Jadwiga Pożycka

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.

Alteration of human serum albumin binding properties induced by modifications: A review

Albumin, a major transporting protein in the blood, is the main target of modification that affects the binding of drugs to Sudlows site I and II. These modification of serum protein moderates its physiological function, and works as a biomarker of some diseases. The main goal of the paper was to explain the possible alteration of human serum albumin binding properties induced by modifications such as glycation, oxidation and ageing, their origin, methods of evaluation and positive and negative meaning described by significant researchers.

Effect of Temperature on Tolbutamide Binding to Glycated Serum Albumin

Glycation process occurs in protein and becomes more pronounced in diabetes when an increased amount of reducing sugar is present in bloodstream. Glycation of protein may cause conformational changes resulting in the alterations of its binding properties even though they occur at a distance from the binding sites. The changes in protein properties could be related to several pathological consequences such as diabetic and nondiabetic cardiovascular diseases, cataract, renal dysfunction and Alzheimer’s disease. The experiment was designed to test the impact of glycation process on sulfonylurea drug tolbutamide-albumin binding under physiological (T = 309 K) and inflammatory (T = 311 K and T = 313 K) states using fluorescence and UV-VIS spectroscopies. It was found in fluorescence analysis experiments that the modification of serum albumin in tryptophanyl and tyrosyl residues environment may affect the tolbutamide (TB) binding to albumin in subdomain IIA and/or IIIA (Sudlow’s site I and/or II), and also in subdomains IB and IIB. We estimated the binding of tolbutamide to albumin described by a mixed nature of interaction (specific and nonspecific). The association constants Ka (L∙mol−1) for tolbutamide at its high affinity sites on non-glycated albumin were in the range of 1.98–7.88 × 104 L∙mol−1 (λex = 275 nm), 1.20–1.64 × 104 L∙mol−1 (λex = 295 nm) and decreased to 1.24–0.42 × 104 L∙mol−1 at λex = 275 nm (T = 309 K and T = 311 K) and increased to 2.79 × 104 L∙mol−1 at λex = 275 nm (T = 313 K) and to 4.43–6.61 × 104 L∙mol−1 at λex = 295 nm due to the glycation process. Temperature dependence suggests the important role of van der Waals forces and hydrogen bonding in hydrophobic interactions between tolbutamide and both glycated and non-glycated albumin. We concluded that the changes in the environment of TB binding of albumin in subdomain IIA and/or IIIA as well as in subdomains IB and IIB influence on therapeutic effect and therefore the studies of the binding of tolbutamide (in diabetes) to transporting protein under glycation that refers to the modification of a protein are of great importance in pharmacology and biochemistry. This information may lead to the development of more effective drug therapy in people with diabetes.

In Vitro Investigation of the Interaction of Tolbutamide and Losartan with Human Serum Albumin in Hyperglycemia States

Serum albumin is exposed to numerous structural modifications which affect its stability and activity. Glycation is one of the processes leading to the loss of the original properties of the albumin and physiological function disorder. In terms of long lasting states of the hyperglycemia, Advanced Glycation End-products (AGEs) are formed. AGEs are responsible for cellular and tissue structure damage that cause the appearance of a number of health consequences and premature aging. The aim of the present study was to analyze the conformational changes of serum albumin by glycation—“fructation”—using multiple spectroscopic techniques, such as absorption (UV-Vis), fluorescence (SFM), circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy and evaluate of possible alteration of binding and competition between tolbutamide (TB, a first-generation sulfonylurea oral hypoglycemic drug) and losartan (LOS, an angiotensin II receptor (AT1) blocker used in hypertension (1st line with a coexisting diabetes)) in binding to non-glycated (HSA) and glycated (gHSAFRC) human serum albumin in high-affinity binding sites. The studies allowed us to indicate the structural alterations of human serum albumin as a result of fructose glycation. Changes in binding parameters, such as association (Ka) or Stern-Volmer (KSV) constants suggest that glycation increases the affinity of TB and LOS towards albumin and affects interactions between them. The process of albumin glycation influences the pharmacokinetics of drugs, thus monitored pharmacotherapy is reasonable in the case of diabetes and hypertension polypharmacy. This information may lead to the development of more effective drug treatments based on personalized medicine for patients with diabetes. Our studies suggest the validity of monitored polypharmacy of diabetes and coexisting diseases.

Fluorometric Investigation on the Binding of Letrozole and Resveratrol with Serum Albumin.

Breast cancer is the most common cancer in women worldwide. Combination of drugs during long-therm cancer therapy can increase free, biological active form of the drug and cause dangerous side effects. The 21st century is a period of searching for a progress in cancer chemotherapy. The simultaneous dosage of drugs and natural agents isolated from fruits and vegetables used in breast cancer treatment could be more effective and less toxic. The aim of the study was to determine the binding sites of both letrozole (LET) and polyphenol product, resveratrol (RES) in tertiary structure of human serum albumin (HSA) based on the fluorescence spectroscopy. The binding of LET and RES to HSA was studied by monitoring the changes in emission fluorescence spectra of albumin in the presence of ligands at 280 nm and 295 nm excitation wavelengths. To identify the binding sites of LET and RES on HSA, warfarin (WAR) and 5-(Dimethylamino)-1-naphthalenesulfonamide (DNSA) were used as site probes for binding site I, while dansyl-L-proline (DP) was studied as a marker for binding site II. The binding sites for LET and RES in HSA were found to be located in subdomain IIIA. Based on the Stern - Volmer and binding isotherm using non-linear regression methods, the formation of complexes of LET and RES with HSA and association constants were obtained. The binding analysis showed that the association constants indicated a stronger interaction of HSA with RES than LET and the presence of RES in the tertiary system alters the stability of LET-albumin complex. This conclusion points to the necessity of precaution and monitoring therapy when resveratrol as a natural compound and letrozole are used together.

Effect of Encapsulation on Phase Transition Temperature of Liposomes. Binding Sites in HSA

The degree of encapsulation of 5-fluorouracil (5-FU) and vinorelbine (VIN) to dipalmitoylphosphatidylcholine (DPPC) liposomes was estimated using UV-vis spectroscopy. The phase transition temperature was determined by the differential scanning calorimetry (DSC). 5-FU incorporates in more extend to liposomes than VIN and affects phase transition temperature of phospholipide forming liposomal membrane. There is only a slight influence of VIN on the 5-FU encapsulation in liposome vesicle while 5-FU affects significantly the VIN content in the liposome vesicles. The binding sites of VIN and 5-FU in HSA determined by fluorescence quenching method were confirmed by computer simulation.