Jasminka Brnjas-Kraljević

FT-IR spectroscopy of lipoproteins—A comparative study

FT-IR spectra, in the frequency region 4000-600 cm(-1), of four major lipoprotein classes: very low density lipoprotein (VLDL), low density lipoprotein (LDL) and two subclasses of high density lipoproteins (HDL(2) and HDL(3)) were analyzed to obtain their detailed spectral characterization. Information about the protein domain of particle was obtained from the analysis of amide I band. The procedure of decomposition and curve fitting of this band confirms the data already known about the secondary structure of two different apolipoproteins: apo A-I in HDL(2) and HDL(3) and apo B-100 in LDL and VLDL. For information about the lipid composition and packing of the particular lipoprotein the well expressed lipid bands in the spectra were analyzed. Characterization of spectral details in the FT-IR spectrum of natural lipoprotein is necessary to study the influence of external compounds on its structure.

Investigation of Spermatozoa and Seminal Plasma by Fourier Transform Infrared Spectroscopy

Fourier transform infrared (FT-IR) spectra of human spermatozoa and seminal plasma were recorded and analyzed. The procedure that was established for sample preparation enabled acquisition of reproducible spectra. The parameter I1087/I966 for controlling spectra reproducibility was defined. The assignment of bands was carried out using an empirical approach and the origin of the “sperm specific doublet”, the bands at 968 cm−1 and 981 cm−1, was determined. The principal component regression (PCR) algorithm was used to define the specific spectral regions correlating to characteristics of spermatozoa, such as concentration, straight-line velocity (VSL), and beat cross frequency (BCF). Then, simple spectral parameters, such as band intensities and band ratios, were tested to determine which one best correlates to characteristics of spermatozoa. The region of the amide I band, between 1700 cm−1 and 1590 cm−1, was defined as a specific spectral region that correlates to the concentration of spermatozoa. The parameter that gave the linear dependence to the concentration of spermatozoa was the intensity of the amide I band. For VSL, the bands between 1119 cm−1 and 943 cm−1 were defined as the specific spectral region. The relative amount of nucleic acids with respect to proteins showed linear dependence on the straight-line velocity of spermatozoa. BCF showed the best correlation to the bands between 3678 cm−1 and 2749 cm−1, which largely represent lipids and proteins. These results suggest that FT-IR spectroscopy can serve as an adjunct to conventional histopathology studies.

Surface-core correlations in serum lipoproteins

It has been found that the capacity of lipoproteins for binding Mn(II) ions is dependent on the arrangement of the Iipoprotein core. A change in the molecular organization of the LDL core (thermotropic transition) is associated with the change on the lipoprotein surface. In HDL there is no thermotropic transition and no abrupt change of the binding capacity.. The observed surface-core correlation might be an important link in understanding how dietary fat influences the lipoprotein metabolism.

Causal relationship between the transitions in the core and the surface in porcine low-density lipoproteins

Two independent parameters, two characteristic temperatures, one indicating the change in the molecular organization of the core, Tc, and the other in the surface layer, Ts, were measured for a number of natural and triglyceride-enriched porcine low-density lipoprotein (LDL1 (buoyant density 1.020--1.063 g/ml) and LDL2 (buoyant density 1.063--1.080 g/ml) samples. Tc was determined by differential scanning calorimetry (DSC), whereas Ts was measured by Mn(II) binding to the lipoprotein surface followed by electron spin resonance (ESR) spectroscopy. A significant causal relationship between Tc and Ts in both LDL subfractions demonstrates the surface-core interaction in LDL. The significance of that interaction is emphasized as a possible link in the chain diet----lipoprotein changes----atherosclerosis.

An ESR study of the effect of an electrostatic field on binding of divalent cations to the surface of serum low-density lipoproteins

The ESR technique has been used to study binding of Mn(II) ions to low-density lipoprotein (LDL) in solutions of various electrolyte ionic strengths. A model of the binding has been proposed which describes all the observations in electrolytes of ten different concentrations in terms of two types of binding sites and two corresponding sets of intrinsic binding parameters (n1 = 8, Kd1 = 1.31 X 10(-3) mol X l-1 and n2 = 170, Kd2 = 5.71 X 10(-2) mol X l-1). These parameters, together with the values of the potential (phi 0) responsible for binding of the ions to specific charged sites on the surface, reproduce the observed binding curves well in all the systems studied. The phi 0 values are obtained as an appropriate solution of the Poisson-Boltzmann equation.

Hydration and selfassociation of haemoglobin in solution.

Abstract The hydration of haemoglobin was determined by the nmr-freezing technique. There is a hydration-threshold around 15 mM(haem)/L, with the maximum hydration of ∼ 0.46 g H2O/g Hb. The phosphate buffered solutions show a completely different behaviour from the other ionic and deionized solutions. The results in ordinary water and in the presence of 80% 2H2O are indistin-guishable. The hydration is neither a function of the extremes in the ionic double layer structure nor of the type of ions. The threshold Hb-concentration does not depend on the absolute amount of hydration and the type of ion. The results corroborate the selfassociation of haemoglobin in concentrated solutions.

Proton relaxation study of molecular motions in low density lipoproteins

Abstract Molecular motion and molecular organization of human serum low-density lipoprotein (LDL) has been studied in the temperature range − 30 to 30°C by proton magnetic relaxation. LDL in deuterated Tris-HCl buffer exhibit two mobile phases. The slow-relaxing phase ( T 1 ⋍ 1.5 s ) is assigned to the incompletely deuterated water of the buffer, and the fast-relaxing phase ( T 1 ⋍ 60 ms ) to the fatty acid chains of the lipoprotein core. It has been established that there is a correlation between the state of the outer surface and the interior of the LDL particle: the number of fast-relaxing protons is significantly altered by cooling the system through the freezing point of the buffer or by selecting buffers of different ionic strengths. At room temperature, ∼ 30% of the lipid protons of LDL in the 0.1 m buffer and ∼ 40% of the lipid protons of LDL in the 0.01 m buffer relax quickly within the time-scale of n.m.r. frequency (24 MHz).

Self-association of oxyhaemoglobin. A nuclear magnetic relaxation study in H2O/D2O solutions.

The proton and deuterium longitudinal relaxation rates were studied at room temperature up to the highest protein concentrations in oxyhaemoglobin solutions of different H2O/D2O composition. The deuterium relaxation rates followed the experimentally well known single linear dependence on protein concentration, the slopes being little influenced by solvent (D2O/H2O) composition. The proton relaxation rates show two different linear dependences on haemoglobin concentration. The entire concentration range is described by two straight lines with the threshold concentration about 11 mM (in haem). The ratio of the slopes is 1.6 (high-to-low HB-conc). Only in the higher concentration range two T1s were observed if the solvent contained more than half of D2O. The slow relaxation phase of protons has T1s similar to those measured in solutions with less than half of D2O. The relaxation of the other phase was ten times faster. The ratio of the proton populations in these two phases was equal to 2 (slow-to-fast) and independent of protein concentration. The fast relaxing protons are attributed to water molecules encaged within two or more haemoglobin molecules which associate for times long enough on the PMR time-scale.

Location of PRODAN in lipid layer of HDL particle: a Raman study

FT Raman spectroscopy has been applied to determine the location of PRODAN within HDL and to investigate its influence on the structure of the particle. The complex spectra of HDL and HDL labeled with PRODAN were divided into three regions according to the wave numbers, and adherent spectra were compared separately. Additionally, recorded spectra of protein and lipid fractions of HDL were used as a support for the assignment of particular vibrations in intact particles. In high frequency region, the shift in vibrational frequencies of CH3 groups but almost negligible shift of CH2 groups suggests that PRODAN is situated at the water/lipid interface in the vicinity of the protein. The statement is supported by the observed influence of PRODAN on particular lipid vibrations of phospholipids head-groups. In the fingerprint region, the influence of PRODAN is observed as the slight change in β-strand secondary structure of apolipoprotein and strongly reduced vibrations of the acyl chain in lipids. That additionally confirms that PRODAN mainly interacts with the lipid domain of the particle. In the low frequency region, the lack of change in Tyr Fermi resonance doublet and only slight differences in the pattern of CS and SS stretching vibrations in labeled HDL confirms that PRODAN has no influence on structure of apolipoprotein embedded in lipid domain. The main conclusions drawn from the vibrational spectra of HDL with and without PRODAN clearly confirm that PRODAN induces negligible changes in HDL structure and hence is reliable fluorescent label for the structural analysis.

EPR evidence for the oxidation-induced formation of negatively charged species on the low-density lipoprotein surface.

Oxidation-induced increase of the net negative charge on low-density lipoprotein was studied by electrophoretic mobility and by electron paramagnetic resonance. The negative-charge increase is associated not only with neutralization of the lysine residues of apoprotein B, but also with the exposition of the excessive negatively charged residues on the lipoprotein surface. The accumulation of the negatively charged residues is believed to be brought about by the conformational change of apoprotein B, triggered by neutralization of lysines and cleavage of peptide bonds. Alternatively, reactive oxygen species could also convert histidine to aspartic acid and proline to glutamic acid.