A spectroscopic study of phenylbutazone and aspirin bound to serum albumin in rheumatoid diseases.

Interaction of phenylbutazone (PBZ) and aspirin (ASA), two drugs recommended in rheumatoid diseases (RDs), when binding to human (HSA) and bovine (BSA) serum albumins, has been studied by quenching of fluorescence and proton nuclear magnetic resonance ((1)HNMR) techniques. On the basis of spectrofluorescence measurements high affinity binding sites of PBZ and ASA on albumin as well as their interaction within the binding sites were described. A low affinity binding site has been studied by proton nuclear magnetic resonance spectroscopy. Using fluorescence spectroscopy the location of binding site in serum albumin (SA) for PBZ and ASA was found. Association constants K(a) were determined for binary (i.e. PBZ-SA and ASA-SA) and ternary complexes (i.e. PBZ-[ASA]-SA and ASA-[PBZ]-SA). PBZ and ASA change the affinity of each other to the binding site in serum albumin (SA). The presence of ASA causes the increase of association constants K(aI) of PBZ-SA complex. Similarly, PBZ influences K(aI) of ASA-SA complex. This phenomenon shows that the strength of binding and the stability of the complexes increase in the presence of the second drug. The decrease of K(aII) values suggests that the competition between PBZ and ASA in binding to serum albumin in the second class of binding sites occurs. The analysis of (1)HNMR spectral parameters i.e. changes of chemical shifts and relaxation times of the drug indicate that the presence of ASA weakens the interaction of PBZ with albumin. Similarly PBZ weakens the interaction of ASA with albumin. This conclusion points to the necessity of using a monitoring therapy owning to the possible increase of uncontrolled toxic effects.

The spectroscopic studies of humic acid extracted from sediment collected at different seasons.

Goczalkowice Reservoir is the biggest water reservoir in the south of Poland. For our studies bottom sediments were collected from eight different places of the reservoir at various seasons of the year. EPR spectroscopy was applied to analyse both quantitatively and qualitatively the humic acids (HA) present in the samples. EPR spectra of the extracted HA exhibited broad lines from the paramagnetic metal ions and narrow lines from free radicals. The values of the free radical concentration obtained for HA amounted to 1.14-13.6 × 1016 spin g(-1) depending on the season and the place of sample collection. The values of the g factor obtained for HA were of the range 2.0027-2.0035. The EPR studies show that HA extracted from bottom sediment collected at various points of the Goczalkowice Reservoir exhibit similar physical-chemical properties. It was also observed that the depth of the reservoir affects the content of the oxygen functional groups as well as the free radical concentration in HA. The identification of the functional groups was done by means of IR. UV/VIS spectroscopy was used to estimate the maturity of the HA.

Influence of myristic acid on furosemide binding to bovine serum albumin. Comparison with furosemide-human serum albumin complex.

Fluorescence studies on furosemide (FUR) binding to bovine serum albumin (BSA) showed the existence of three or four binding sites in the tertiary structure of the protein. Two of them are located in subdomain IIA, while the others in subdomains IB and/or IIIA. Furosemide binding in subdomain IB is postulated on the basis of run of Stern-Volmer plot indicating the existence of two populations of tryptophans involved in the interaction with FUR. In turn, the significant participation of tyrosil residues in complex formation leads to the consideration of the subdomain IIIA as furosemide low-affinity binding site. The effect of increasing concentration of fatty acid on FUR binding in all studied binding sites was also investigated and compared with the previous results obtained for human serum albumin (HSA). For BSA the lesser impact of fatty acid on affinity between drug and albumin was observed. This is probably a result of more significant role of tyrosines in the complex formation and different polarity of microenvironment of the fluorophores when compared HSA and BSA. The most distinct differences between FUR-BSA and FUR-HSA binding parameters are observed when third fatty acid molecule is bound with the protein and rotation of domains I and II occurs. However these structural changes mostly affect FUR low affinity binding sites.

Alterations of furosemide binding to serum albumin induced by increased level of fatty acid.

Localization of high and low affinity binding sites of furosemide in human serum albumin (HSA) as well as the influence of myristic acid on the drug binding to the albumin using fluorescence quenching method was investigated. Two independent classes of binding site in subdomain IIA of HSA structure were found. Alteration of protein affinity towards the drug and the participation of tryptophanyl and tyrosil residues in drug-albumin interaction for the determined binding sites were studied. It was concluded that association of myristic acid in its low affinity binding sites which corresponds to elevated fatty acid level in vivo, significantly decreases albumin affinity towards furosemide.

The influence of dietary habits and pathological conditions on the binding of theophylline to serum albumin.

The influence of fatty acids (FA) on theophylline (Th) binding to human serum albumin (HSA) in its high and low affinity binding sites was investigated. The content of studied FA solutions corresponds to the ones associating with different dietary habits and pathological states in vivo. Using fluorescence and (1)H NMR spectroscopy two high and two low affinity binding sites of Th in HSA structure were found. For each site several binding parameters in the absence and presence of FA were estimated. The results showed that the impact of FA on the affinity of HSA towards Th in high affinity binding sites is negligible whereas binding of the drug in low affinity sites decreases significantly in the presence of FA. It was observed that this effect is dependent on the number of fatty acid molecules bound to the protein while the chemical structure of fatty acids contained in the solution plays a minor role.

Binding of 6-propyl-2-thiouracil to human serum albumin destabilized by chemical denaturants.

We compared the binding affinity of 6-propyl-2-thiouracil (PTU) with native and destabilized human serum albumin (HSA) as a model to assess the binding ability of albumin in patients suffering from chronic liver or renal diseases. Urea (U) and guanidine hydrochloride (Gu.HCl) at a concentration of 3.0M were used as denaturation agents. Increasing the concentration of PTU from 0.8x10(-5) to 1.20x10(-4)M in the systems with HSA causes a decrease in fluorescence intensity of the protein excited with both 280 and 295nm wavelengths. The results indicate that urea and Gu.HCl bind to the carbonyl group and then to the NH-group. To determine binding constants we used the Scatchard plots. The presence of two classes of HSA-PTU binding sites was observed. The binding constants (K(b)) are equal to 1.99x10(4)M(-1) and 1.50x10(4)M(-1) at lambda(ex)=280nm, 5.20x10(4)M(-1) and 1.65x10(4)M(-1) at lambda(ex)=295nm. At lambda(ex)=280nm the number of drug molecules per protein molecule is a(I)=1.45 and a(II)=1.32 for I and II binding sites, respectively. At lambda(ex)=295nm they are a(I)=0.63 and a(II)=1.54 for the I and II binding sites. The estimation of the binding ability of changed albumin in the uremic and diabetic patients suffering from chronic liver or renal diseases is very important for safety and effective therapy.

Interaction of phenylbutazone and colchicine in binding to serum albumin in rheumatoid therapy: 1H NMR study.

The monitoring of drug concentration in blood serum is necessary in multi-drug therapy. Mechanism of drug binding with serum albumin (SA) is one of the most important factors which determine drug concentration and its transport to the destination tissues. In rheumatoid diseases drugs which can induce various adverse effects are commonly used in combination therapy. Such proceeding may result in the enhancement of those side effects due to drug interaction. Interaction of phenylbutazone and colchicine in binding to serum albumin and competition between them in gout has been studied by proton nuclear magnetic resonance ((1)H NMR) technique. The aim of the study was to determine the low affinity binding sites, the strength and kind of interaction between serum albumin and drugs used in combination therapy. The study of competition between phenylbutazone and colchicine in binding to serum albumin points to the change of their affinity to serum albumin in the ternary systems. This should be taken into account in multi-drug therapy. This work is a subsequent part of the spectroscopic study on Phe-COL-SA interactions [A. Sulkowska, et al., J. Mol. Struct. 881 (2008) 97-106].

Comparison of humification processes occurring during sewage purification in treatment plants with different technological processes.

The course of the humification process of sewage sludge collected from three biologic-mechanical treatment plants with different treatment technologies was studied. The maturity of sewage sludge and its usefulness for agricultural purposes was also discussed. The physical-chemical properties of humic acids extracted from sewage sludge received from comparable stages of sludge purification were described. Changes of the sludge properties during sewage purification and the progress of the humification process were investigated with EPR, IR and UV/VIS spectroscopic methods. The content of the elements and the carboxylic groups in humic acids extracted from each stage of the sewage treatment were also determined. It was found that the humification processes take place in all three treatment plants but with different intensities resulting from the differences in the individual cleaning processes in these plants. The most intensive changes of physical-chemical parameters in the extracted humic acids were observed in the anaerobic digester where mesophilic fermentation occurs. The sludge oxygenation processes also significantly affect the course of the humification process during sewage treatment.

NMR study of the humification process during sewage sludge treatment.

Humic acids, extracted from sludge at the biologic-mechanical sewage treatment plant in Jastrzebie Zdroj, have been investigated by means of (1)H, (13)C and (31)P NMR spectroscopy. Sludge samples for studies were taken from the primary settling tank, the nitrification chamber, the digestion chamber and the sludge drying beds. The (1)H NMR analysis of humic acid extracted from sludge at various stages of sewage treatment confirmed the presence of the functional groups that are characteristic for humic substances, and the analysis showed changes in their relative intensities. The (13)C NMR indicated that the aromatisation of the humic acid increased during sewage treatment. Moreover, the analysis of the (31)P NMR spectra allowed us to observe the changes in the phosphorus groups of the studied humic acids.

Changes of serum albumin affinity for aspirin induced by fatty acid.

Saturated fatty acids such as myristic acid play an important role in the pathogenesis of cardiovascular disorders. Using the quenching fluorescence method we examined the influence of myristate on the changes of transporting protein affinity towards aspirin-the most popular anticoagulant. Our results showed that the presence of the myristic acid alters the stability of the anticoagulant-albumin complex. The ranges of [myristate]/[albumin] molar ratio at which the stability of drug-protein complex increases or decreases were determined. The differences in interaction between ligands and human or bovine serum albumins were identified. The competition in binding of ligands with these albumins was also described.

Primary and secondary relaxations in bis-5-hydroxypentylphthalate.

Broadband dielectric spectroscopy was used to study the relaxation dynamics in bis-5-hydroxypentylphthalate (BHPP) under both isobaric and isothermal conditions. The relaxation dynamics exhibit complex behavior, arising from hydrogen bonding in the BHPP. At ambient pressure above the glass transition temperature T(g), the dielectric spectrum shows a broad structural relaxation peak with a prominent excess wing toward higher frequencies. As temperature is decreased below T(g), the excess wing transforms into two distinct peaks, both having Arrhenius behavior with activation energies equal to 58.8 and 32.6 kJmol for slower (beta) and faster (gamma) processes, respectively. Furthermore, the relaxation times for the beta process increase with increasing pressure, whereas the faster gamma relaxation is practically insensitive to pressure changes. Analysis of the properties of these secondary relaxations suggests that the beta peak can be identified as an intermolecular Johari-Goldstein (JG) process. However, its separation in frequency from the alpha relaxation, and both its activation energy and activation volume, differ substantially from values calculated from the breadth of the structural relaxation peak. Thus, the dynamics of BHPP appear to be an exception to the usual correlation between the respective properties of the structural and the JG secondary relaxations.

Effect of pressure on dynamic heterogeneity in dendrimeric alkyd resin.

Broadband dielectric spectroscopy is employed to investigate the non-Debye relaxation behavior in a dendrimeric alkyd resin. From temperature-dependent measurements at ambient pressure, we found a very broad distribution of relaxation times. This is attributed to the complex geometrical topology of the molecule. However, compression significantly reduces the non-Debye character of the dielectric response; thus, pressure induces dynamic homogeneity in the dendrimeric alkyd resin.