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PubMed2005

Influence of macromolecular crowding upon the stability and state of association of proteins: predictions and observations.

The concept of excluded volume and possible effects of excluded volume on the reactivity of macromolecules in highly volume-occupied or "crowded" media are introduced and briefly summarized. Theoretical and experimental studies of the effect of crowding on protein folding and unfolding, and on the effect of crowding on protein association and aggregation, are reviewed. Possible effects of the effect of crowding on an initially native protein that can undergo unfolding, self-association of native protein, and/or aggregation of non-native protein are considered.

Biopharmaceutics↗
PubMed2006

Rapid and efficient enzyme encapsulation in a dendrimer silica nanocomposite.

We report the entrapment of horseradish peroxidase and quantitative encapsulation of glucose oxidase within silica nanoparticles by utilizing an amine-terminated dendritic template. Our improved strategy employs a water-soluble biomimetic template which is able to catalyze the condensation of Si(OH)(4) to silica nanoparticles while trapping an enzyme inside the mesoporous material. Kinetic analysis shows enzyme functionality to be mostly unchanged. Also, the role of pI and ionic strength within the encapsulation environment was found to strongly influence encapsulation. These results suggest that the electrostatic manipulation of a strong supramolecular silica-precipitating complex of enzyme and dendrimer has the potential of adding a vast array of chemical and biological activity to hybrid materials. [image: see text] Enzyme immobilization within a silica nanocomposite.

Biocompatible Materials↗
PubMed1999

Purification and characterization of thermostable aspartase from Bacillus sp. YM55-1.

A thermostable aspartase was purified from a thermophile Bacillus sp. YM55-1 and characterized in terms of activity and stability. The enzyme was isolated by a 5-min heat treatment at 75 degrees C in the presence of 11% (w/v) ammonium sulfate and 100 mM aspartate, followed by Q-Sepharose anion-exchange and AF-Red Toyopearl chromatographies. The native molecular weight of aspartase determined by gel filtration was about 200,000, and this enzyme was composed of four identical monomers with molecular weights of 51,000 determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Unlike Escherichia coli aspartase, the enzyme was not activated by the presence of magnesium ion at alkaline pH. At the optimum pH, the Km and Vmax were 28.5 mM and 700 units/mg at 30 degrees C and 32.0 mM and 2200 units/mg at 55 degrees C, respectively. The specific activity was four and three times higher than those of E. coli and Pseudomonas fluorescens enzymes at 30 degrees C, respectively. Eighty percent of the activity was retained after a 60-min incubation at 55 degrees C, and the enzyme was also resistant to chemical denaturants; 80% of the initial specific activity was detected in assay mixtures containing 1.0 M guanidine hydrochloride. The purified enzyme shared a high sequence homology in the N-terminal region with aspartases from other organisms.

Amino Acid Sequence↗
PubMed1995

Halophilic protein stabilization by the mild solubilizing agents nondetergent sulfobetaines.

In this work, experiments performed on pig heart and halophilic malate dehydrogenase as well as halophilic elongation factor Tu demonstrate a protein stabilization property from the recently described mild solubilizing agents nondetergent sulfobetaines. A practical application is given by the separation of halophilic bacteria elongation factor Tu and halophilic malate dehydrogenase by high-performance ion-exchange chromatography achieved at reduced salt levels without significant loss of activity.

Animals↗
PubMed2002

Introduction of functional groups onto polypropylene and polyethylene surfaces for immobilization of enzymes.

Polypropylene and polyethylene surfaces are activated by introducing an active functional group through 1-fluoro-2 nitro-4-azidobenzene by UV irradiation. Horseradish peroxidase and glucose oxidase are immobilized onto the activated surfaces, simply by incubating the enzymes at 37 degrees C. When untreated surfaces are used, insignificant immobilization of the enzymes is observed.

Enzyme Stability↗
PubMed1999

Pressure/temperature effects on protein flexibilty from acrylamide quenching of protein phosphorescence.

Pressure is an effective modulator of protein structure and biological function. The influence of hydrostatic pressure (</=3 kbar, 10-50 degrees C) on conformational dynamics was assessed from the rate of migration of acrylamide through the protein interior. Migration rates in apoazurin, alcohol dehydrogenase and alkaline phosphatase were obtained from the phosphorescence quenching rate constant (kq) of the deeply buried Trp residues. The dominant effect of applied pressure is to slow the diffusion process, although at low temperature, high pressure may also accelerate it. For apoazurin, alcohol dehydrogenase and alkaline phosphatase the activation free volumes, DeltaV(obs), derived from the pressure-dependence of kq, ranges from +10, +16 and +20 ml mol(-1)at 50 degrees C to -20, +5 and 0 ml mol(-1)at 10 degrees C, respectively. Analysing DeltaV(obs) in terms of a positive contribution from cavity expansion and a negative one from peptide hydration, the results emphasise that whereas at warm temperature the formation of cavities plays a dominant role in the migration process, at low temperature the required flexibility may be conferred by internal protein hydration. The relatively small magnitude of both DeltaV(obs) and the activation enthalpy (DeltaH=10-20 kcal mol(-1)) indicates that acrylamide diffusion jumps inside these proteins appear to involve relatively small amplitude structural fluctuations not requiring major unfolding-like transitions. The implication of these findings for the thermodynamic stability of proteins under pressure is discussed.

Acrylamide↗
PubMed1999

Realistic modeling of the denatured states of proteins allows accurate calculations of the pH dependence of protein stability.

Computational techniques based on continuum electrostatics treatments have been successful in predicting and interpreting the pKa values of ionizable amino acids in folded proteins. Despite this progress, efforts to reproduce the pH-dependence of protein stability have met with only limited success: agreement with experimental results has been only qualitative. It has been argued previously that the most likely reason for discrepancies is the presence of residual electrostatic interactions in the unfolded state, which cause pKa values to be shifted from their model compound values. Here we show that by constructing atomistic models of the unfolded state with a simple molecular mechanics protocol that uses the native state as a starting point, much improved reproduction of pH effects on protein stability can be obtained. In contrast, when a fully extended model of the unfolded state is used, no such improvement is obtained, a result that suggests that local interactions with residues nearby in the sequence are not sufficient to properly account for the pKa shifts in the unfolded state. In comparison to model compound values, the pKa values of acidic residues in "native-like" unfolded states are typically found to be shifted downwards by approximately 0.3 pH unit, in good agreement with the average downward shift deduced from experimental measurements. Given its success in the present situation, the protocol employed here for developing simple models of the unfolded state may prove useful in other computer simulation applications.

Animals↗
PubMed2001

Native-state energetics of a thermostabilized variant of ribonuclease HI.

Escherichia coli RNase HI is a well-characterized model system for protein folding and stability. Controlling protein stability is critical for both natural proteins and for the development of engineered proteins that function under extreme conditions. We have used native-state hydrogen exchange on a variant containing the stabilizing mutation Asp10 to alanine in order to determine its residue-specific stabilities. On average, the DeltaG(unf) value for each residue was increased by 2-3 kcal/mol, resulting in a lower relative population of partially unfolded forms. Though increased in stability by a uniform factor, D10A shows a distribution of stabilities in its secondary structural units that is similar to that of E. coli RNase H, but not the closely related protein from Thermus thermophilus. Hence, the simple mutation used to stabilize the enzyme does not recreate the balance of conformational flexibility evolved in the thermophilic protein.

Amides↗
PubMed2001

Detection of errors of interpretation in experiments in enzyme kinetics.

Although modern statistical computing will often be the method of choice for analyzing kinetic data, graphic methods provide an important supplement that ought not to be neglected. Residual plots, or plots of differences between observed and calculated values against variables not expected to be correlated with these differences, permit a rapid judgment of whether data have been correctly interpreted and analyzed. The rapid increase in the frequency with which artificially modified or mutated enzymes are studied is making it less and less safe to assume that enzymes are stable under assay conditions, and there is thus an increased need for methods to check for enzyme stability, and a method for doing this is briefly described. Finally, the Scatchard plot (together with the Eadie-Hofstee plot) is used as an example to discuss the dangers of publishing derived information unaccompanied by any primary data.

Biochemistry↗
PubMed1988

Implication of protein oxidation in protein turnover, aging, and oxygen toxicity.

It is evident from the results summarized here that a variety of MFO systems catalyze the oxidation inactivation of enzymes. This likely involves site-directed Fenton-chemistry in which Fe(II) bound to metal binding sites on the protein undergoes peroxidation to form active oxygen species that convert proximal amino acid residues to carbonyl derivatives. Such oxidation is likely involved in the accumulation of altered enzymes during aging, in premature aging diseases, in the killing of bacteria by neutrophils and in protein turnover. In view of these results, the possibility that protein oxidation is implicated in various diseases, viz, arthritis, pulmonary dysfunction, and carcinogenesis deserves consideration.

Aging↗
PubMed1991

Incorporation and activation of a membrane-bound enzyme in bilayers of liposomes.

The effects of lipid composition and fluidity of lipid bilayers on incorporation and activation of membrane-bound D-fructose dehydrogenase are described in this study. The incorporation of the enzyme into bilayers of small unilamellar vesicles (SUV) made of several phospholipids resulted in enzyme activation with magnitudes higher than that observed in the presence of Triton X-100, indicating that this higher activation is due to lipid-protein interaction. The activity was highest in the presence of SUV formed by the addition of 10% DL-alpha-dipalmitoylphosphatidylethanolamine to L-alpha-dimyristoylphosphatidylcholine, which resulted in eightfold higher activation compared with that of the enzyme in its free state. This activation did not appear to be due to the degree of incorporation of the enzyme, indicating that incorporation is distinct from the activation event. Thus, it is probably the lipid environment that leads to higher activation of the enzyme. A break in the Arrhenius plot of the activity of the membrane-bound enzyme at temperatures close to the phase transition of the phospholipid implies that changes in the physical state of the lipid bilayer influence the enzyme activity. Furthermore, immobilization of D-fructose dehydrogenase, previously adsorbed to SUV, on urethane prepolymer also resulted in about eightfold higher activation than that of the free enzyme.

Carbohydrate Dehydrogenases↗
PubMed1993

Characterization of immobilized enzymes in polyurethane foams in a dynamic bed reactor.

beta-D-Galactosidase (E 3.2.1.23) from Aspergillus oryzae was immobilized with polyurethane foam (PUF). Among several immobilization methods attempted in this work, the immobilized enzyme preparation by in-situ co-polymerization between enzyme and prepolymer HYPOL 3000 showed the highest activity. The intrinsic kinetics of PUF-immobilized enzyme was determined in a dynamic bed reactor, used to increase transport rates. The immobilization mechanism in PUF was studied by measurements of immobilized enzyme kinetics and by using scanning electron microscopy combined with immuno-gold labeling techniques. The results showed that immobilization was predominantly by covalent bonding between primary amino groups of beta-D-galactosidase and isocyanate groups of the prepolymers. Entrapment in the PUF micropores assisted the immobilization of enzymes, and adsorption on the surface of macropores was not important for immobilization. The bicinchoninic acid method was applied for the determination of PUF loading capacity and specific enzyme activity and used to determine enzyme deactivation during immobilization.

Adsorption↗
PubMed1978

Studies on the glycosidases of semen: purification and properties of alpha-D-mannopyranosidase from goat seminal plasma.

Alpha D-mannosidase activity in goat semen was observed to be distributed in sperm and seminal plasma. In sperm the enzyme, present in soluble and bound forms, was located within the acrosome. The bound enzyme was associated with the denuded sperm. Seminal plasma alpha-mannosidase was purified 100-fold and the final preparation was shown to be homogeneous by polyacrylamide and SDS gel electrophoresis and on isoelectric focusing. The molecular weight of the enzyme, determined by gel filtration and disc electrophoresis in the presence of SDS, was 220,000. The isoelectric pH was 7.42 and the amino acid composition is reported. alpha-Mannosidase catalyzed the hydrolysis of both synthetic and natural substrates. The Km of p-nitrophenyl alpha-D-mannoside and alpha-methyl D-mannoside were 0.695 mM and 71.9 mM at pH 4.0, the optimum pH. The natural substrates were hydrolysed to varying degrees. Zn2+ was not essential though it activated the enzyme activity over longer incubations. The enzyme was observed to be more stable at wider pH range in the presence of Zn2+ than in its absence. EDTA which did not affect the enzyme activity has effect on enzyme stability similar to Zn.2+ Seminal alpha-mannosidase is not a zinc metalloenzyme but is activated by Zn2+.

Acrosome↗
PubMed1984

Cloning of a Bacillus subtilis restriction fragment complementing auxotrophic mutants of eight Escherichia coli genes of arginine biosynthesis.

Following shotgun cloning of EcoRI fragments of Bacillus subtilis 168 chromosomal DNA in pBR322 a hybrid plasmid, pUL720, was isolated which complements Escherichia coli K12 mutants defective for argA, B, C, D, E, F/I, carA and carB. Restriction analysis revealed that the insert of pUL720 comprises four EcoRI fragments, of sizes 12.0, 6.0, 5.0 and 0.8 kbp. Evidence was obtained from subcloning, Southern blot hybridisation, enzyme stability studies and transformation of B. subtilis arginine auxotrophs that the 12 kbp EcoRI fragment carries all the arg genes. It proved impossible to subclone the intact fragment in isolation in the multicopy vectors pBR322, pBR325 or pACYC184, and although it could be subcloned in the low copy vector pGV1106, propagation of the hybrid rapidly resulted in the selection of stable derivatives carrying, near one end, an insertion of 1 kbp of DNa originating from the E. coli chromosome. These and other stable derivatives resulting from subcloning the 12 kbp EcoRI fragment have lost only the ability to complement for E. coli argC, and it is suggested that sequences located close to the equivalent of argC are involved in destabilising plasmids bearing the 12 kbp fragment in E. coli in a copy number dependent manner.

Arginine↗
PubMedDate not supplied

Comparative beta-lactamase hydrolysis of and inhibition by 7-aminothiazolyl alpha-methoxyimino cephalosporins.

Six 7-aminothiazolyl alpha-methoxyimino cephalosporins were found to be most variable in their ability to resist Type IV beta-lactamases (ceftizoxime, most stable and ceftriaxone, least stable), to inhibit Type I beta-lactamases (cefotaxime, the best inhibitor and desacetyl-cefotaxime, the least inhibition), to inhibit Enterobacteriaceae as measured by their minimum inhibitory concentrations (MICs), and in their published serum half-lives. The 3-position substituents appear to exert significant physical-chemical effects that influence the pharmacology, molecular enzyme stability and the antimicrobial activity of these compounds. However, we conclude that these differences are of minimal clinical consequence and that other more relevant factors for in vivo drug selection should be considered, including proven clinical efficacy and cost.

Cefmenoxime↗
PubMed1996

Early detection of anastomotic leaks after low anterior resection of the rectum.

PURPOSE: Lysozyme destroys the mucopolysaccharide chains of the cell wall of gram-negative bacteria. It is a component of local defense and is formed in macrophages. Determination of lysozyme content in the wound seems to be the most reliable method for early recognition of wound infection. METHODS: In a prospective randomized study on the efficacy of single vs. double staple technique in anterior rectum resection, the effluent from the pelvic drain was examined with regard to its lysozyme activity. RESULTS: Lysozyme activity in drained secretion remained stable for more than 24 hours at room temperature. When the single staple technique was used, enzyme activity was sharply increased (mean, 9.6 mg/dl on the first postoperative day) compared with the double staple technique (mean, 5.5 mg/dl on the first postoperative day). The difference was statistically significant (P < 0.0001). Mean lysozyme activity was increased in those patients with clinically (18 mg/dl on the first postoperative day) and radiologically (15.3 mg/dl on the first postoperative day) detected dehiscence (P < 0.0001). CONCLUSION: Lysozyme determination may be reproduced by detection of enzyme stability in drained secretion. Determination of lysozyme content seems to be a new possibility for early recognition of anastomotic dehiscence.

Aged↗