A new cleavable reagent for cross-linking and reversible immobilization of proteins.

We have prepared a new bifunctional reagent for the cross-linking and reversible immobilization of proteins through their amine groups. This compound, ethylene glycolyl bis(succinimidyl succinate), reacts rapidly with proteins, at pH 7 and at high dilution. The resulting protein cross-links are readily cleaved at pH 8.5 using hydroxylamine for 3–6 hr. at 37°C. Substantial enzymatic activity was observed with lactic dehydrogenase after such reversible cross-linking. Trypsin immobilized on agarose using this reagent retains full specific activity, is stable for weeks in the cold, and may be released with hydroxylamine at 25°C. This compound appears suitable for studies involving proteins with essential disulfide linkages.     

Stabilization of enzymes by multipoint attachment via reversible immobilization on phenylboronic activated supports

In this work, we have used supports activated with m-amino-phenylboronic groups to “reversibly” immobilize proteins under very mild conditions. Most of the proteins contained in a crude extract from E. coli could be immobilized on Eupergit C-250 L activated with phenylboronic and then fully desorbed from the support by using mannitol or SDS. This suggested that the immobilization of the proteins on these supports was not only via sugars interaction, but also by other interaction/s, quite unspecific, that might be playing a key role in the immobilization of the proteins. Penicillin acylase from E. coli (PGA) was also immobilized in Eupergit C activated with m-amino-phenylboronic groups. The enzyme could be fully desorbed with mannitol immediately after being immobilized on the support. However, longer incubation times of the immobilized preparation caused a reduction of protein elution from the boronate support in presence of mannitol. Moreover, these immobilized preparations showed a higher stability in the presence of organic solvents than the soluble enzyme; the stability also improved when the incubation time was increased (to a factor of 100). By desorbing the weakest bound enzyme molecules, it was possible to correlate adsorption strength with stabilization; therefore, it seems that this effect was due to the rigidification of the enzyme via multipoint attachment on the support.     

The immobilization of enzymes on nylon structures and their use in automated analysis

1. Glucose oxidase (EC 1.1.3.4) and urease (EC 3.5.1.5) were covalently attached through glutaraldehyde to low-molecular-weight nylon powder. 2. Immobilized derivatives of glucose oxidase and urease were prepared by cross-linking the respective enzymes within the matrix of a nylon membrane. 3. An improved process is described for the immobilization of glucose oxidase and urease on the inside surface of partially hydrolysed nylon tube. 4. Automated analytical procedures are described for the determination of glucose with each of the three immobilized glucose oxidase derivatives and for the determination of urea with each of the three immobilized urease derivatives. 5. The efficiencies of the three immobilized enzyme structures as reagents for the automated determination of their substrates were compared.     

Theoretical studies on the heats of formation, densities, and detonation properties of substituted s-tetrazine compounds

Substituted s-tetrazine compounds were designed and investigated in order to find comprehensive relationships between the structures and performances of high-nitrogen energetic compounds. Density functional theory (DFT) was used to predict the optimized geometries, electronic structures, heats of formation and densities, and the detonation properties were evaluated by using the VLW equation of state (EOS). Calculation results show that there are good linear relationships between heats of formation, densities, detonation properties and the number of N atom in all designed high-nitrogen compounds. Furthermore, several designed high-nitrogen compounds show good detonation velocities and pressures compared with octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocane (HMX), making them potential candidates for high-energy-density materials (HEDM).     

DNA-Directed immobilization: efficient, reversible, and site-selective surface binding of proteins by means of covalent DNA-streptavidin conjugates

Covalent DNA-streptavidin conjugates have been utilized for the reversible and site-selective immobilization of various biotinylated enzymes and antibodies by DNA-directed immobilization (DDI). Biotinylated alkaline phosphatase, beta-galactosidase, and horseradish peroxidase as well as biotinylated anti-mouse and anti-rabbit immunoglobulins have been coupled to the DNA-streptavidin adapters by simple, two-component incubation and the resulting preconjugates were allowed to hybridize to complementary, surface-bound capture oligonucleotides. Quantitative measurements on microplates indicate that DDI proceeds with a higher immobilization efficiency than conventional immobilization techniques, such as the binding of the biotinylated proteins to streptavidin-coated surfaces or direct physisorption. These findings can be attributed to the reversible formation of the rigid, double-stranded DNA spacer between the surface and the proteins. Moreover, BIAcore measurements demonstrate that DDI allows a reversible functionalization of sensor surfaces with reproducible amounts of proteins. Ultimately, the simultaneous immobilization of different compounds using microstructured oligonucleotide arrays as immobilization matrices demonstrate that DDI proceeds with site selectivity due to the unique specificity of Watson-Crick base pairing.     

Genetic modification of the penicillin G acylase surface to improve its reversible immobilization on ionic exchangers

A new mutant of the industrial enzyme penicillin G acylase (PGA) from Escherichia coli has been designed to improve its reversible immobilization on anionic exchangers (DEAE- or polyethyleneimine [PEI]-coated agarose) by assembling eight new glutamic residues distributed homogeneously through the enzyme surface via site-directed mutagenesis. The mutant PGA is produced and processed in vivo as is the native enzyme. Moreover, it has a similar specific activity to and shows the same pH activity profile as native PGA; however, its isoelectric point decreased from 6.4 to 4.3. Although the new enzyme is adsorbed on both supports, the adsorption was even stronger when supports were coated with PEI, allowing us to improve the enzyme stability in organic cosolvents. The use of restrictive conditions during the enzyme adsorption on anionic exchangers (pH 5 and high ionic strength) permitted us to still further increase the strength of adsorption and the enzyme stability in the presence of organic solvents, suggesting that these conditions allow the penetration of the enzyme inside the polymeric beds, thus becoming fully covered with the polymer. After the enzyme inactivation, it can be desorbed to reuse the support. The possibility to improve the immobilization properties on an enzyme by site-directed mutagenesis of its surface opens a promising new scenario for enzyme engineering.     

Studies on ras proteins. Catalytic properties of normal and activated ras proteins purified in the absence of protein denaturants

Normal (Gly12) and activated (Val12) Ha-ras proteins were produced in Escherichia coli, and purified to an apparent homogeneity without using any protein denaturants. The purified proteins contained an equimolar amount of GDP. They were stable in the presence of 5 mM Mg2+ and 25% (v/v) glycerol when incubated at 60 degrees C for 5 min. The binding of GDP to the protein was greatly stabilized by Mg2+. In the presence of 10 mM Mg2+, the bound GDP hardly exchanged with external guanine nucleotides, even at 30 degrees C. The exchange reaction was markedly enhanced in the presence of 10 mM EDTA or 120 mM ammonium sulfate. The rate-limiting step of the exchange reaction was the dissociation of the bound GDP from the ras protein, and this step was facilitated 40- to 100-fold by the addition of EDTA or ammonium sulfate. The dissociation rate of the normal (Gly12) ras protein was 2- to 3-fold faster than that of the activated (Val12) protein. The dissociation constants (Kd) for GDP of the normal and activated ras proteins were 1.2 X 10(-8) and 3.1 X 10(-9) M, respectively. The overall turnover rate of GTPase activity of the normal ras protein (10.8 mmol.mol-1.min-1) was about 10-fold higher than that of the activated protein (1.1 mmol.mol-1.min-1) in the absence of Mg2+ (less than 10(-8) M).     

Evolution of oligomeric proteins. The unusual case of a dimeric ribonuclease.

The model system made up of a monomeric and a dimeric ribonuclease of the pancreatic-type superfamily has recently attracted the attention of investigators interested in the evolution of oligomeric proteins. In this system, bovine pancreatic ribonuclease (RNase A) is the monomeric prototype, and bovine seminal ribonuclease (BS-RNase) is the dimeric counterpart. However, this evolutionary case is unusual, as BS-RNase is the only dimeric member of the whole large superfamily comprising more than 100 identified members from amphibia, aves, reptilia and mammalia. Furthermore, although the seminal-type RNase gene can be traced back to the divergence of the ruminants, it is expressed only in a single species (Bos taurus). These unusual findings are discussed, as well as previous hypotheses on the evolution of seminal RNase. Furthermore, a new 'minimalist' hypothesis is proposed, in line with basic principles of structural biology and molecular evolution.     

The effect of immobilization on carbon flow through Anabaena variabilis and A. azollae

Immobilization in polyvinyl foam resulted in an increased carbon fixation and release of fixed carbon in Anabaena variabilis while in A. azollae both processes decreased. This revised version was published online in June 2006 with corrections to the Cover Date.     

The effect of immobilization on carbon flow through Anabaena variabilis and A. azollae

Immobilization in polyvinyl foam resulted in an increased carbon fixation and release of fixed carbon in Anabaena variabilis while in A. azollae both processes decreased.     

Oligonucleotide derivatives in the hybridization analysis of nucleic acids. I. Covalent immobilization of oligonucleotide probes on nylon

The characteristics of the UV-induced immobilization of oligonucleotides on nylon membranes and the efficiency of the enzymatic labeling of immobilized probes in heterophase identifying specific DNA sequences were studied. Oligonucleotides bound to short terminal oligothymidylates (up to 10 nt) through a flexible linker based on diethylene glycol phosphodiester are proposed as probes for immobilization on nylon. The presence of this fragment allows one to enhance the immobilization efficiency and reduce the UV-dependent degradation of the sequence-specific part of the probe by decreasing the irradiation dose needed for DNA immobilization. The optimal dose of UV irradiation is evaluated to be ∼0.4 J/cm² at 254 nm, which provides a high level of the hybridization signal for immobilized probes of various nucleotide sequences. It was found that nylon amide groups play a key role in the photoinduced fixation of oligonucleotides to the polymer surface, while its primary amino groups were not as responsible for the covalent binding of DNA as previously thought. Various additives in the membrane wetting solution were demonstrated to influence both the efficiency of the UV-induced immobilization and the functional integrity of immobilized probes. Other radical generating systems alternative to UV irradiation are shown to provide the immobilization of oligonucleotides on nylon membranes.     

The reversible DNA-alkylating activity of duocarmycin and its analogues.

Intact drugs with spirocyclopropylhexadienone moieties can be regenerated from the covalent DNA adducts induced by antitumor antibiotics duocarmycin (DUM) A, SA and some DUMA analogues in neutral aqueous solution. We detected the reversible nature of DUMs by determination of the antimicrobial activity and cytotoxicity of DUM-DNA adducts. All of the adducts selectively inhibited the growth of a sensitive strain of Bacillus but not that of the wild type strain, a property of parent DUM and its analogues. Most of the DNA adducts were also cytotoxic to HeLa S3. These results suggested that active drugs can be released from their covalent DNA adducts under these biological assay conditions. Regeneration of intact drugs was quantitatively analyzed by HPLC and the amount of free drug released from DNA adducts revealed that the rate and efficiency of this reversal were dependent on structural variables among the drugs. The differences in rates of reversibility were correlated with the biological activity of DUMs. The effect of pH, temperature and salt concentration on the regeneration of drugs from their DNA adducts suggest a catalytic role of double-helical DNA on the reversal pathway.     

Studies on nitrate reductase of Clostridium perfringens. Purification, some properties, and effect of tungstate on its formation.

Nitrate reductase (NaR) linked to reduced methyl viologen from Clostridium perfringens was purified by ammonium sulfate precipitation. DEAE-cellulose chromatography, disc electrophoresis on polyacrylamide gel, and triple DEAE-Sephadex chromatography. The specific activity was increased 1,200-fold with a yield of 9%. The purified preparation was nearly homogeneous in disc electrophoresis. It was brown, and its spectrum showed a slight shoulder near 420 nm as well as a peak at 280 nm. The molecular weight was found to be 90,000 based on s020,w (5.8S) and 80,000 by Sephadex G-100 gel filtration. In SDS-polyacrylamide electrophoresis, it showed only a single band with a molecular weight of 90,000; it had no subunit structure. The isoelectric point was pH 5.5, and the optimum pH was 9. Mn2+, Fe2+, Mg2+, and Ca2+ stimulated the activity. Km for nitrate was 0.10 mM, and nitrate was stoichiometrically reduced to nitrite in the presence of 2 mM Mn2+. Ferredoxin fraction obtained from extracts of the bacterium was utilizable as an electron donor at pH 8. Cyanide and azide inhibited the enzyme. The formation of NaR was induced by nitrate and inhibited by 0.5 mM tungstate, but recovered in the presence of 0.1 mM molybdate; NaR of C. perfringens appears to be a molybdo-iron-sulfur protein.     

Synthesis of a mesoporous functional copolymer bead carrier and its properties for glucoamylase immobilization

A series of mesoporous and hydrophilic novel bead carriers containing epoxy groups were synthesized by modified inverse suspension polymerization. Glycidyl methacrylate and acryloyloxyethyl trimethyl ammonium chloride were used as the monomers, and divinyl benzene, allyl methacrylate, and ethylene glycol dimethacrylate as crosslinking agents, respectively. The resulting carriers were employed in the immobilization of glucoamylase (Glu) with covalent bond between epoxy groups and enzymes. The activity recovery of the three series of immobilized Glus could reach 76%, 79%, and 86%, respectively. The immobilized Glus exhibit excellent stability and reusability than that of the free ones.     

Polyhydroxyalkanoate chip for the specific immobilization of recombinant proteins and its applications in immunodiagnostics

In this study, a novel strategy was developed for the highly selective immobilization of proteins, using the polyhydroxyalkanoate (PHA) depolymerase substrate binding domain (SBD) as an active binding domain. In order to determine the appropriacy of this method for immunodiagnostic assays, the single-chain antibody (ScFv) against the hepatitis B virus (HBV) preS2 surface protein and the severe acute respiratory syndrome coronavirus (SARS-CoV) envelope protein (SCVe) were fused to the SBD, then directly immobilized on PHA-coated slides via microspotting. The fluorescence-labeled HBV antigen and the antibody against SCVe were then utilized to examine specific interactions on the PHA-coated surfaces. Fluorescence signals were detected only at the spotted positions, thereby indicating a high degree of affinity and selectivity for their corresponding antigens/antibodies. Furthermore, we detected small amounts of ScFv-SBD (2.7 ng/mL) and SCVe-SBD fusion proteins (0.6 ng/mL). Therefore, this microarray platform technology, using PHA and SBD, appears generally appropriate for immunodiagnosis, with no special requirements with regard to synthetic or chemical modification of the biomolecules or the solid surface.     

Evaluation of the antioxidant properties of propofol and its nitrosoderivative. comparison with homologue substituted phenols

Propofol (2,6-diisopropylphenol), some substituted phenols (2,6-dimethylphenol and 2,6-ditertbutylphenol) and their 4-nitrosoderivatives have been compared for their scavenging ability towards 1,1-diphenyl-2-picrylhydrazyl and for their inhibitory action on lipid peroxidation. These products were also compared to the classical antioxidants butylated hydroxytoluene and butylated hydroxyanisole. When measuring the reactivity of the various phenolic derivatives with 1,1-diphenyl-2-picrylhydrazyl the following order of effectiveness was observed: butylated hydroxyanisole > propofol > 2,6-dimethylphenol > 2,6-di-tertbutylphenol > butylated hydroxytoluene. In cumene hydroperoxide-dependent microsomal lipid peroxidation, propofol acts as the most effective antioxidant, while butylated hydroxyanisole, 2,6-di-tertbutylphenol and butylated hydroxytoluene exhibit a rather similar effect, although lower than propofol. In the iron/ascorbate-dependent lipid peroxidation propofol, at concentrations higher than 10 microM, exhibits antioxidant properties comparable to those of butylated hydroxytoluene and butylated hydroxyanisole, 2,6-Dimethylphenol is scarcely effective in both lipoperoxidative systems. The antioxidant properties of the various molecules depend on their hydrophobic characteristics and on the steric and electronic effects of their substituents. However, the introduction of the nitroso group in the 4-position almost completely removes the antioxidant properties of the examined compounds. The nitrosation of the aromatic ring of antioxidant molecules and the consequent loss of antioxidant capacity can be considered a condition potentially occurring in vivo since nitric oxide and its derivatives are continuously formed in biological systems.     

The use of ninhydrin as a reagent for the reversible modification of arginine residues in proteins.

A simple technique was developed for the specific reversible modification of guanidino groups in proteins involving reaction with ninhydrin. The extent of the reaction is easily determined non-destructively by spectrophotometric analysis. The reagent can also be used for the titration of sterically unhindered thiol groups in proteins.