Immobilization of proteins in immunochemical microarrays fabricated by electrospray deposition.

Electrospray (ES) deposition has been applied to fabricate protein microarrays for immunochemical assay. Protein antigens were deposited as arrays of dry spots on a surface of aluminized plastic. Deposition was performed from water solutions containing a 10-fold (w/w of dry protein) excess of sucrose.

Electrospray deposition as a method for mass fabrication of mono- and multicomponent microarrays of biological and biologically active substances.

Electrospray of protein and DNA solutions is currently used to generate ions for mass spectrometric analysis of these molecules. Deposition of charged electrospray products on certain areas of a substrate under control of electrostatic forces is suggested here as a method for fabrication of multiple deposits of any size and form. For example, multiple dots of protein, DNA, or other organic substances can be deposited simultaneously through an array of holes in a dielectric mask covering any slightly conductive substrate (membrane, wet glass, semiconductor, etc.

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.

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.

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.

Survey of small molecule and ion binding to beta 2-microglobulin--possible relation to BEN.

Using a new method which permits rapid detection of ligand binding to cross linked protein films and crystals, we evaluated a spectrum of ligand binding to cross linked amorphous films of beta 2-microglobulin purified from urine of Balkan endemic nephropathy patients. Among more than 50 substances studied, including amino acids, fatty acids, sugars, nucleotides, nucleosides, and different inorganic ions, only polyvalent cations, such as Cu2+, Ca2+, Zn2+, Be2+ and La3+, were found to strongly bind to human beta 2-microglobulin with dissociation constants in the range 10(-6) to 10(-4) M. These cations can release beta 2-microglobulin from HLA complexes leading to an increased beta 2-microglobulin level in serum.

Comparison of structures of dry and wet hen egg-white lysozyme molecule at 1.8 A resolution.

A high resolution structure of hen egg-white lysozyme containing 36 +/- 1 mol H2O per mol of protein has been obtained using triclinic (P1) crystals cross-linked with glutaraldehyde. Analysis of dehydration-induced structural changes has revealed displacement in relative position of domains and numerous small displacements in positions of individual atoms with r.m.

Thermal motion of whole protein molecules in protein solids.

A preliminary analysis is presented of whether and to what extent thermal motion of protein molecules as rigid bodies contributes to the mobility found in X-ray and Mössbauer studies. A simple theory is advanced enabling the estimation of mean-square amplitudes of translational and librational motion of the protein molecules in crystals and amorphous glasses from the experimental data on the elastic properties of these solids. The values calculated and their dependence on the crystal packing, temperature and hydration level were found to be in good accord with the data of X-ray analysis and Mössbauer spectroscopy.

A protein molecule as a bioanalytical device. Chemomechanical protein sensors.

Cross-linked protein solids are proposed as new types of chemomechanical sensors for identification of biological molecules and measurement of their concentration. New sensors make use of the ability of proteins to specifically bind its ligands and to alter their conformation upon the binding. The use of immobilized proteins enables these conformational changes to be detected through the changes in mechanical properties of protein solid samples (stress, stain or the Young's modulus).