Roland Krivanek

Immunosensors based on supported lipid membranes, protein films and liposomes modified by antibodies

Abstract The comparison of physical properties, sensitivity and reproducibility of various detection parameters of several newly developed affinity biosensors for determination of human IgE and herbicide 2,4-D is presented. The protein film based biosensors were composed of antibody (swine anti-human IgE (Q-SwaHU/IgE) or monoclonal antibody (MAb) against herbicide 2,4-D) attached to thin gold support through cysteamine or cysteamine–bovine serum albumin. The antigen (Ag)–antibody (Ab) interaction was detected by measurement of conductivity. The detection limit for human IgE was ∼1 nM, however, the sign of response depends on the method of antibody attachment to the gold support. This type of design and detection method was not appropriate for detecting small molecules, like 2,4-D. In the case of metal (stainless steel or gold) supported lipid films (s-BLM), the sensor was constructed by means of binding of avidin-modified antibody to s-BLM contained biotinylated phospholipids. Measurement of electrical capacitance, C , and elasticity modulus in direction perpendicular to the membrane plane, E ⊥ , allowed to detect the Ag–Ab reaction, that resulted in decrease of C and increase of E ⊥ . Most reproducible results have been obtained with lipid films supported on thin gold layers with detection limit of determination of 2,4-D∼1 μM. The best reproducibility and sensitivity (0.1–1 nM) have been obtained in liposome immunoassay. The Ag–Ab reaction was monitored by means of measurement the changes of ultrasound velocity in liposome suspension.

Cholesterol-Induced Variations in the Volume and Enthalpy Fluctuations of Lipid Bilayers

The sound velocity and density of suspensions of large unilamellar liposomes from dimyristoylphosphatidylcholine with admixed cholesterol have been measured as a function of temperature around the chain melting temperature of the phospholipid. The cholesterol-to-phospholipid molar ratio xc has been varied over a wide range (0 </= xc </= 0.5). The temperature dependence of the sound velocity number, of the apparent specific partial volume of the phospholipid, and of the apparent specific adiabatic compressibility have been derived from the measured data. These data are particularly discussed with respect to the volume fluctuations within the samples. A theoretical relation between the compressibility and the excess heat capacity of the bilayer system has been derived. Comparison of the compressibilities (and sound velocity numbers) with heat capacity traces display the close correlation between these quantities for bilayer systems. This correlation appears to be very useful as it allows some of the mechanical properties of membrane systems to be calculated from the specific heat capacity data and vice versa.

Interaction of the antimicrobial peptide gramicidin S with dimyristoyl^phosphatidylcholine bilayer membranes: a densitometry and sound velocimetry study

We determined changes in the volume and adiabatic compressibility of large multi- and unilamellar vesicles composed of dimyristoylphosphatidylcholine containing various concentrations of the antimicrobial peptide gramicidin S (GS) by applying densitometry and sound velocimetry. Gramicidin S incorporation was found to progressively decrease the phase transition temperature of DMPC vesicles as well as to decrease the degree of cooperativity of the main phase transition and to increase the volume compressibility of the vesicles. GS probably enhanced thermal fluctuations at the region of main phase transition and provide more freedom of rotational movement for the phospholipid hydrocarbon chains. The ability of GS to increase the membrane compressibility and to decrease the phase transition temperature is evidence for regions of distorted membrane structure around incorporated gramicidin S molecules. At relatively high GS concentration (10 mol%), more significant changes of specific volume and compressibility appear. This might suggest changes in the integrity of the lipid bilayer upon interaction with high concentrations of GS.

Interaction of crystalline bacterial cell surface proteins with lipid bilayers in liposomes. A sound velocity study

Abstract Sound velocity and density measurements have been used to study physical properties of plain unilamellar liposomes and covered by a crystalline cell surface layer (S-layer) of Bacillus coagulans, strain E38-66. The liposomes were composed of dipalmitoylphosphatidylcholine (DPPC), cholesterol and hexadecylamine. The measured parameters — velocity number [ u l and density ρ — decrease with increasing temperature for both suspensions of liposomes with S-layers (S-DPPC) and without S-layers. The values of [ u ] at T T >29°C larger for S-DPPC than those of plain liposomes. The density of the S-DPPC system was slightly greater than those of plain bilayer suspensions. The specific adiabatic compressibility, ϕ k / β 0 , calculated from the values of [ u ] and of the specific volume, ϕ k , increases with temperature for both liposome systems. The comparison of the parameter ϕ k / β 0 for S-DPPC and DPPC indicates that the adiabatic compressibility of S-DPPC at T T >20°C was lower in comparison with that of plain DPPC. This is evidence of an interesting phenomenon of softening and condensing effects of S-proteins on the lipid bilayer depending on the temperature.

A study of the interaction of some neuropeptides and their analogs with bilayer lipid membranes and liposomes

Abstract Several biophysical methods have been used to study the interaction of the amphiphilic hormone derivative ACTHt-24, the wasp venom peptide mastoparan and the synthetic neuropeptide galparan with planar bilayer lipid membranes (BLM) and liposomes. Addition of ACTH1–24, galparan and mastoparan at different concentrations (0.1 μM to 0.1 mM) to BLM from soybean phosphatidylcholine leads to an increase of membrane electrical conductance to an extent that correlates with the activity of the different peptides on mast cells. Addition of ACTH1–24 to a suspension of DMPC + DMPG liposomes leads to an increase of dielectric relaxation time, however the less charged peptides did not influence the dielectric relaxation properties of liposomes. The velocity number [u], measured by ultrasonic velocimetry in suspensions of liposomes from DMPC, decreased after addition of the peptides. This reveals an increase of the adiabatic volume compressibility of the liposomes. The presence of ACTH1–24, galparan and mastoparan in liposomes of DMPC (mole fraction peptide/DMPC=7.7 × 10−4) resulted in different changes of absorption number Δ[αλ] of ultrasound at temperatures below and above the phase transition of DMPC. The results obtained are consistent with the idea that the lipid bilayer may function as a mediator for the receptor-independent stimulation of G-proteins.

Affinity interactions on a liposome surface detected by ultrasound velocimetry

In this work, we performed targeted immobilization of immunoglobulins by means of bacterial S-layer proteins from Bacillus coagulans E38-66/V1 recrystallized on liposomes, which were exploited as immobilization matrix for antibody (Ab)-human IgG. The study of interaction of rabbit or swine anti-human IgG as antigens (Ag) was performed by means of measuring changes of ultrasound velocity. We showed that at a temperature of 25 degrees C, the increment of ultrasound velocity [u] linearly decreased following an increase of concentration of Ag. The decrease of [u] was presumably due to changes of hydration of the membrane due to the binding process. Approximately 10 times lower changes of [u] were observed at 45 degrees C for Ag-Ab interaction as well as for nonspecific interaction of Ag with liposomes covered by S-layer without Ab. No substantial differences in the behaviour of [u] were observed for interactions of human IgG with rabbit or swine anti-human IgG.

Local anesthetic tetracaine influences the physical properties of globular proteins

Abstract The interaction of local anesthetic tetracaine (TTC) with globular protein-bovine serum albumin (BSA) was studied by the measurement of sound velocity and density. We showed, that in its native form, at pH 7, the TTC in a concentration of 0.1 mmol l –1 resulted in an increase of adiabatic compressibility of BSA, while practically no changes of adiabatic compressibility was observed, when native form has been lost or changed (at pH 3). The binding of TTC on BSA was shown by measurement fluorescence quenching. Addition of TTC resulted BSA tryptophan fluorescence quenching that saturates at TTC concentration 1–3 mmol l –1 . The dissociation constant, K d =1.18±0.54 mmol l –1 , related to the binding of TTC to BSA was determined from fluorescence quenching experiments. We assume that TTC incorporates into hydrophobic core of BSA. This incorporation caused increase of BSA specific volume and consequently the conformational freedom of polypeptid chain could be the main reason of increase of BSA adiabatic compressibility.

Specific volume and compressibility of bilayer lipid membranes with incorporated Na,K-ATPase

Ultrasound velocimetry and densitometry methods were used to study the interactions of the Na,K-ATPase with the lipid bilayer in large unilamellar liposomes composed of dioleoyl phosphatidylcholine (DOPC). The ultrasound velocity increased and the specific volume of the phospholipids decreased with increasing concentrations of protein. These experiments allowed us to determine the reduced specific apparent compressibility of the lipid bilayer, which decreased by approx. 11% with increasing concentrations of the Na,K-ATPase up to an ATPase/DOPC molar ratio = 2 × 10⁻⁴. Assuming that ATPase induces rigidization of the surrounding lipid molecules one can obtain from the compressibility data that 3.7 to 100 times more lipid molecules are affected by the protein in comparison with annular lipids. However, this is in contradiction with the current theories of the phase transitions in lipid bilayers. It is suggested that another physical mechanisms should be involved for explanation of observed effect.