Victor N. Morozov

Electrospray deposition as a method to fabricate functionally active protein films

Electrospray ionization is a routine method in MS analysis of proteins and other biopolymers. Deposition of the electrospray products onto a conductive electrode is suggested here as a means to manufacture functionally active protein films. Recovery of the specific hydrolytic activity of the electrosprayed alkaline phosphatase (AP) was used as a probe for preservation of protein intactness in the electrospray deposition (ESD). It was shown that protein inactivation upon ESD is highly dependent on voltage and current used. Humidity and the presence of protective substances in solution also affect the process. Complete preservation of the enzyme activity was observed when the ESD was performed at low current and humidity in the presence of disaccharides.

Elasticity of globular proteins. The relation between mechanics, thermodynamics and mobility

An analysis of elasticity of lysozyme and myoglobin crystals in terms of thermodynamics has revealed a direct relation between entropy and enthalpy of deformation and delta S* and delta H* terms in the standard free energy change in proteins, delta G(o), (K.P. Murphy, P.L. Privalov, S.J. Gill (1990) Science 247, 559-561), so that at any temperature (between the glass-transition and denaturation temperatures) free energy of deformation is proportional to the hydration independent part of delta G(o). Both energies are characterized with large enthalpy-entropy compensation and tend to zero at the same temperature, Tm = (delta H*/delta S*) = 353 +/- 20 K. Large positive entropy contribution to deformation energy causes large linear decrease in protein elasticity, and increase in thermal mobility of protein atoms with temperature. Being plotted in inverse coordinates, temperature dependence of the mean-square amplitudes, obtained in neutron and mossbauer experiments as well as in molecular dynamic simulations, gives the same 353 +/- 10 K for the temperature, where the amplitudes tend to infinity. Mechanism explaining large positive entropy contribution in deformation energy of native protein molecules presumably involves emergence of more room for motion of protein side-chain groups squeezed between alpha-helices and other rigid skeleton elements, when precise packing of atoms in native protein molecule is distorted as a result of deformation.

Direct detection of isotopically labeled metabolites bound to a protein microarray using a charge-coupled device.

A charge-coupled device (CCD) was used to quantitatively detect isotope-labeled ligands bound to a protein microarray. Protein microarrays with protein dots, 10-50 microm in diameter, were fabricated on an aluminized Mylar film using an electrospray deposition technique. Proteins in dots were immobilized by cross-linking in glutaraldehyde vapor. After contact with solutions of isotope-labeled metabolites, the protein microarrays were washed, dried and placed face down onto the surface of a standard B/W video CCD chip with the protective window removed. We show here that such a simple inexpensive CCD detector can be used to quantify distribution of 14C and other radioactive isotopes on microarrays.

Viscoelasticity of protein crystal as a probe of the mechanical properties of a protein molecule: Hen egg-white lysozyme

Abstract Here we present a new approach to studying the anisotropy of the elastic properties of a protein globule based on an analysis of the elastic properties of a protein monocrystal. The anisotropy of the elasticity of hen egg-white lysozyme triclinic crystals, as well as its changes because of the formation of a lysozyme—N-acetyl- d -glucosamine complex, were investigated. The data can be explained on the assumption that the lysozyme molecule consists of two rigid domains connected by a flexible link. The binding of the inhibitor in the active-site cleft is accompanied by an increase of about 40% in the interdomain rigidity.

Are Reactive Oxygen Species Generated in Electrospray at Low Currents

It was demonstrated that electrospraying (ES) of solvents from a glass capillary proceeds without emission of light provided that the current is kept below a certain critical level (<100 nA at positive potential and <25 nA at negative potential for 96% ethanol; < 40 nA at positive potential for water). Though the onset of corona, as detected by the appearance of light, was always accompanied by a break in the current-voltage slope, such breaks also happened before the onset of corona, so they cannot be used as an adequate indicator of corona ignition. Of four ROS studied (hydrogen peroxide, ozone, hydroxyl radicals, and superoxide anions), only H2O2 and ozone were found to be generated at a current of 150-200 nA in detectable quantities: with a yield of 0.5-1 H2O2 molecules per electron at positive potential and 1.5-3 at negative potential. Despite the low yield of the ROS, jack bean urease was shown to be inactivated when the enzyme solution with a concentration below 20 μg/mL was electrosprayed at a current of 200 nA. Addition of 0.1 mM EDTA totally protected the activity of the electrosprayed urease.

Ionic conductivity, transference numbers, composition and mobility of ions in cross-linked lysozyme crystals

Micromethods for measurements of electric conductivity, transference numbers and concentrations of inorganic ions within immobilized protein crystals have been developed and applied to study tetragonal lysozyme crystals cross-linked with glutaraldehyde. Donnan equilibria and mobilities of ions in this crystal were calculated using the data of these methods and the data of crystal pH titration. Taken together these results characterize the lysozyme crystal as an ion exchanger whose electrical properties and ion composition differ greatly from those of the external solution. Although anions transfer most of the current in the crystals, anion mobility is considerably lower than that of cations. Mobility of all ions in the crystal is considerably lower than in solution (3.5-50 times for cations and 120-330 times for anions) and depends on steric restrictions and charges of both ions and lysozyme molecules. Similar features in behavior of crystalline and biological channels are discussed.

Mechanical detection of interaction of small specific ligands with proteins and DNA in cross-linked samples.

Cross-linked crystalline and amorphous films of different proteins and cross-linked DNA gels were found to change their mechanical properties when soaked in solutions of specific ligands at nearly physiological concentrations. This chemomechanical effect may be used to rapidly (within a few minutes) detect the ability of macromolecules to bind small (less than 1 kDa) ligand molecules, to measure concentrations of ligands (higher than 10 nM), and to estimate binding constants (lower than 10(7) M-1). Only 0.1-1 mg of protein or DNA is needed to prepare more than 10 samples sufficient for a large number of tests, provided binding is reversible. The method is recommended for rapid primary screening in search of new drugs, in biochemical studies, and as a basis for designing biosensors and other analytical instruments.

Detection of microarray-hybridized oligonucleotides with magnetic beads.

The efficiency of hybridization analysis with oligonucleotide microarrays depends heavily on the method of detection. Conventional methods based on labeling nucleic acids with fluorescent, chemiluminescent, enzyme, or radioactive reporters suffer from a number of serious drawbacks which demand development of new detection techniques. Here, we report two new approaches for detection of hybridization with oligonucleotide microarrays employing magnetic beads as active labels. In the first method streptavidin-coated magnetic beads are used to discover biotin-labeled DNA molecules hybridized with arrayed oligonucleotide probes. In the second method biotin-labeled DNA molecules are bound first to the surface of magnetic beads and then hybridized with arrayed complementary strands on bead-array contacts. Using a simple low-power microscope with a dark-field illumination and a pair of complementary primers as a model hybridization system we evaluated sensitivity, speed, and cost of the new detection method and compared its performance with the detection techniques employing enzyme and fluorescent labels. It was shown that the detection of microarray-hybridized DNA with magnetic beads combines low cost with high speed and enhanced assay sensitivity, opening a new way to routine hybridization assays which do not require precise measurements of DNA concentration.

Carboxymethyl cellulose film as a substrate for microarray fabrication.

Magnetic beads (MB) are widely used for quick and highly sensitive signal detection in microarray-based assays. However, this technique imposes stringent requirements for smoothness and adhesive properties of the surface, which most common substrates do not satisfy. We report here a new type of substrate for microarrays with a low adhesion to MB-thermally cross-linked carboxymethyl cellulose (CMC) film. This substrate can be readily fabricated on a conventional glass slide. A highly cross-linked CMC film (∼1 cross-link per monomer unit) possesses a surface smooth on a nanometer scale and a low adhesion to protein-coated MB, which partly originates from electrostatic repulsion of MB from negatively charged CMC surface. The efficiency of the CMC substrate is demonstrated hereby in fabrication of microarrays for the detection of three bacterial toxins: cholera toxin, staphylococcal enterotoxin A, and toxic shock syndrome toxin. The assay employing a primary antibodies arrayed on a CMC surface and detection of the bound bacterial toxins with a biotinylated secondary antibodies and streptavidin-coated MB resulted in a limits of detection as low as 0.1 ng/mL. The CMC-based microarrays demonstrated very high storage stability; their activity did not change after one year storage at room temperature.

A magnetic micromethod to measure Young's modulus of protein crystals and other polymer materials

A new micromethod has been developed to measure the elastic modulus of polymer materials. It is based on measurements of bending of a polymer sample in a periodic uneven magnetic field acting on a small permanent magnet attached to the sample free end. As compared to the other methods known, it combines simplicity of resonant methods with a possibility to perform measurements at different frequencies in liquids under normal and high pressures. The method is specifically designed to measure the temperature dependence of cross-linked protein crystals under different conditions.