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, Hill's 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.

Alteration of human serum albumin tertiary structure induced by glycation. Spectroscopic study.

The modification of human serum albumin (HSA) structure by non-enzymatic glycation is one of the underlying factors that contribute to the development of complications of diabetes and neurodegenerative diseases. The aim of the present work was to estimate how glycation of HSA altered its tertiary structure. Changes of albumin conformation were investigated by comparison of glycated (gHSA) and non-glycated human serum albumin (HSA) absorption spectra, red edge excitation shift (REES) and synchronous spectra. Effect of glycation on human serum albumin tertiary structure was also investigated by (1)H NMR spectroscopy. Formation of gHSA Advanced Glycation End-products (AGEs) caused absorption of UV-VIS light between 310 nm and 400 nm while for non-glycated HSA in this region no absorbance has been registered. Analysis of red edge excitation shift effect allowed for observation of structural changes of gHSA in the hydrophobic pocket containing the tryptophanyl residue. Moreover changes in the microenvironment of tryptophanyl and tyrosyl residues brought about AGEs on the basis of synchronous fluorescence spectroscopy have been confirmed. The influence of glycation process on serum albumin binding to 5-dimethylaminonaphthalene-1-sulfonamide (DNSA), 2-(p-toluidino) naphthalene-6-sulfonic acid (TNS), has been studied. Fluorescence analysis showed that environment of both binding site I and II is modified by galactose glycation.

The influence of fatty acids on theophylline binding to human serum albumin. Comparative fluorescence study.

Theophylline, popular diuretic, is used to treat asthma and bronchospasm. In blood it forms complexes with albumin, which is also the main transporter of fatty acids. The aim of the present study was to describe the influence of fatty acids (FA) on binding of theophylline (Th) to human serum albumin (HSA) in the high affinity binding sites. Binding parameters have been obtained on the basis of the fluorescence analysis. The data obtained for the complex of Th and natural human serum albumin (nHSA) obtained from blood of obese patients qualified for surgical removal of stomach was compared with our previous studies on the influence of FA on the complex of Th and commercially available defatted human serum albumin (dHSA).

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.

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].