significantly lower than the corresponding values recorded in the C group. These results suggest a mitigate effect of GTE supplementation on post-training muscle damage. DSC results did not reveal clear effects of training nor GTE supplementation on serum denaturation transition. However, interesting dependences of thermodynamic parameters describing this transition have been observed in different phases of the experiment. Statistically significant negative correlations have been found between post-training VO2max and post-exercise thermodynamic parameters associated with haptoglobin contribution to serum denaturation transition

Whole-body cryostimulation impact on blood serum thermal denaturation profiles of cross-country skiers

Blood plasma and serum are important diagnostic materials as they clearly reflect an individual’s metabolism. The study has evaluated the effect of whole-body cryostimulation (WBC) on the blood serum of professional cross-country skiers. The experiment involved eight athletes (two women and six men) who underwent a series of ten WBC treatments. Aqueous solutions of human blood serum samples before WBC procedures, after one treatment and after a series of ten treatments were measured by means of differential scanning calorimetry (DSC), a relatively novel diagnostic tool. DSC results showed rather little impact of cryostimulation on heat capacity changes accompanying the process of thermal denaturation of blood serum proteins in elite athletes. However, the statistically significant reduction in the intensity of the serum denaturation transition in its low temperature range has been observed after ten WBC treatments. The results have been interpreted by changes in the serum proteome profile, notably in the ratio of ligated to unligated albumin molecules. As a side result, the relationships between the relative change in body fat mass after ten WBC treatments and the levels of alpha2-globulins and beta2-globulins fractions have been found

Calorimetric and spectroscopic studies characterization of newborn rat’ blood serum after maternal administration of cyclophosphamide

Differential scanning microcalorimetry (DSC) and UV–VIS absorption spectroscopy were used to obtain the characteristics of blood serum from newborn rat’ after maternal treatment with cyclophosphamide in comparison with control. The obtained DSC curves reveal a complex endothermic peak due to the unfolding process of various serum proteins. Thermal profiles and absorption spectra of blood serum are sensitive to the age of newborns as well as to effect of maternal administration of cyclophosphamide. The most significant disturbances in serum proteome were observed for 14-day old newborns. The thermodynamic parameters: enthalpy change (DH), the normalized first moment (M1) of the thermal transition with respect to the temperature axis and the ratio of Cp ex at 70 and 60 C describing denaturation contributions of globulin forms in respect to unliganded albumin with haptoglobin was estimated. Moreover, the second derivative spectroscopy in the UV region was used to resolve the complex protein spectrum. The differences in blood serum detected by DSC and UV–VIS confirm a potential usefulness of these methods for diagnostic and monitoring changes with age as well as the pathological state of blood serum