Feride Severcan

FTIR spectroscopic characterization of protein structure in aqueous and non-aqueous media

Abstract With increasing use of proteins in many different applications, ranging from phramaceuticals to biosensors and biomaterials, there has emerged a need for protein structural characterisation in diverse environments. In many cases it is not sufficient to just have the three-dimensional structure of a protein in H 2 O or in the crystalline state. Often information on the structural properties of a protein is required in the presence of organic solvents, detergent micelles, phospholipid membranes and so on. Fourier transform infrared spectroscopy (FTIR) has been identified as one of the few techniques that can be applied for structural characterisation of proteins in such environments. Here we discuss how this technique is being used to obtain information on protein structure and stability in both aqueous and non-aqueous media. Examples are drawn from our studies of water soluble proteins and membrane proteins.

Early alterations in myocardia and vessels of the diabetic rat heart: an FTIR microspectroscopic study

Diabetes mellitus is associated with a high incidence and poor prognosis of cardiovascular disease. The aim of the present study was to examine the effect of relatively short-term (5 weeks) Type I diabetes on the left ventricle, the right ventricle and the vessel (vein) on the left ventricle of the myocardium at molecular level by FTIR (Fourier-transform infrared) microspectroscopy. The rats were categorized into two groups: control group (for the left ventricle myocardium, n=8; for the right ventricle myocardium, n=9; for the vein, n=9) and streptozotocin-induced diabetic group (for the left ventricle myocardium, n=7; for the right ventricle myocardium, n=9; for the vein, n=8). Two adjacent cross-sections of 9 microm thickness were taken from the ventricles of the hearts in two groups of rats by using a cryotome. The first sections were used for FTIR microspectroscopy measurements. The second serial sections were stained by haematoxylin/eosin for comparative purposes. Diabetes caused an increase in the content of lipids, an alteration in protein profile with a decrease in alpha-helix and an increase in beta-sheet structure as well as an increase in glycogen and glycolipid contents in both ventricles and the vein. Additionally, the collagen content was found to be increased in the vein of the diabetic group. The present study demonstrated that diabetes-induced alterations in the rat heart can be detected by correlating the IR spectral changes with biochemical profiles in detail. The present study for the first time demonstrated the diabetes-induced alterations at molecular level in both ventricle myocardia and the veins in relatively short-term diabetes.

FT-IR spectroscopic analysis of rainbow trout liver exposed to nonylphenol.

Nonylphenol (NP) is a biodegradation product of nonylphenol ethoxylates (NPEs) belonging to the alkylphenol ethoxylates (APEs) group. APEs are widely used nonionic surfactants in detergents, herbicides, pesticides, paints, and cosmetics. The present work investigates the effects of NP on rainbow trout (Oncorhynchus mykiss) livers at the molecular level using Fourier transform infrared (FT-IR) spectroscopy. The FT-IR spectra revealed dramatic differences between the NP-treated and control tissues, which mainly indicated that the level of triglycerides increased, the lipid order increased, and the protein concentration decreased in the treated samples. Moreover, it was also found that glycogen levels significantly decreased and the relative content of nucleic acids increased in NP-treated fish. The 17β-Estradiol-treated fish liver spectra were found to be quite similar to those of NP-treated fish. All these results implied that rainbow trout may offer considerable promise to be used as a bioindicator for NP in the future.

Chronic hypoperfusion alters the content and structure of proteins and lipids of rat brain homogenates: a Fourier transform infrared spectroscopy study

Arteriovenous malformations (AVMs), masses of abnormal blood vessels which grow in the brain, produce high flow shunts that steal blood from surrounding brain tissue, which is chronically hypoperfused. Hypoperfusion is a condition of inadequate tissue perfusion and oxygenation, resulting in abnormal tissue metabolism. Fourier transform infrared (FTIR) spectroscopy is used in this study to investigate the effect of hypoperfusion on homogenized rat brain samples at the molecular level. The results suggest that the lipid content increases, the protein content decreases, the lipid-to-protein ratio increases, and the state of order of the lipids increases in the hypoperfused brain samples. FTIR results also revealed that, owing to hypoperfusion, not only the protein synthesis but also the protein secondary structure profile is altered in favor of β-sheets and random coils. These findings clearly demonstrate that, FTIR spectroscopy can be used to extract valuable information at the molecular level so as to have a better understanding of the effect of hypoperfusion on rat brain.

Fourier transform infrared study of the effect of diabetes on rat liver and heart tissues in the CH region.

Diabetes mellitus is characterized by hyperglycemia, a relative lack of insulin. The metabolic disturbances in diabetic patients are often associated with cardiac and liver dysfunctions. Generally, experimental diabetic models in animals have been used to study diabetes-related changes in organ function, but the complexity of intact tissues can cause contradictory results. For this reason, different techniques have been used to understand the mechanisms of these dysfunctions in diabetic organs. The purpose of the present study is to investigate the effects of streptozotocin (STZ)-induced diabetes on rat liver and heart tissues at the molecular level by Fourier Transform Infrared (FTIR) spectroscopy. Wistar rats of both sexes, weighing 200-250 g, were made diabetic by a single injection of 50 mg kg(-1) intraperitoneal (i.p.) streptozotocin dissolved in 0.05 M citrate buffer (pH 4.5) and they were kept for 4-5 weeks. The diabetes status was checked by measuring the blood glucose level. In the complex FTIR spectra, the bands in the CH region for example the CH(2) antisymmetric and symmetric stretching, the CH(3) symmetric and asymmetric stretching vibrations due to lipids and proteins in the 3000-2800 cm(-1) region and CH(2) scissoring around 1464 cm(-1) and the CH(3) scissoring at 1454 cm(-1) were analyzed. Characteristic spectral bands of these diabetic samples were compared with those of control group to confirm the effect of diabetes on liver and heart tissues. The FTIR spectra revealed dramatic differences in the band positions and bandwidths, signal intensity values and signal intensity ratios between diabetic and control tissues. Similar differences were observed for diabetic liver and heart tissues. A significant increase in the bandwidth of the CH(2) symmetric and antisymmetric stretching and the CH(3) symmetric and asymmetric stretching bands has been observed for both tissue types. The wavenumber of the CH(3) asymmetric stretching band shifts to lower values, indicating an increase in the order in the deep interior part of the lipid chains. The ratio of the CH(2) symmetric to CH(3) symmetric stretching band (lipid/protein ratio) decreases by 13% for diabetic heart and liver tissues. A decrease is also detected in the ratio of the CH(2) scissoring to the CH(3) scissoring mode. The overall intensity of these bands is seen to increase for diabetic tissues.

Competitive effect of vitamin D2 and Ca2+ on phospholipid model membranes: an FTIR study.

The interaction of Ca(2+), with dipalmitoyl phosphatidylcholine (DPPC) model membranes was studied in the presence and absence of vitamin D(2) by using Fourier transform infrared spectroscopy. Addition of vitamin D(2) and/or Ca(2+) into pure DPPC liposomes shifts the phase transition to higher temperature, orders and decreases the dynamics of the acyl chains in both phases and does not induce hydrogen bond formation in the interfacial region. Moreover, the dynamics of the head group of the phospholipid decreases in both phases. The addition of vitamin D(2) into DPPC liposomes containing Ca(2+), decreases the effect of Ca(2+) at all the functional groups under investigation. Similarly, the effect of vitamin D(2) also decreases in the presence of Ca(2+). This behavior is dominant at high Ca(2+) concentrations. Our results show how simultaneous presence of vitamin D(2) and Ca(2+) alter the behavior of each other, which is reflected as a decrease in the interactions between the ions and vitamin D(2) within the membrane.

VITAMIN E DECREASES THE ORDER OF THE PHOSPHOLIPID MODEL MEMBRANES IN THE GEL PHASE : AN FTIR STUDY

The effect of α-tocopherol on the frequency of the CH2 stretching bands of infrared spectra of dipalmitoylphosphotidylcholine multibilayers has been investigated, both in H2O and 2H2O buffer, to determine the reason for the discrepancy in the literature between the results of different spectroscopic techniques relating to the effect of α-tocopherol on membrane order in the gel phase. In contrast to previous FTIR studies, the present FTIR results indicate that αT increases the frequencies of the CH2 stretching bands in the gel phase, which implies an increase in the number of gauche conformers (increase in disordering), which is in agreement with other ESR and NMR spectroscopic studies.

Differentiation of Anatolian honey samples from different botanical origins by ATR-FTIR spectroscopy using multivariate analysis

Botanical origin of the nectar predominantly affects the chemical composition of honey. Analytical techniques used for reliable honey authentication are mostly time consuming and expensive. Additionally, they cannot provide 100% efficiency in accurate authentication. Therefore, alternatives for the determination of floral origin of honey need to be developed. This study aims to discriminate characteristic Anatolian honey samples from different botanical origins based on the differences in their molecular content, rather than giving numerical information about the constituents of samples. Another scope of the study is to differentiate inauthentic honey samples from the natural ones precisely. All samples were tested via unsupervised pattern recognition procedures like hierarchical clustering and Principal Component Analysis (PCA). Discrimination of sample groups was achieved successfully with hierarchical clustering over the spectral range of 1800-750 cm(-1) which suggests a good predictive capability of Fourier Transform Infrared (FTIR) spectroscopy and chemometry for the determination of honey floral source.

Screening of Protective Effect of Amifostine on Radiation-Induced Structural and Functional Variations in Rat Liver Microsomal Membranes by FT-IR Spectroscopy

In this study, the protective effect of amifostine, which is the only FDA-approved radioprotective agent, was investigated against the deleterious effects of ionizing radiation on rat liver microsomal membranes at molecular level. Sprague-Dawley rats, which were either administered amifostine or not, were whole-body irradiated with a single dose of 800 cGy and decapitated after 24 h. The microsomal membranes isolated from the livers of these rats were investigated using FT-IR spectroscopy. The results revealed that radiation caused a significant decrease in the lipid-to-protein ratio and the degradation of lipids into smaller fragments that contain less CH(2) and more carbonyl esters, olefinic═CH and CH(3) groups, which could be interpreted as a result of lipid peroxidation. Radiation altered the secondary structure of proteins by inducing a decrease in the β-sheet structures and an increase in the turns and random coil structures. Moreover, a dramatic increase in lipid order and a significant decrease in the membrane dynamics were observed in the irradiated group. The administration of amifostine before ionizing radiation inhibited all the radiation induced compositional, structural, and functional damages. In addition, these results suggest that FT-IR spectroscopy provides a novel approach to monitoring radiation-induced damage on biological membranes.

FT-IR spectroscopy in diagnosis of diabetes in rat animal model

In recent years, Fourier Transform Infrared (FT-IR) spectroscopy has had an increasingly important role in the field of pathology and diagnosis of disease states. In the current study, FT-IR spectroscopy together with cluster analysis were used as a diagnostic tool in the discrimination of diabetic samples from control ones in rat kidney plasma membrane apical sides (brush-border membranes), liver microsomal membranes and Extensor digitorum longus (EDL) and Soleus (SOL) skeletal muscle tissues. A variety of alterations in the spectral parameters, such as frequency and signal intensity/area was observed in diabetic tissues and membranes compared to the control samples. Based on these spectral variations, using cluster analysis successful differentiation between diabetic and control groups was obtained in different spectral regions. The results of this current study further revealed the power and sensitivity of FT-IR spectroscopy in precise and automated diagnosis of diabetes.