A. Sułkowska

Interaction of anticancer drugs with human and bovine serum albumin

Abstract The properties of 5-fluorouracil (5FU), 6-azauracil (6AU), 6-mercaptopurine (6MP), thiouracil (2TU) and thioimidazole (TI), as a quencher of fluorescence of tryptophanyl Trp 214 residue in serum albumin were studied. For the molar ratio [ligand]: [BSA]=30: 1 the decrease of protein fluorescence due to the presence of thiouracil as a quencher attains 90%, while imidazole derivative-thiamazole quenches the BSA fluorescence only by 25%. To estimate the character of the binding between studied quencher and human and bovine serum albumin the Scatchard and Stern–Volmer methods were used. The binding stoichiometry of allopurinol–mercaptopurine–serum albumin complex was studied.

Effect of urea on serum albumin complex with antithyroid drugs: fluorescence study

Abstract Comparative study of the ligand effect on the conformational stability of human (HSA) and bovine (BSA) serum albumin was performed using fluorescence spectroscopy. Tryptophan fluorescence emission spectra of serum albumin in the presence of mercaptopyrimidine (MPI) were recorded at the excitation wavelengths of 280 and 295 nm. The quenching of the fluorescence of protein in the presence of MPI resulted from the interactions with the drug in the subdomain IIA of α-helix where Trp 214 is located. The unfolding process of serum albumin on increasing the concentration of urea (1÷8 M) was studied. It was suggested that the destabilization of serum albumin structure in the presence of increasing urea concentration runs probably through intermediate state at 4÷6 M urea. The binding and quenching constants MPI–HSA and MPI–BSA has been calculated and compared with those obtained under urea-denatured conditions. An unfolding of serum albumin, induced by urea (within the concentration range 1÷5 M), involved diminishing of binding constant by ∼27% for HSA and by ∼49% for BSA. When concentration of urea exceeded 6 M, the binding constant raised by 21 and 27% for HSA and BSA, respectively. Anti-denaturant action of MPI was shown.

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.

Effective polyelectrolytes synthesised from expanded polystyrene wastes

Chemical modification of used polymer plastics to useful products is a way of effective waste management. To obtain the effective polyelectrolytes the synthesis of sulphonated derivatives of expanded polystyrene wastes was performed. Modification was conducted by means of known methods and products having various contents of sulpho- groups in polymer chain were obtained. Flocculation properties of the anionic type polyelectrolytes obtained were tested. Studies were conducted for coal mine water from the “Kleofas” coal mine and for water from the “Huta Czȩstochowa” plant circulation system. It was found that the polyelectrolytes have good flocculation properties similar to those of anionic type commercial polyelectrolyte. They could be used with good effect for aiding industrial water treatment processes. The optimal concentration of obtained polyelectrolytes was 0.066 mg/dm3. Application of synthesised polyelectrolytes caused a decrease of turbidity and concentration of solved contamination, and improved quality parameter of purified water.

Spectroscopic study of the humification process during sewage sludge treatment

The aim of this work was to study the free radical transition of organic materials during the sewage treatment process. Investigations of sludge from biologic–mechanical sewage treatment plant in Sosnowiec Zagorze were carried out. The course of the humification processes during sewage treatment was studied by electron paramagnetic resonance (EPR) technique. The concentration of free radicals at each process stage and the value g were determined. Sludge samples and extracted fractions of humic acids were examined. Humic acids were extracted from sludge by means of conventional methods elaborated by Stevenson. For study of humic acids structures, besides EPR, the UV–Vis and IR spectroscopy were used.

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 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.

Effects of non-enzymatic glycation in human serum albumin. Spectroscopic analysis

Human serum albumin (HSA), transporting protein, is exposed during its life to numerous factors that cause its functions become impaired. One of the basic factors --glycation of HSA--occurs in diabetes and may affect HSA-drug binding. Accumulation of advanced glycation end-products (AGEs) leads to diseases e.g. diabetic and non-diabetic cardiovascular diseases, Alzheimer disease, renal disfunction and in normal aging. The aim of the present work was to estimate how non-enzymatic glycation of human serum albumin altered its tertiary structure using fluorescence technique. We compared glycated human serum albumin by glucose (gHSA(GLC)) with HSA glycated by fructose (gHSA(FRC)). We focused on presenting the differences between gHSA(FRC) and nonglycated (HSA) albumin used acrylamide (Ac), potassium iodide (KI) and 2-(p-toluidino)naphthalene-6-sulfonic acid (TNS). Changes of the microenvironment around the tryptophan residue (Trp-214) of non-glycated and glycated proteins was investigated by the red-edge excitation shift method. Effect of glycation on ligand binding was examined by the binding of phenylbutazone (PHB) and ketoprofen (KP), which a primary high affinity binding site in serum albumin is subdomain IIA and IIIA, respectively. At an excitation and an emission wavelength of λex 335nm and λem 420nm, respectively the increase of fluorescence intensity and the blue-shift of maximum fluorescence was observed. It indicates that the glycation products decreases the polarity microenvironment around the fluorophores. Analysis of red-edge excitation shift method showed that the red-shift for gHSA(FRC) is higher than for HSA. Non-enzymatic glycation also caused, that the Trp residue of gHSA(FRC) becomes less accessible for the negatively charged quencher (I(-)), KSV value is smaller for gHSA(FRC) than for HSA. TNS fluorescent measurement demonstrated the decrease of hydrophobicity in the glycated albumin. KSV constants for gHSA-PHB systems are higher than for the unmodified serum albumin, while KSV values for gHSA-KP systems are only slightly lower than that obtained for HSA-KP. The affinity of PHB to the glycated HSA is stronger than to the non-glycated in the first class binding sites within subdomain IIA, in the vicinity of Trp-214. Ketoprofen bound to unmodified human serum albumin stronger than for glycated albumin and one class of binding sites is observed (Scatchard linear plots).

Alteration of methotrexate binding to human serum albumin induced by oxidative stress. Spectroscopic comparative study

Changes of oxidative modified albumin conformation by comparison of non-modified (HSA) and modified (oHSA) human serum albumin absorption spectra, Red Edge Excitation Shift (REES) effect and fluorescence synchronous spectra were investigated. Studies of absorption spectra indicated that changes in the value of absorbance associated with spectral changes in the region from 200 to 250nm involve structural alterations related to variations in peptide backbone conformation. Analysis of the REES effect allowed for the observation of changes caused by oxidation in the region of the hydrophobic pocket containing the tryptophanyl residue. Synchronous fluorescence spectroscopy confirmed changes of the position of the tryptophanyl and tyrosil residues fluorescent band. Effect of oxidative stress on binding of methotrexate (MTX) was investigated by spectrofluorescence, UV-VIS and (1)HNMR spectroscopy. MTX caused the fluorescence quenching of non-modified (HSA) and modified (oHSA) human serum albumin molecule. The values of binding constants, Hills coefficients and a number of binding sites in the protein molecule in the high affinity binding site were calculated for the binary MTX-HSA and MTX-oHSA systems. For these systems, qualitative analysis in the low affinity binding sites was performed with the use of the (1)HNMR technique.

Effect of temperature on liposome structures studied using EPR spectroscopy

The effect of temperature on liposome structures has been investigated by means of electron paramagnetic resonance spectroscopy with the use of the spin labelling technique. The EPR spectra were recorded on a Bruker EMX spectrometer at the X band in the temperature range 300–340 K. Liposomes were prepared from L?a?phosphatidylcholine dipalmitoyl (1,2?dihexadecanoyl?sn?glycerol?3?phosphocholine) 99% (DPPC), DL?a?phosphatidylcholine dimyristoyl (1,2?ditetradecanoyl?rac?glycerol?3?phosphocholine) 99% (DMPC) and cholesterol (5?cholesten?3s?ol) 99