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Biomedical subjects

A M Klibanov

Publications and source records attributed to A M Klibanov.

At least 37 records · Page 2Linked to original sources
PubMed1999

Structure, thermostability, and conformational flexibility of hen egg-white lysozyme dissolved in glycerol.

Hen egg-white lysozyme dissolved in glycerol containing 1% water was studied by using CD and amide proton exchange monitored by two-dimensional 1H NMR. The far- and near-UV CD spectra of the protein showed that the secondary and tertiary structures of lysozyme in glycerol were similar to those in water. Thermal melting of lysozyme in glycerol followed by CD spectral changes indicated unfolding of the tertiary structure with a Tm of 76.0 +/- 0.2 degreesC and no appreciable loss of the secondary structure up to 85 degreesC. This is in contrast to the coincident denaturation of both tertiary and secondary structures with Tm values of 74.8 +/- 0.4 degreesC and 74.3 +/- 0.7 degreesC, respectively, under analogous conditions in water. Quenched amide proton exchange experiments revealed a greater structural protection of amide protons in glycerol than in water for a majority of the slowly exchanging protons. The results point to a highly ordered, native-like structure of lysozyme in glycerol, with the stability exceeding that in water.

Animals↗
PubMed1998

Comparison of x-ray crystal structures of an acyl-enzyme intermediate of subtilisin Carlsberg formed in anhydrous acetonitrile and in water.

The x-ray crystal structures of trans-cinnamoyl-subtilisin, an acyl-enzyme covalent intermediate of the serine protease subtilisin Carlsberg, have been determined to 2.2-A resolution in anhydrous acetonitrile and in water. The cinnamoyl-subtilisin structures are virtually identical in the two solvents. In addition, their enzyme portions are nearly indistinguishable from previously determined structures of the free enzyme in acetonitrile and in water; thus, acylation in either aqueous or nonaqueous solvent causes no appreciable conformational changes. However, the locations of bound solvent molecules in the active site of the acyl- and free enzyme forms in acetonitrile and in water are distinct. Such differences in the active site solvation may contribute to the observed variations in enzymatic activities. On prolonged exposure to organic solvent or removal of interstitial solvent from the crystal lattice, the channels within enzyme crystals are shown to collapse, leading to a drop in the number of active sites accessible to the substrate. The mechanistic and preparative implications of our findings for enzymatic catalysis in organic solvents are discussed.

Acetonitriles↗
PubMed1998

Organic solvent binding to crystalline subtilisin1 in mostly aqueous media and in the neat solvents.

The X-ray crystal structures of the protease subtilisin Carlsberg in 40% acetonitrile and in 20% dioxane have been determined to at least 2.3 A resolution, and their solvent binding patterns have been compared to those observed in the neat organic solvents. The structures of the protein in the two aqueous-organic mixtures are essentially the same as in pure water, acetonitrile, and dioxane. Interestingly, the enzyme-bound organic solvent molecules tend to congregate in the active site. Three of the five bound acetonitrile molecules observed in the structure of subtilisin in 40% acetonitrile are situated in the enzyme active site, as is the single enzyme-bound dioxane molecule observed in 20% dioxane (whose location is distinct from that of any bound acetonitrile molecule). Furthermore, the organic solvent molecules detected in the enzyme active site in the aqueous-organic mixtures are in the same locations as in the structures in the corresponding neat organic solvents.

Acetonitriles↗
PubMed1998

Molecular-modeling calculations of enzymatic enantioselectivity taking hydration into account.

A new molecular-modeling methodology has been applied to explain enzymatic enantioselectivity in water. This methodology, which combines vacuum molecular mechanics and the continuum solvation method, should provide a more realistic view of the solvent-enzyme and solvent-substrate interactions than the heretofore used approaches involving the vacuum molecular mechanics only. The methodology described herein has been validated using the experimental data on alpha-chymotrypsin's enantioselectivity in the hydrolysis of four chiral substrates. The reasons why the vacuum molecular mechanics, although not taking hydration into account, still in most cases provide a satisfactory approximation of reality are discussed.

Chymotrypsin↗
PubMed1998

On enzymatic activity in organic solvents as a function of enzyme history.

Catalytic activities of alpha-chymotrypsin and subtilisin Carlsberg in various hydrous organic solvents were measured as a function of how the enzyme suspension had been prepared. In one method, lyophilized enzyme was directly suspended in the solvent containing 1% water. In another, the enzyme was precipitated from its aqueous solution by a 100-fold dilution with an anhydrous solvent. In most cases, the reaction rate in a given nonaqueous enzymatic system strongly (up to an order of magnitude) depended on the mode of enzyme preparation. The magnitude of this dependence was markedly affected by the nature of the solvent and enzyme. A mechanistic hypothesis proposed to explain the observed dependencies was verified in additional experiments in which the water contents and enzyme history were further varied.

Chymotrypsin↗
PubMed1998

Deterioration of lyophilized pharmaceutical proteins.

The successful use of proteins in pharmaceutical and other commercial applications requires close examination of their relative fragility. Because of the resultant enhanced stability, proteins are often formulated in the solid state, even though dehydration tends to alter their structure. Even in the solid form, however, proteins may become inactivated due to various deleterious processes, e.g., aggregation. This review focuses on such mechanisms, with an emphasis on case studies conducted in our laboratory. Proteins which have both disulfide bonds and free thiols may aggregate via thiol-disulfide exchange, and this process may be facilitated by lyophilization-induced structural perturbations. For proteins possessing disulfides but not free thiols, aggregation also may occur when native disulfides are beta-eliminated, thus giving rise to thiol species which can catalyze disulfide scrambling. Other deleterious processes have also been uncovered, including a formaldehyde-mediated aggregation of formalinized vaccines. It is illustrated how knowledge of such deterioration pathways makes possible the rational development of stable solid protein formulations.

Drug Stability↗
PubMed1997

Correct protein folding in glycerol.

Water is the natural medium for protein folding, which is also used in all in vitro studies. In the present work, we posed, and answered affirmatively, a question of whether it is possible to fold correctly a typical protein in a nonaqueous solvent. To this end, unfolded and reduced hen egg-white lysozyme was refolded and reoxidized in glycerol containing varying amounts of water. The unfolded/reduced enzyme was found to regain spontaneously substantial catalytic activity even in the nearly anhydrous solvent; for example, the refolding yield in 99% glycerol was still some one-third of that in pure water, and one-half of that was regained even in 99.8% glycerol. The less than full recovery of the enzymatic activity in glycerol is, as in water, because of competing protein aggregation during the refolding. Lysozyme reoxidation in glycerol was successfully mediated by two dissimilar oxidizing systems, and the refolding yield was markedly affected by the pH of the last aqueous solution before the transfer into glycerol. No recovery of the lysozyme activity was observed when the refolding/reoxidation reaction was carried out in the denaturing solvent dimethyl sulfoxide. This study paves the way for a systematic investigation of the solvent effect on protein folding and demonstrates that water is not a unique milieu for this process.

Animals↗
PubMed1997

The crystal structure of subtilisin Carlsberg in anhydrous dioxane and its comparison with those in water and acetonitrile.

The x-ray crystal structure of the serine protease subtilisin Carlsberg in anhydrous dioxane has been determined to 2.6-A resolution. The enzyme structure is found to be nearly indistinguishable from the structures previously determined in water and acetonitrile. Small changes in the side-chain conformations between the dioxane and water structures are of the same magnitude as those observed between two structures in different aqueous systems. Seven enzyme-bound dioxane molecules have been detected, each potentially forming at least one hydrogen bond with a subtilisin hydrogen-bond donor or bound water. Two of the bound dioxane molecules are in the active-site region, one in the P2 and another bridging the P1' and P3' pockets. The other five dioxane molecules are located on the surface of subtilisin at interprotein crystal contacts. The locations of the bound solvent in the dioxane structure are distinct from those in the structures in acetonitrile and in water.

Acetonitriles↗
PubMed1997

Why are enzymes less active in organic solvents than in water?

In order to exploit fully the biotechnological opportunities afforded by nonaqueous enzymology, the issue of often drastically diminished enzymatic activity in organic solvents compared with that in water must be addressed and resolved. Recent studies have made great strides towards elucidating causes of this phenomenon of activity loss. None of these causes is insurmountable; by designing strategies that systematically target them, enzymatic activity in organic solvents can be readily enhanced by multiple orders of magnitude and ultimately brought to the aqueous-like level.

Biomechanical Phenomena↗
PubMed1996

The secondary structure and aggregation of lyophilized tetanus toxoid.

Tetanus toxoid (TT), the vaccine for tetanus, is an important protein antigen and candidate for sustained release from polymeric matrices. During administration from the latter, the solid (e.g., lyophilized) protein will be exposed to elevated levels of temperature and moisture, conditions which trigger its aggregation. To examine the connection between this aggregation and the structure of the TT molecule in the solid state, Fourier-transform infrared (FTIR) spectroscopy was employed to determine the secondary structure of TT in the presence of various excipients. We found that excipient-free TT undergoes a significant alteration (mostly reversible) in the secondary structure during lyophilization. Specifically, more than half the total alpha-helix content was lost with a concomitant increase in beta-sheet structure. The extent of structural alterations in the presence of 1:5 (g:g protein) NaCl, sorbitol, or poly-(ethylene glycol), did not correlate with stability conferred towards moisture-induced aggregation. These results suggest that the degree of retention of the native protein structure in the dry state is not a general predictor of stability for the "wetted" solid within polymer controlled-release vehicles.

Excipients↗
PubMed1995

Lyophilization-induced reversible changes in the secondary structure of proteins.

Changes in the secondary structure of some dozen different proteins upon lyophilization of their aqueous solutions have been investigated by means of Fourier-transform infrared spectroscopy in the amide III band region. Dehydration markedly (but reversibly) alters the secondary structure of all the proteins studied, as revealed by both the quantitative analysis of the second derivative spectra and the Gaussian curve fitting of the original infrared spectra. Lyophilization substantially increases the beta-sheet content and lowers the alpha-helix content of all proteins. In all but one case, proteins become more ordered upon lyophilization.

Amides↗
PubMed1995

Stabilization of tetanus and diphtheria toxoids against moisture-induced aggregation.

The progress toward single-dose vaccines has been limited by the poor solid-state stability of vaccine antigens within controlled-release polymers, such as poly(lactide-co-glycolide). For example, herein we report that lyophilized tetanus toxoid aggregates during incubation at 37 degrees C and elevated humidity--i.e., conditions relevant to its release from such systems. The mechanism and extent of this aggregation are dependent on the moisture level in the solid protein, with maximum aggregation observed at intermediate moisture contents. The main aggregation pathway is consistent with formaldehyde-mediated cross-linking, where reactive electrophiles created and stored in the vaccine upon formalinization (exposure to formaldehyde during vaccine preparation) react with nucleophiles of a second vaccine molecule to form intermolecular cross-links. This process is inhibited by the following: (i) succinylating the vaccine to block reactive amino groups; (ii) treating the vaccine with sodium cyanoborohydride, which presumably reduces Schiff bases and some other electrophiles created upon formalinization; and (iii) addition of low-molecular-weight excipients, particularly sorbitol. The moisture-induced aggregation of another formalinized vaccine, diphtheria toxoid, is also retarded by succinylation, suggesting the generality of this mechanism for formalinized vaccines. Hence, mechanistic stability studies of the type described herein may be important for the development of effective single-dose vaccines.

Bacterial Vaccines↗
PubMed1995

Fourier-transform infrared spectroscopic investigation of protein stability in the lyophilized form.

Upon the removal of water, proteins undergo a major, reversible rearrangement of their secondary structure, as revealed by FTIR spectroscopy. We have found herein that for recombinant human albumin (rHA) the extent of this structural change does not depend significantly either on the composition of the aqueous solution prior to lyophilization (protein concentration, pH, and the presence of excipients such as dextran or NaCl) or on the mode of dehydration (lyophilization, spray drying, or rotary evaporation), even though these factors profoundly affect rHA's solid-state stability against moisture-induced aggregation. In all cases, the alpha-helix content of rHA drops from 58% in solution to 25-35% in the dehydrated state, the beta-sheet content rises from 0 to 10-20%, and unordered structures increase from 40% to 50-60%. We have also investigated another model protein, hen egg-white lysozyme, and confirmed that it too undergoes a significant alteration of the secondary structure upon lyophilization. The extent of this structural reorganization has been found to be insensitive to the pH of the aqueous solution prior to lyophilization from pH 1.9 to 5.1, even though the thermal transition temperature (Tm) in aqueous solution over this range varies by 30 degrees C.

Dextrans↗
PubMed1995

Cloning of an organic solvent-resistance gene in Escherichia coli: the unexpected role of alkylhydroperoxide reductase.

Although bacterial strain able to grow in the presence of organic solvents have been isolated, little is known about the mechanism of their resistance. In the present study, 1,2,3,4-tetrahydronaphthalene (tetralin), a solvent with potential applications in industrial biocatalysis, was used to select a resistant mutant of Escherichia coli. The resultant mutant strain was tested for resistance to a wide range of solvents of varying hydrophobicities and was found to be resistant not only to tetralin itself but also to cyclohexane, propylbenzene, and 1,2-dihydronaphthalene. A recombinant library from mutant DNA was used to clone the resistance gene. The sequence of the cloned locus was determined and found to match the sequence of the previously described alkylhydroperoxide reductase operon ahpCF. The mutation was localized to a substitution of valine for glycine at position 142 in the coding region of ahpC, which is the gene encoding the catalytic subunit of the enzyme. The ahpC mutant was found to have an activity that was three times that of the wild type in reducing tetralin hydroperoxide to 1,2,3,4-tetrahydro-1-naphthol. We conclude that the toxicity of such solvents as tetralin is caused by the formation of toxic hydroperoxides in the cell. The ahpC mutation increases the activity of the enzyme toward hydrophobic hydroperoxides, thereby conferring resistance. The ahpC mutant was sensitive to the more hydrophilic solvents xylene and toluene, suggesting that there are additional mechanisms of solvent toxicity. Mutants resistant to a mixture of xylene and tetralin were isolated from the ahpC mutant but not from the wild-type strain.

Base Sequence↗
PubMed1995

Transport of proteins dissolved in organic solvents across biomimetic membranes.

Using lipid-impregnated porous cellulose membranes as biomimetic barriers, we tested the hypothesis that to afford effective transmembrane transfer of proteins and nucleic acids, the vehicle solvent should be able to dissolve both the biopolymers and the lipids. While the majority of solvents dissolve one or the other, ethanol and methanol were found to dissolve both, especially if the protein had been lyophilized from an aqueous solution of a pH remote from the protein's isoelectric point. A number of proteins, as well as RNA and DNA, dissolved in these alcohols readily crossed the lipidized membranes, whereas the same biopolymers placed in nondissolving solvents (e.g., hexane and ethyl acetate) or in those unable to dissolve lipids (e.g., water and dimethyl sulfoxide) exhibited little transmembrane transport. The solubility of biopolymers in ethanol and methanol was further enhanced by complexation with detergents and poly(ethylene glycol); significant protein and nucleic acid transport through the lipidized membranes was observed from these solvents but not from water.

Biological Transport↗
PubMed1995

Aggregation of a lyophilized pharmaceutical protein, recombinant human albumin: effect of moisture and stabilization by excipients.

In the presence of water vapor at 37 degrees C, lyophilized recombinant human albumin (rHA) undergoes intermolecular thiol-disulfide interchange, eventually forming high-molecular-weight, water-insoluble aggregates. The relationship between the extent of aggregation and the water content of the lyophilized protein was bell-shaped, with maximum aggregation (over 80% after one day) at approximately 50 g water per 100 g dry protein, corresponding to incubation at 96% relative humidity. Nineteen different excipients were co-lyophilized with rHA to test their ability to inhibit aggregation under these conditions. These compounds included low- and high-molecular-weight sugars, as well as various organic acids (amino, hydroxy, and aliphatic), and the simple inorganic salt sodium chloride. Seven of them afforded complete stabilization of rHA against moisture-induced aggregation. The stabilizing potency of the excipients correlated with their water-sorbing capability, presumably due to increasing the moisture level in the vicinity of rHA.

Albumins↗
PubMed1994

The solvent dependence of enzyme specificity.

The discovery that enzymes possess catalytic activity in organic solvents has made it possible to address the question of the influence of the reaction medium on enzymatic specificity. Recently, the substrate specificity, enantioselectivity, prochiral selectivity, regioselectivity, and chemoselectivity of enzymes have been found to dramatically depend on the nature of the solvent. This review discusses the scope, possible mechanisms, and implications of this phenomenon, as well as directions of future research in the area.

Animals↗