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Biomedical subjects

A M Klibanov

Publications and source records attributed to A M Klibanov.

At least 55 records · Page 3Linked to original sources
PubMed1994

X-ray crystal structure of cross-linked subtilisin Carlsberg in water vs. acetonitrile.

The crystal structure of subtilisin Carlsberg lightly cross-linked with glutaraldehyde was solved in aqueous solution by X-ray crystallography at 2.3 A resolution. It was found to be virtually identical to the recently determined (Fitzpatrick, P.A., Steinmetz, A.C.U., Ringe, D.A. & Klibanov, A.M. (1993) Proc. Natl. Acad. Sci. USA 90, 8653) structure of the cross-linked enzyme in anhydrous acetonitrile. The latter structure was found to be significantly more rigid than in water, as reflected by their average B factors. The numbers of subtilisin-bound water molecules in the two structures are similar (114 and 99 in water and in acetonitrile, respectively), but the locations of some half of these bound waters are distinct.

Acetonitriles↗
PubMed1994

Detergent-enabled transport of proteins and nucleic acids through hydrophobic solvents.

It is demonstrated that proteins and nucleic acids can be transported through hydrophobic organic solvents (liquid membranes) via nonspecific complex formation with detergents, whereas no macromolecule transport is observed without the latter. A protein (or a nucleic acid) first interacts with an oppositely charged detergent due to hydrophobic ion pairing in the aqueous feed phase. The resultant hydrophobic complex readily partitions into an organic solvent and then into the aqueous receiver phase, where it dissociates. Experiments with (i) different detergent/protein molar ratios, (ii) a range of unrelated organic solvents as liquid membranes, and (iii) homologous detergents with hydrophobic tails of varying lengths indicate that the protein flux through the membrane directly correlates with the partitioning of the protein-detergent complexes from the aqueous feed into the organic phase. Very little protein transport was detected at detergent concentrations above the critical micelle concentration, suggesting that individual detergent molecules, rather than micelles, play the key role. The rate of the detergent-enabled protein transport is not a function of the protein molecular weight, provided that enough detergent molecules bind to make the complex sufficiently hydrophobic; e.g., bovine serum albumin can be transported faster than insulin, which is less than 1/10th of its size.

Amino Acid Sequence↗
PubMed1994

Solid-phase aggregation of proteins under pharmaceutically relevant conditions.

In order to successfully employ proteins as pharmaceuticals, it is essential to understand mechanistically the stability issues relevant to their formulation and delivery. Various deleterious processes may occur in protein formulations, thereby diminishing their therapeutic value. This review focuses upon one aspect of this problem, namely aggregation of solid proteins under pharmaceutically relevant conditions (elevated temperature and water activity). Strategies to pursue such studies are presented with an emphasis on a mechanistic analysis of aggregate formation. Both covalent and noncovalent aggregation pathways have been elucidated. Proteins that contain disulfide bonds as well as free thiol residues may aggregate via thiol-disulfide interchange. For proteins which contain disulfides but not free thiol residues, intermolecular disulfide bonding may still occur when intact disulfides undergo beta-elimination, yielding free thiols which can catalyze disulfide scrambling. Finally, proteins containing no cysteine/cystine residues may aggregate by other covalent pathways or by noncovalent routes. On the basis of these pathways, some rational stabilization strategies have been proposed and verified. Ultimately, application of this knowledge should lead to more stable and effective pharmaceutical protein formulations.

Chemical Phenomena↗
PubMed1994

Moisture-induced aggregation of lyophilized insulin.

A critical problem in the storage and delivery of pharmaceutical proteins is aggregation in the solid state induced by elevated temperature and moisture. These conditions are particularly relevant for studies of protein stability during accelerated storage or for proteins loaded in polymeric delivery devices in vivo. In the present investigation, we have found that, when exposed to an environment simulating these conditions, lyophilized insulin undergoes both covalent and noncovalent aggregation. The covalent process has been elucidated to be intermolecular thiol-catalyzed disulfide interchange following beta-elimination of an intact disulfide bridge in the insulin molecule. This process is accelerated by increasing the temperature and water content of the insulin powder or by performing lyophilization and/or dissolution of insulin in alkaline media. The aggregation can be ameliorated by the presence of Cu2+, which presumably catalyzes the oxidization of free thiols. The water sorption isotherm for insulin reveals that the extent of aggregation directly correlates with the water uptake by the lyophilized insulin powder, thus pointing to the critical role of protein conformational mobility in the aggregation process.

Animals↗
PubMed1993

Enzyme crystal structure in a neat organic solvent.

The crystal structure of the serine protease subtilisin Carlsberg in anhydrous acetonitrile was determined at 2.3 A resolution. It was found to be essentially identical to the three-dimensional structure of the enzyme in water; the differences observed were smaller than those between two independently determined structures in aqueous solution. The hydrogen bond system of the catalytic triad is intact in acetonitrile. The majority (99 of 119) of enzyme-bound, structural water molecules have such a great affinity to subtilisin that they are not displaced even in anhydrous acetonitrile. Of the 12 enzyme-bound acetonitrile molecules, 4 displace water molecules and 8 bind where no water had been observed before. One-third of all subtilisin-bound acetonitrile molecules reside in the active center, occupying the same region (P1, P2, and P3 binding sites) as the specific protein inhibitor eglin c.

Acetonitriles↗
PubMed1992

Solid-state NMR assessment of enzyme active center structure under nonaqueous conditions.

By using solid-state NMR spectroscopy, the integrity of the active center of alpha-chymotrypsin was investigated under a variety of nonaqueous conditions. Specifically, 13C cross-polarization/magic angle spinning NMR was used to analyze the ability of alpha-chymotrypsin to stabilize a transition state intermediate analog after freezing, drying, and addition of organic solvents (both anhydrous and hydrated) to the resultant powder. Lyophilization disrupted 42 +/- 5% of the active centers; it was determined that this occurred during drying, as opposed to freezing. Seven anhydrous solvents caused 0-50% additional disruption, which occurred immediately on addition of the solvent to the enzyme powder. The extent of structural integrity loss correlated with the solvent hydrophobicity, indicating that further dehydration, i.e. stripping of water retained by the enzyme during lyophilization, was the cause. Enzyme samples prepared with lyoprotecting additives, sucrose and ammonium sulfate, exhibited varying degrees of stabilization against the drying step of lyophilization. Moreover, when hydrophilic anhydrous solvents, which had the highest propensity to strip bound water, were added to the resultant enzyme powders, no additional damage occurred.

Amino Acid Sequence↗
PubMed1992

Alterations in the structure of proteins that cause their irreversible inactivation.

A variety of changes in environmental conditions (temperature, pH, salts, solvents, etc.) can cause protein inactivation. However, the mechanisms of irreversible protein inactivation often follow common pathways. Using heat stress as an example, the conformational and covalent processes leading to the irreversible thermo-inactivation of enzymes will be described. In particular, work in our laboratory has identified several chemical reactions which contribute to enzyme thermo-inactivation: cystine destruction, thiol-catalysed disulfide interchange, oxidation of cysteine residues, deamidation of asparagine and glutamine residues, and hydrolysis of peptide bonds at aspartic acid residues. Implications of this work for the stability of proteins during, and following, the lyophilization process will be addressed.

Amino Acids↗
PubMed1991

Kinetics of insulin aggregation in aqueous solutions upon agitation in the presence of hydrophobic surfaces.

The stability of protein-based pharmaceuticals (e.g., insulin) is important for their production, storage, and delivery. To gain an understanding of insulin's aggregation mechanism in aqueous solutions, the effects of agitation rate, interfacial interactions, and insulin concentration on the overall aggregation rate were examined. Ultraviolet absorption spectroscopy, high-performance liquid chromatography, and quasielastic light scattering analyses were used to monitor the aggregation reaction and identify intermediate species. The reaction proceeded in two stages; insulin stability was enhanced at higher concentration. Mathematical modeling of proposed kinetic schemes was employed to identify possible reaction pathways and to explain greater stability at higher insulin concentration.

Adsorption↗
PubMed1991

Protein separation and purification in neat dimethyl sulfoxide.

Pure DMSO (instead of water) is used as the reaction medium for protein separations. It is shown that common extracellular proteins (i) have high solubility in DMSO (1-50 mg/ml), (ii) do not irreversibly inactivate in this solvent, and (iii) can adsorb onto carboxymethyl cellulose in DMSO and be subsequently fully desorbed in this solvent by inorganic salts. Ion-exchange chromatography on this resin in DMSO has been used to purify bovine pancreatic trypsin and to separate it from hen egg-white lysozyme in their mixture. Another approach to protein separation in DMSO, fractional precipitation with ethyl acetate (which does not dissolve proteins), has been verified with a mixture of bovine pancreatic chymotrypsinogen and chicken egg ovalbumin.

Animals↗
PubMed1991

Regioselective modification of Lysine's amino groups by proteases.

The regioselectivity of six serine proteases for the amino groups of lysine was investigated. alpha-Chymotrypsin showed a preference for the alpha-amino group, although the selectivity can be varied 10-fold depending on the reaction medium. Subtilisin Carlsberg and other bacterial proteases were highly specific for the epsilon-amino group, regardless of the reaction medium: they were used as catalysts for the preparative synthesis of isopeptides in anhydrous tert-amyl alcohol.

Amino Acid Sequence↗
PubMed1990

Production of abiotic receptors by molecular imprinting of proteins.

When a protein is dissolved in a concentrated aqueous solution of a multifunctional organic compound, freeze-dried, and washed with an anhydrous organic solvent to remove the ligand, the resultant "imprinted" protein preparation binds up to 30-fold more of the template compound in anhydrous solvents than the nonimprinted protein in the same solvent (and both proteins in water). These artificial receptors exhibit marked ligand selectivity as well as stability in anhydrous media. This phenomenon of molecular imprinting, demonstrated for several unrelated proteins and ligands, may be helpful in the development of unique bioadsorbents and, potentially, new biocatalysts.

Carboxylic Acids↗
PubMed1989

Stabilization of poliovirus against heat inactivation.

Fatty acids and related compounds, as well as many salts, stabilize poliovirus against heat inactivation. Addition of myristate to poliovirus prevents heat-induced conformational changes which are detected by trypsin degradation of the virion. Using equilibrium dialysis, we found that several molecules of myristate bind per virion. The relative stabilizing potencies of the salts can be explained by the Hofmeister effect.

Fatty Acids, Nonesterified↗
PubMed1989

Antibody-antigen binding in organic solvents.

We describe, for the first time, the action of antibodies in anhydrous organic solvents. It has been demonstrated that the binding of a hapten, 4-aminobiphenyl, to the immobilized monoclonal antibody 2E11 is strong and specific not only in water but also in a variety of non-aqueous media. Further, the strength of interaction between antibody and hapten has been related to the hydrophobicity of the solvent: the more hydrophobic the solvent, the weaker the protein-ligand interaction.

Aminobiphenyl Compounds↗
PubMed1989

A colorimetric method for the enzymatic analysis of gases: the determination of ethanol and formaldehyde vapors using solid alcohol oxidase.

A novel enzymatic approach to the direct determination of ethanol vapors in the gas phase is described. The system is composed of alcohol oxidase, peroxidase, and the color indicator 2,6-dichloroindophenol dispersed on microcrystalline cellulose (avicel). Simple devices are developed for the semiquantitative determination of ethanol in the breath. The devices are optimized to produce a sharp color change at a set time of 1 min for ethanol concentrations above the legal limit for driving (kinetic method) or a stable final color after 5 min (equilibrium method). Such color changes are detectable by simple visual observation. Using TLC plastic sheets and a transmittance densitometer, the system can also be used as a quantitative method for the determination of ethanol or formaldehyde vapors. Dehydrated enzymes may be useful for the analysis of hazardous gases.

2,6-Dichloroindophenol↗
PubMed1989

Enzymatic catalysis in anhydrous organic solvents.

Not only do enzymes work vigorously in anhydrous organic media, but in this unnatural milieu they acquire remarkable properties such as greatly enhanced stability, radically altered substrate and enantiomeric specificities, molecular memory, and the ability to catalyse unusual reactions.

Catalysis↗
PubMed1988

Inhibitor-induced enzyme activation in organic solvents.

The enzymatic activity of the protease subtilisin in anhydrous organic solvents can be dramatically increased by pretreating the enzyme before it is placed in the nonaqueous medium. For instance, lyophilization of subtilisin from aqueous solution containing competitive inhibitors (followed by their removal) created an enzyme which was up to 100 times more active than the enzyme lyophilized in the absence of such ligands. This phenomenon of ligand-induced "enzyme memory" also extends to the stability, affinity, and substrate specificity of subtilisin in organic solvents.

Binding, Competitive↗
PubMed1988

The effect of water on enzyme action in organic media.

Three model, unrelated enzymes (yeast alcohol oxidase, mushroom polyphenol oxidase, and horse liver alcohol dehydrogenase) were found to be catalytically active in a variety of organic solvents. For all enzymes and solvents tested, the enzymatic activity greatly increased upon an increase in the water content in the solvents (which always remained below the solubility limit). Much less water was required to reach the maximal activity in hydrophobic solvents than in their hydrophilic counterparts. However, when the catalytic activity was plotted versus the amount of water bound to the enzymes, a common pattern emerged for different solvents. These data suggest that the effect of organic solvents on an enzyme is primarily due to interactions with the enzyme-bound, essential layer of water rather than with the enzyme itself. At optimal water contents, enzymatic activities in organic solvents were in the range from 20 to 40% of those in aqueous solutions. From experiments on (i) replacement of water with other hydrogen bond-forming additives and (ii) titration of enzyme amino groups in an organic medium, as well as the literature data on dehydrated enzymes, it is concluded that the water required by enzymes in nonaqueous solvents provides them with sufficient conformational flexibility needed for catalysis.

1-Butanol↗