ABSTRACT
PURPOSE: Albumin is an universal transport protein. Plasma pool of free fatty acids arising from triglyceride hydrolysis, critical in energy metabolism and etiology of metabolic disorders is transported by albumin. According to various studies albumin has from seven to nine binding sites with diverse affinity to long chain fatty acids. X-ray diffraction crystallography measurements have provided data only for pure human serum albumin or albumin with fully saturated binding sites. These results have shown that amount of ï¡-helices is higher after fatty acids binding. Molecular mechanics simulations suggest that binding of fatty acids in two high-affinity sites leads to major conformational changes in albumin structure. The aim of this research was to investigate albumin secondary structure upon gradually increasing fatty acids to protein mole ratio. METHODS: Fourier transform infrared spectroscopy was applied to study changes of bovine serum albumin (as an analogue of human serum albumin) ï¡-helical structures after binding palmitic acid in a range of 0-20 palmitic acid: albumin molar ratios representing pure protein, partial, full saturation and excess binding sites capacity. RESULTS: Amount of ï¡-helices was increasing along with the amount of palmitic acid: bovine serum albumin molar ratio and reached maximum value around 2 mol/mol. CONCLUSIONS: Our studies confirmed molecular mechanics simulations and crystallographic studies. Palmitic acid binding in two high-affinity sites leads to major structural changes, filling another sites only slightly influenced bovine serum albumin secondary structure. The systematic study of fatty acids and albumin interactions, using an experimental model mimicking metabolic disorders, may results in new tools for personalized nanopharmacotherapy.
Subject(s)
Palmitic Acid/chemistry , Serum Albumin, Bovine/chemistry , Animals , Calibration , Cattle , Least-Squares Analysis , Ligands , Protein Binding , Protein Structure, Secondary , Spectroscopy, Fourier Transform InfraredABSTRACT
During a haemodialysis (HD), because of the contact of blood with the surface of the dialyser, the immune system becomes activated and reactive oxygen species (ROS) are released into plasma. Particularly exposed to the ROS are lipids and proteins contained in plasma, which undergo peroxidation. The main breakdown product of oxidized lipids is the malondialdehyde (MDA). A common method for measuring the concentration of MDA is a thiobarbituric acid reactive substances (TBARS) method. Despite the formation of MDA in plasma during HD, its concentration decreases because it is removed from the blood in the dialyser. Therefore, this research proposes the Fourier Transform Infrared Attenuated Total Reflectance (FTIR-ATR) spectroscopy, which enables determination of primary peroxidation products. We examined the influence of the amount of hydrogen peroxide added to lipid suspension that was earlier extracted from plasma specimen on lipid peroxidation with use of TBARS and FTIR-ATR methods. Linear correlation between these methods was shown. The proposed method was effective during the evaluation of changes in the extent of lipid peroxidation in plasma during a haemodialysis in sheep. A measurement using the FTIR-ATR showed an increase in plasma lipid peroxidation after 15 and 240 minutes of treatment, while the TBARS concentration was respectively lower.
Subject(s)
Lipid Peroxidation , Lipid Peroxides/blood , Malondialdehyde/blood , Renal Dialysis , Animals , Humans , Hydrogen Peroxide/metabolism , Reactive Oxygen Species/metabolism , Sheep , Spectroscopy, Fourier Transform Infrared , Thiobarbituric Acid Reactive Substances/metabolismABSTRACT
Among the currently used methods of monitoring human tissues and their components many types of research are distinguished. These include spectroscopic techniques. The advantage of these techniques is the small amount of sample required, the rapid process of recording the spectra, and most importantly in the case of biological samples - preparation of tissues is not required. In this work, vibrational spectroscopy: ATR-FTIR and Raman spectroscopy will be used. Studies are carried out on tissues: tendons, blood vessels, skin, red blood cells and biological components: amino acids, proteins, DNA, plasma, and deposits.
Subject(s)
Organ Specificity , Spectrum Analysis, Raman , Animals , Biomedical Technology , DNA/analysis , Humans , Hydrogen-Ion Concentration , Male , Proteins/analysis , Spectroscopy, Fourier Transform InfraredABSTRACT
Among the currently used methods of monitoring human tissues and their components many types of research are distinguished. These include spectroscopic techniques. The advantage of these techniques is the small amount of sample required the rapid process of recording the spectra, and most importantly in the case of biological samples - preparation of tissues is not required. In this work vibrational spectroscopy: ATR-FTIR and Raman spectroscopy will be used. Studies are carried out on tissues: tendons, blood vessels, skin, red blood cells and biological components: amino acids, proteins, DNA, plasma, and deposits.
Subject(s)
Blood Vessels/chemistry , Bone and Bones/chemistry , DNA/chemistry , DNA/genetics , Skin/chemistry , Spectrum Analysis, Raman/methods , DNA/analysis , Humans , Hydrogen-Ion ConcentrationABSTRACT
Modifications of phenylalanine amino acid after its exposure to near-infrared (NIR) radiation have been investigated using ATRFTIR (Attenuated Total Reflectance-Fourier Transform Infrared Spectroscopy). The process of amino acid aggregation after its exposure to NIR has been observed. A possible mechanism of amino acid dimer formation has been proposed with the help of theoretical calculations of quantum mechanics (MP2 and B3LYP/6-31 G* level) using the GAUSSIAN 03 package. The usefulness of spectroscopy for biomedical engineering is discussed. ATR-FTIR appears to be a powerful tool for measuring tissue damage in aqueous environments.
