Preparative isolation of polyphosphoinositides and other anionic phospholipids from natural sources using chromatography on adsorbents containing primary amino groups.

A new method for the preparative isolation of anionic phospholipids with the use of chromatography on adsorbents containing primary amino groups has been developed. The method combines elements of bioaffinity and ion-exchange chromatography. Synthesis of adsorbents based on Spheron and silica supports with immobilized neomycin, L-lysine, or aminoalkyl groups was carried out. Optimal conditions for the separation of phospholipid mixtures on aminosorbents were determined. Separation of rat brain bis- and trisphosphoinositides and preparative isolation of bovine heart cardiolipin and baker's yeast phosphatidylinositol are described. The chromatographic behavior of anionic phospholipids on three types of adsorbent was studied. The contribution of biospecific interactions to the adsorption of polyphosphoinositides on aminosorbents is noted.     

Use of monolithic sorbents modified by directly synthesized peptides for affinity separation of recombinant tissue plasminogen activator (t-PA)

Present report demonstrates the examples of practical application of sorbents obtained via direct solid phase peptide synthesis (SPPS) on GMA-EDMA monoliths (CIM Disks, BIA Separations, d.o.o., Ljubljana, Slovenia). Several peptidyl complementary to recombinant tissue plasminogen activator (t-PA) ligands have been synthesized using Fmoc-chemistry. This approach affords to get directly sorbents for affinity chromatography avoiding a cleavage of synthesized peptides from a carrier following by their isolation, analysis and purification. The affinity binding parameters were found from experimental frontal analysis data. The results have been compared with those established for CIM affinity sorbents obtained by immobilization of the same but preliminarily synthesized on convenient resin, cleaved and purified ligands on the disks using one step reaction with epoxy groups of monolithic material. It has been shown that the affinity constants of these two kinds of sorbent did not vary significantly. Directly obtained affinity sorbents have been used for fast and efficient on-line analysis as well as semi-preparative isolation of recombinant t-PA from crude cellular supernatant.     

Solid phase peptide synthesis on epoxy-bearing methacrylate monoliths.

Monoliths based on a copolymer of glycidyl methacrylate (GMA) and ethylene dimethacrylate (EDMA) can be used directly as sorbents for affinity chromatography after solid phase peptide synthesis. The quality of the synthesized products, the amount of grown peptides on a support and the reproducibility of the process must be considered. A determination of the quantity of the introducing beta-Ala (and, consequently, the total amount of synthesized peptide) was carried out. Three peptides complementary to recombinant tissue plasminogen activator (t-PA) have been synthesized using Fmoc-chemistry on GMA-EDMA disks. The peptidyl ligands were analysed by amino acid analysis, ES-MS and HPLC methods. The affinity binding parameters were obtained from frontal elution data. The results were compared with those established for GMA-EDMA affinity sorbents formed by the immobilization of the same but separately synthesized and purified ligands. The immobilization on GMA-EDMA disks was realized using a one-step reaction between the amino groups of the synthetic ligand and the original epoxy groups of monolithic material. The affinity constants found for two kinds of sorbent did not vary significantly. Finally, the directly obtained affinity sorbents were tested for t-PA separation from a cellular supernatant.     

The development of methods for obtaining monospecific ingredients for immunoenzyme analysis

The methods of the modification of Salmonella O- and H-antigens and the preparation of biologically active sorbents on their basis have been developed. The use of these sorbents has permitted the isolation of affinity antibodies with strictly defined specific activity. The work shows the possibility of the successful use of carriers obtained on the basis of porous glass, chemically modified by acrylic copolymers containing activated carboxylic groups, and intended for the immobilization of antigens of both protein and carbohydrate nature.     

Composite fluorine polymer-containing sorbents for isolation and purification of biopolymers

Composite fluoropolymer-containing sorbents based on porous silicas were synthesized for the isolation and purification of biopolymers under nondenaturing conditions. Examples of the application of these sorbents in the separation of various mixtures of peptides and proteins and purification of nucleic acids from various sources (plasmid DNA and DNA from nucleated human blood cells) using the cartridge, column, and batch (sorption in a stirred volume) methods are presented. It was shown that the sorbents can be used in laboratory practice because they are selective to nucleic acids (DNA and RNA) and proteins. These materials combine the mechanical properties of the inorganic matrix with the specific sorption properties of the polymer phase and exhibit enhanced stability to alkaline hydrolysis. Alternative methods of preparing sorbents containing polytetrafluoroethylene, polytrifluorostyrene, and polyfluorobutadiene are described. By the example of polyfluorobutadiene-containing sorbents, a completely new method for obtaining fluorinated polymer phases was developed: the polymer phase was preliminarily formed on the surface of porous disperse carriers and was fluorinated with xenon difluoride.     

Preparation of affinity sorbents with immobilized synthetic ligands for therapeutic apheresis

The practical aspects of preparation and stability of medical sorbents are considered. A simple and convenient technique has been developed for synthesis of highly effective biospecific autoclavable sorbents based on the polysassharide matrix; synthetic ligands (amino acid, oligopeptide, or oligosaccharide) containing primary amino group were immobilized to the matrix via a spacer. The developed approach may be used for preparation of various affinity sorbents suitable for application in medicine and biotechnology.     

Sensitive analysis of ethanolamine- and serine-containing phosphoglycerides by high-performance liquid chromatography.

A highly sensitive method for the separation and quantitative measurement of phospholipids containing primary amino groups, such as phosphatidylethanolamine, phosphatidylserine and lysophosphatidylethanolamine, is described. The method involves a simple and quantitative derivative formation of the phospholipids containing amino groups to their u.v.-absorbing biphenylcarbonyl derivatives. These have molar extinction coefficients of about 23,000 at 268nm. The phospholipid derivatives are then separated and non-destructively determined by high-performance liquid chromatography. The amino phospholipids containing vinyl ether bonds (plasmalogens) can be determined separately from the diacyl- and alkylacyl-amino phospholipids. The lower limit of detection by high-performance liquid-chromatographic analysis of the phospholipid derivatives is about 10-13pmol or 0.3-0.4ng of phospholipid P. The quantitative range of derivative formation and analysis by high-performance liquid chromatography of the phospholipids containing amino groups was shown to be 10-500nmol. The method was shown to be applicable to the analysis of phospholipids containing amino groups in tissue samples.     

In situ preparation of peptidylated polymers as ready-to-use adsorbents for rapid immunoaffinity chromatography

Summary The preparation method of peptide ligands employing polymer-supported solid-phase synthesis and leading to biospecific sorbents has been designed and optimized. This approach directly affords porous polymer sorbents for biospecific chromatography and avoids the cleavage of the synthesized peptide moieties from the carrier and their isolation. The specifics of both peptide synthesis and biospecific chromatography using hydrophilic macroporous polymer supports based on porous poly(glycidyl methacrylate-co-ethylene dimethacrylate) beads and discs were also investigated. The protecting groups can be removed from the target peptide (bradykinin) attached to the polymer support by trifluoromethylsulfonic acid without any significant loss of the attached peptide from the polymer carrier. Introduction of styrene as a comonomer into the copolymer structure improves the reactivity of the support. However, no nonspecific adsorption of proteins in the course of the biospecific isolation of antibradykinin antibodies was observed with these media. In contrast, the nonspecific sorption of proteins increases as a result of increasing peptide loading.     

In situ preparation of peptidylated polymers as ready-to-use adsorbents for rapid immunoaffinity chromatography

The preparation method of peptide ligandsemploying polymer-supported solid-phase synthesisand leading to biospecific sorbents has beendesigned and optimized. This approach directlyaffords porous polymer sorbents for biospecificchromatography and avoids the cleavage of thesynthesized peptide moieties from the carrier andtheir isolation. The specifics of both peptidesynthesis and biospecific chromatography usinghydrophilic macroporous polymer supports based onporous poly(glycidyl methacrylate-co-ethylenedimethacrylate) beads and discs were alsoinvestigated. The protecting groups can be removed from the target peptide (bradykinin) attached tothe polymer support by trifluoromethylsulfonic acidwithout any significant loss of the attached peptidefrom the polymer carrier. Introduction of styreneas a comonomer into the copolymer structureimproves the reactivity of the support. However, nononspecific adsorption of proteins in the course ofthe biospecific isolation of antibradykininantibodies was observed with these media. Incontrast, the nonspecific sorption of proteinsincreases as a result of increasing peptide loading.     

Mass spectrometry analysis of oxidized phospholipids

The evidence that oxidized phospholipids play a role in signaling, apoptotic events and in age-related diseases is responsible for the increasing interest for the study of this subject. Phospholipid changes induced by oxidative reactions yield a huge number of structurally different oxidation products which difficult their isolation and characterization. Mass spectrometry (MS), and tandem mass spectrometry (MS/MS) using the soft ionization methods (electrospray and matrix-assisted laser desorption ionization) is one of the finest approaches for the study of oxidized phospholipids. Product ions in tandem mass spectra of oxidized phospholipids, allow identifying changes in the fatty acyl chain and specific features such as presence of new functional groups in the molecule and their location along the fatty acyl chain. This review describes the work published on the use of mass spectrometry in identifying oxidized phospholipids from the different classes.     

Sorption of lysine by molecularly imprinted carboxyl sorbents

Tuned (molecularly imprinted) and nontuned, with respect to lysine amino acid, carboxylic heteroreticular sorbents based on methacrylic acid and ethylene glycol dimethacrylate, were synthesized. Study of sorption of lysine within wide pH range and ionic strength indicated significant dissimilarities in amino acid sorption by tuned sorbents, which were expressed as an increase in the contribution of nonionic interaction, and resulted in a decrease in the ionic strength effect on the sorption capacity, as well as an increase in amino acid sorption selectivity.     

High-performance liquid chromatography of phospholipids with UV detection: optimization of separations on silica

Chromatography of phospholipids was performed on silica columns with detection by absorbance at 205 nm using mixtures of hexane—isopropanol—water in which the role of water and isopropanol in elution was investigated. One system was developed which provided adequate separation of most major phospholipid species. However, lipids with several ionizable groups were not well separated and gave multiple broad peaks. A second system was developed utilizing sulfuric acid for ion suppression. The behavior of phospholipids in this system was found to be dependent on the presence of quaternary ammonium, amino, or hydroxyl groups. Except for plasmalogen, phospholipids were recovered intact. This system was optimized to provide baseline resolution of essentially all phospholipid species commonly found in mammalian tissues.     

Reaction of 1-fluoro-2,4-dinitrobenzene and 2,4,6-trinitrobenzenesulphonic acid with membranes of sarcoplasmic reticulum.

Membranes of sarcoplasmic reticulum were labelled with 1-fluoro-2,4-dinitro[3H]benzene at pH 6.5 and with 2,4,6-trinitrobenzenesulphonate at pH 9.2. Conditions were chosen to restrict reaction to amino groups, and the effect of blockings of these groups by methyl acetimidate was determined. All proteins were labelled to some extent by both reagents, but, whereas the trinitrophenylation of both lipid and protein amino groups was almost completely blocked by methyl acetimidate, the dinitrophenylation of the ATPase at pH 6.5 was much less affected. The seven amino groups on the ATPase that were labelled under these conditions did not react with methyl acetimidate. This reagent can therefore be used to enhance the specificity of fluorodinitrobenzene for amino groups in a hydrophobic environment. The amino groups on the minor proteins and on the phospholipids that reacted with fluorodinitrobenzene at pH 6.5 were probably in an aqueous environment, since the reaction was blocked by methyl acetimidate.     

Methods and prospects of development of the affinitive sorbents for plasminogen isolation from human blood plasma

The review paper was dedicated to development of the promising photochemical synthesis of affine sorbents for plasminogen isolation from the human blood plasma. Some most interesting, from the viewpoint of practice, types of sorbents and carriers based on high-molecular compounds of natural (organic) or synthetic origin have been considered. The advantages of the use of photochemical synthesis of biospheric sorbent as compared with thermochemical method have been shown. The most promising methods of creation of affine sorbents on the basis of oligomer-polymeric photoinitiators and oligomerpolymeric photosensibilizing (donor-acceptor) systems are presented.     

Controlled porous glass (CPG) with reactive epoxy groups as support for affinity chromatography I. Optimization of CPG modification and the binding of glucose with modified surface

The selective and complementary interaction between the ligand bound to support surface and isolated substance is the key of the affinity chromatography. This is the reason of the research in the field of certain sorbents. They are obtained by chemical reaction of matrix with proper ligands. It is carried out mainly by using bifunctional substances which make the reaction with the ligand in question possible. This paper deals with the preparation of controlled porosity glass with reactive epoxy groups. In order to obtain such support and avoid application of a proper epoxy silane compound, the double-step reaction using simple agents was employed. The sorbent syntheses were optimized. The prepared sorbents were applied for coupling some carbohydrates and amino acids.     

A Soil and Rhizosphere Microorganism Isolation and Enumeration Medium That Inhibits Bacillus mycoides

A new solid medium has been developed for the enumeration and isolation of soil and rhizosphere microorganisms. This medium, named rhizosphere isolation medium, contains glucose and 15 of the 20 common amino acids. The absence of five other amino acids, namely, aspartic acid, asparagine, cysteine, proline, and threonine, inhibits the growth of Bacillus mycoides, a commonly encountered bacterium that rapidly spreads on agar media and complicates the isolation and enumeration of other microorganisms. Compared with a similar medium containing Casamino Acids, rhizosphere isolation medium had half as many colonies of B. mycoides, with each colony approximately half the diameter. The two media had similar total numbers of bacterial colonies. Isolates were divided into taxononomic groups, roughly corresponding to species and genus, by fatty acid methyl ester analysis and numerical methods. There were 24 genera and 41 species found in the isolates from rhizosphere isolation medium, while 19 genera and 35 species were found in the isolates from the medium prepared with Casamino Acids. No major group of bacteria was found to occur only on one medium or on the other, indicating that the five missing amino acids had no great effect on organisms other than B. mycoides. This medium may prove useful in soil and rhizosphere studies in which the growth of B. mycoides is undesirable.     

Amino-modified silicate gels prepared by sol-gel processing as metal-ion sorbents

Two series of amino-modified silicate gels prepared by sol-gel processing were used to absorb Cu(II), Ni(II), Co(II), Mn(II) and Cr(III) from aqueous solutions. These easily prepared sorbents with various content of primary amino groups in series (A) or primary and secondary amino groups in series (AA) have reasonable stability. The gel composition, time and concentration dependence of the uptake of the metal ions by these materials were studied systematically. These materials would be further used as supports to disperse catalytically active phases by conventional wet chemical procedures. Apart from this they demonstrate potential for the preconcentration aid for transition metal analysis.     

Isolation of nisin from native solution and its purification on cationites]

It was found possible to use organic sorbents and in particular carboxylic cationites for isolation of nisin from the fermentation broth filtrate and its purification. Nisin is known as a polypeptide antibiotic applied as a preservative. The sorbents were shown to have high exchange capacities by the isolated substance and mechanical strength and resistance. They also proved to be highly stable.     

The oxidized form of cholesterol 3β-hydroxy-5-oxo-5,6-secocholestan-6-al induces structural and thermotropic changes in phospholipid membranes

The effect of an oxidized form of cholesterol, 3β-hydroxy-5-oxo-5,6-secocholestan-6-al on the thermotropic and structural properties of phospholipid membranes was investigated by differential scanning calorimetry and X-ray diffraction and compared with that of cholesterol. The phospholipids studied included 1-palmitoyl-2-oleoylphosphatidylserine, dipalmitoleoylphosphatidylethanolamine, 1-palmitoyl-2-oleoylphosphatidylethanolamine, dipalmitoleoylphosphatidylcholine, 1-palmitoyl-2-oleoylphosphatidylcholine. Depending on the constituent phospholipids, the oxidized cholesterol is observed to shift phase transitions, disrupt stacking, modify interbilayer spacings and promote increased negative membrane curvature. We determined by absorption spectroscopy that the amino group of phosphatidylserine forms a Schiff base with the aldehyde group of the 3β-hydroxy-5-oxo-5,6-secocholestan-6-al as was previously found for the amino group of phosphatidylethanolamine. This result strengthens the biologically significant finding that not only amino groups of proteins but also amino groups of phospholipids are able to form a Schiff base with oxidized cholesterol. The marked triangular shape of the Schiff base complex with phosphatidylethanolamine may explain how 3β-hydroxy-5-oxo-5,6-secocholestan-6-al can promote increased negative curvature in the hexagonal phase, as compared to cholesterol, even though its increased polarity would favor a location closer to the interface with water.     

Studies on cytochrome c oxidase, VI. Polypeptide IV. the complete primary structure.

The complete primary structure of the cytoplasmically synthesized polypeptide IV from beef heart cytochrome oxidase was determined via isolation and sequencing of overlapping methionine, tryptophan, and arginine fragments. The protein consists of 147 amino acids (Mr 17153). It is characterized as a part of a membrane protein complex by a hydrophobic segment consisting of 19 residues. It is suggested that this segment contacts the lipids of the inner mitochondiral membrane. Additional specific contacts may result from pairwise formation of salt bridges between ionic groups of the protein and the phospholipids. The function of this component of the terminal oxidase is yet unknown.