Josef Chmelík

Capillary isoelectric focusing with electroosmotic zone displacement and on-column multichannel detection

Abstract Isoelectric focusing (IEF) of proteins in uncoated, open-tubular fused-silica capillaries of 75 μm I.D. with on-column multiwavelength detection is reported. Small amounts of hydroxypropylmethylcellulose added to the catholyte are shown to provide column conditioning which allows rapid and high-resolution IEF analysis of proteins to be performed in the presence of an electroosmotic flow along the separation axis. The latter process displaces the developing zone pattern towards and across the point of detection. On-column multichannel zone detection is shown to be an efficient method for the simultaneous monitoring of the eluting proteins and carrier ampholytes. The absorbance profiles monitored at one location towards the capillary end and the temporal behaviour of the current under constant voltage conditions are shown to provide information on the degree of focusing at the time of detection.

Different elution modes and field programming in gravitational field-flow fractionation: I. A theoretical approach

Abstract Gravitational field-flow fractionation utilizes the Earth’s gravitational field as an external force which causes settlement of particles towards channel accumulation wall. It is sometimes considered that gravitational field-flow fractionation is not a versatile technique because the force field cannot be changed as in the other field-flow fractionation techniques. However, there are several possibilities to change the force acting on particles (e.g., by changing the angle between the Earth’s gravitational field and the longitudinal axis of the channel, or the use of carrier liquids having different densities. Hydrodynamic lift forces are also known to act on particles in the carrier liquid flow and give some other means of modulating the force field in this technique by varying the flow-rate. This change can be reached by using programmed pumps or channels with non-constant cross-sections. Some of these possibilities can be exploited for field programming if a unified approach to control the retention variables in gravitational field-flow fractionation can be worked out. Several different elution modes can be indeed observed in gravitational field-flow fractionation, even mixed in some experiments. The equations describing the concentration profiles of analytes and their dependence on the elution mode mechanisms are used to gain insights into the different ways of field programming for each particular elution mode.

Micropreparation of hemopoietic stem cells from the mouse bone marrow suspension by gravitational field-flow fractionation

Gravitational field-flow fractionation is a relatively simple experimental technique. This method was used for the characterization of stem cells from mouse bone marrow. Because these cells are bigger than the other cells in bone marrow, it is possible to separate them from the mixture. The fractions collected after passing through the separation channel were characterized using a Coulter Counter and used for transplantation into irradiated mice.

Behaviour of substituted aminomethylphenol dyes in capillary isoelectric focusing with electroosmotic zone displacement

The behaviour of six substituted aminomethylphenol dyes, having pI values between 5.3 and 10.4, in capillary isoelectric focusing with electroosmotic zone displacement is described. Using untreated fused-silica capillaries and different neutral capillary conditioners in the catholyte, the low-molecular-mass dyes are shown to focus and elute reproducibly in the order of decreasing pI values. In the absence of proteins, the detection times of the dyes are independent of the sampled amount. Hence these substances permit the characterization of the pH gradient produced in this capillary isoelectric focusing method. With concurrent focusing of dyes and test proteins, a macromolecular impact on detection times (reduction of electroosmosis)` is revealed. The effect is shown to be dependent on the type and amount of proteins applied and has been observed with three different capillary conditioners. Nevertheless, mapping of the pH gradient with these dyes and determining the pI values of known proteins is shown to provide values in agreement with those in the literature. Hence the substituted aminomethylphenol dyes can be employed as pI markers in capillary isoelectric focusing with electroosmotic zone displacement. Further, focusing and separation of two of the six dyes by preparative recycling free fluid isoelectric focusing is described, illustrating that the substituted aminomethylphenol dyes are also applicable to other free fluid focusing methods.

Different elution modes and field programming in gravitational field-flow fractionation. IV. Field programming achieved with channels of non-constant cross-sections.

Force field programming provided increased speed of separation with an improved resolution and detection capability in many field-flow fractionation (FFF) techniques. Gravitational field-flow fractionation (GFFF) uses the Earths gravitational field to cause the settlement of particles towards the channel accumulation wall. Although this field is constant and relatively weak, there are different ways to implement force field programming in GFFF. Because hydrodynamic lift forces (HLF) participate in the separation process in focusing (hyperlayer) elution mode, they can control the resulting force field acting on particles via changes in flow-velocity. These changes can be accomplished by a programmable pump or with channels of non-constant cross-sections. This work is focused on flow-velocity programming accomplished with channels of non-constant cross-sections. Three trapezoidal channels of decreasing breadth and two channels of decreasing height (along the longitudinal axis) are tested as tools for optimization of the separation of a model silica gel particle mixture. The trapezoidal channels yielded reduced separation times. However, taking into account both speed of separation and resolution, the optimization effect was lower compared with the flow-rate gradients generated by a programmable pump. The channels of non-constant height did not yield advantageous separations.

Experimental study on the separation of silica gel supports by gravitational field-flow fractionation

The separation and characterization of silica gel particles were investigated by gravitational (1 g) field-flow fractionation. The experiments were performed in a ribbon-like glass-walled channel with a photometric (turbidimetric) detector. Solutions of several surfactants were tested as carrier liquids. Factors affecting the separation process are discussed and the deductions are compared with experimental results. Some aspects concerning the activity of lift forces were confirmed.

Chemical cross-linking and H/D exchange for fast refinement of protein crystal structure.

A combination of chemical cross-linking and hydrogen-deuterium exchange coupled to high resolution mass spectrometry was used to describe structural differences of NKR-P1A receptor. The loop region extended from the compact core in the crystal structure was found to be closely attached to the protein core in solution. Our approach has potential to refine protein structures in solution within a few days and has very low sample consumption.

NMR Structure of the N-Terminal Domain of Capsid Protein from the Mason–Pfizer Monkey Virus

The high-resolution structure of the N-terminal domain (NTD) of the retroviral capsid protein (CA) of Mason-Pfizer monkey virus (M-PMV), a member of the betaretrovirus family, has been determined by NMR. The M-PMV NTD CA structure is similar to the other retroviral capsid structures and is characterized by a six alpha-helix bundle and an N-terminal beta-hairpin, stabilized by an interaction of highly conserved residues, Pro1 and Asp57. Since the role of the beta-hairpin has been shown to be critical for formation of infectious viral core, we also investigated the functional role of M-PMV beta-hairpin in two mutants (i.e., DeltaP1NTDCA and D57ANTDCA) where the salt bridge stabilizing the wild-type structure was disrupted. NMR data obtained for these mutants were compared with those obtained for the wild type. The main structural changes were observed within the beta-hairpin structure; within helices 2, 3, and 5; and in the loop connecting helices 2 and 3. This observation is supported by biochemical data showing different cleavage patterns of the wild-type and the mutated capsid-nucleocapsid fusion protein (CANC) by M-PMV protease. Despite these structural changes, the mutants with disrupted salt bridge are still able to assemble into immature, spherical particles. This confirms that the mutual interaction and topology within the beta-hairpin and helix 3 might correlate with the changes in interaction between immature and mature lattices.

Optimization of experimental conditions for the separation of small and large starch granules by gravitational field-flow fractionation.

Separation of small and large barley starch granules by gravitational field-flow fractionation was investigated from the point of view of sample pre-treatment, amount of injected sample, and elution conditions. The sample pre-treatment study resulted in the conclusion that it is reasonable to soak the starch granules for at least 24 h prior to separation. The experiments with different amounts of injected sample show that it is possible to increase as well as decrease twofold the sample amount usually used without any change in retention ratios. The implementation of flow-rate gradients for elution of the starch granules reduced total separation time. However, the applied flow-rate gradients did not improve the resolution of peaks A and B compared with the generally used constant flow-rate. Thus, for barley starch granules, the constant flow-rates within the range from 0.8 to 1.0 ml/min seem to provide the best compromise of total separation time, peak resolution and instrumental expense.

Soluble recombinant CD69 receptors optimized to have an exceptional physical and chemical stability display prolonged circulation and remain intact in the blood of mice

We investigated the soluble forms of the earliest activation antigen of human leukocyte CD69. This receptor is expressed at the cell surface as a type II homodimeric membrane protein. However, the elements necessary to prepare the soluble recombinant CD69 suitable for structural studies are a matter of controversy. We describe the physical, biochemical and in vivo characteristics of a highly stable soluble form of CD69 obtained by bacterial expression of an appropriate extracellular segment of this protein. Our construct has been derived from one used for CD69 crystallization by further optimization with regard to protein stability, solubility and easy crystallization under conditions promoting ligand binding. The resulting protein is stable at acidic pH and at temperatures of up to 65 °C, as revealed by long‐term stability tests and thermal denaturation experiments. Protein NMR and crystallography confirmed the expected protein fold, and revealed additional details of the protein characteristics in solution. The soluble CD69 refolded in a form of noncovalent dimers, as revealed by gel filtration, sedimentation velocity measurements, NMR and dynamic light scattering. The soluble CD69 proved to be remarkably stable in vivo when injected into the bloodstream of experimental mice. More than 70% of the most stable CD69 proteins is preserved intact in the blood 24 h after injection, whereas the less stable CD69 variants are rapidly taken up by the liver.