Infrared spectroscopic studies of calcium binding to inhibited beta-trypsins.

Fourier transform infrared spectroscopy was used to examine the effect of calcium binding on the secondary structure of two inhibited bovine beta-trypsins. Neither the diisopropyl fluorophosphate- nor benzamidine-inhibited forms showed detectable secondary structure perturbation upon calcium binding at pD 6.9 and 5.0, respectively. Considered in light of the recent assignment of an amide I' band to the autolysis loop of bovine beta-trypsin, these results contradict the generally held hypothesis that calcium slows trypsin autolysis by induction of a conformational change at this site and support the recent contention that the mechanism of action has a specific electrostatic origin. In addition, the appearance of a band at 1699 cm-1 in the benzamidine-inhibited form can be interpreted as resulting from the NC-N stretching vibrations of the amidinium moiety, which the observed crystal structure indicates is hydrogen-bonded to the carboxyl group of active-site Asp-189.

High level expression of human leukemia inhibitory factor (LIF) from a synthetic gene in Escherichia coli and the physical and biological characterization of the protein.

LIF is a multi-functional cytokine that elicits effects on a broad range of cell types. In this report, we present the high level expression of human LIF (hLIF) from a chemically synthesized gene template in Escherichia coli where it comprises up to 25% of the cellular protein. The recombinant hLIF, after purification and folding, was examined using CD, FTIR spectroscopy and light scattering. CD and FTIR spectra showed that the hLIF is an alpha-helical protein and has a distinct tertiary structure. The IFTR spectrum resembles that of other four helical bundle proteins including G-CSF and IL-6. Light scattering analysis indicated that it is a monomeric protein, distinguishing it from M-CSF and interferon gamma, which also belong to the class of four helical bundle proteins but are dimeric. Recombinant hLIF was assayed for its activity on the murine leukemic cell line, M-1 as well as on human leukemic cell line, ML-1. It inhibited the growth of M-1 cells and differentiated them towards macrophages. However, it did not have any differentiation inducing effect on human leukemic cell lines alone or in combination with other cytokines.

Factors affecting short-term and long-term stabilities of proteins.

Proteins are marginally stable and, hence, are readily denatured by various stresses encountered in solution, or in the frozen or dried states. Various additives are known to minimize damage and enhance the stability of proteins. This review discusses the current knowledge of the mechanisms by which these additives stabilize proteins against acute stresses, and also the various factors to be considered for long-term storage of proteins in solution.

Biopharmaceutical powders: particle formation and formulation considerations.

It is well known that protein/peptide-based drug formulations are more stable in the solid state than in the liquid state, thereby offering stability advantages in ambient temperature storage, product shipping/distribution, and long-term shelf life. Novel powder-based drug delivery systems recently emerging for applications in sustained release, inhalation, intradermal delivery, etc, add more value to protein solid dosage forms. Despite great research interests in understanding the drying effects on protein stability and a large collection of publications focusing on this area, systematic accounts of powder formation techniques are lacking. This review is to summarize a number of methods currently available for protein powder preparation. Some are common methods such as lyophilization, spray drying, pulverization, and precipitation, and some methods are more recently developed such as supercritical fluid precipitation, spray-freeze drying, fluidized-bed spray coating and emulsion precipitation. In addition to examining the individual process effect on protein stability that is always the focus of formulation scientists, this review also likes to evaluate each method from a more practical sense in terms of process versatility and scalability. The conclusion is that each method has its own advantages and the use of a method is formulation and application specific. With the understanding of the principles and advantages of these methods, it can benefit our choice on selecting appropriate techniques for preparing a desired protein powder formulation for specific applications.

Activity-stability considerations of trypsinogen during spray drying: effects of sucrose.

The preparation and processing of protein pharmaceuticals into powders may impose significant stresses that could perturb and ultimately denature them. In many cases their stabilization through added excipients is necessary to yield native and active proteins. In this study, the effect of spray drying on the structure and activity of a model protein (trypsinogen) was investigated. In the absence of excipients, spray drying resulted in small losses of its enzymatic activity. Protein conformational rearrangements in the solid state (observed via FTIR) and irreversible aggregation (upon reconstitution) constituted the major degradation pathways. The irreversible unfolding in the solid state was also confirmed by solution calorimetric studies that indicated a decreased thermal stability of the spray-dried protein after reconstitution. The presence of sucrose, a thermal and dehydration stress stabilizer, induced a concentration-dependent protective effect. Protein protection was afforded even at low carbohydrate concentrations, while at specific mass ratios (sucrose-to-protein = 1:1) complete activity preservation was achieved. However, at the high end of sucrose concentrations, a small destabilization was evident, indicating that excluded volume effects may be undesirable during preparation of protein microparticles via spray drying. The profile of both the protein conformational changes and thermal stability in the solid state closely followed that of the incurred activity losses, indicating that protein stabilization during dehydration is crucial during processing of these polypeptides.

Moisture effects on protein-excipient interactions in spray-dried powders. Nature of destabilizing effects of sucrose.

The preparation of stable solid protein formulations presents significant challenges. Ultimately, the interactions between incorporated excipients and the pharmaceutical protein determine the formulation stability. In this study, moisture was utilized to probe the interactions between a model protein, trypsinogen, and sucrose in the solid state, following spray drying. Through investigation of the physical properties of the spray-dried formulations, we attempted to elucidate the mechanisms underlying the previously observed stabilizing and destabilizing effects of the carbohydrate during spray drying. Both dynamic and equilibrium moisture uptake studies indicated the presence of an optimal protein-sugar hydrogen bonding network. At low sucrose contents, a preferential protein-sucrose hydrogen bonding interaction was dominant, resulting in protein stabilization. However, at high carbohydrate concentrations, preferential sugar-sugar interactions prevailed, resulting in a phase separation within the formulation matrix. The preferential incorporation of the sucrose molecules in a sugar-rich phase reduced the actual amount of the carbohydrate available to interact with the protein and thereby decreased the number of effective protein-sucrose contacts. As a consequence, the protein could not be effectively protected during spray drying. We hypothesize that the observed phase separation at this sucrose concentration regime originates from its exclusion from the protein in solution before spray drying, further accompanied by preferential clustering of the sucrose molecules.

Infrared spectroscopic studies of lyophilization- and temperature-induced protein aggregation.

Recent studies have clearly demonstrated that Fourier transform IR spectroscopy can be a powerful tool for the study of protein stabilization during freeze-drying and for optimizing approaches to prevent lyophilization-induced protein aggregation. The purpose of the current review is to provide an overview of these topics, as well as an introduction to the study of protein secondary structure with IR spectroscopy. We will start with a general summary of the theories and practices for processing and interpreting protein IR spectra. We will then review the current literature on the use of IR spectroscopy to study protein structure and the effects of stabilizers during lyophilization. Next we will concentrate specifically on protein aggregation. The bulk of the research and the key assignments of spectral features in protein aggregates come from studies of the effects of high and low temperature on proteins. Therefore, we will first consider this topic. Finally, we will summarize the recent theoretical and applied work on lyophilization-induced aggregation.

Precipitation of proteins in supercritical carbon dioxide.

Supercritical CO2 was used as an antisolvent to form protein particles that exhibited minimal loss of activity upon reconstitution. Organic protein solutions were sprayed under a variety of operating conditions into the supercritical fluid, causing precipitation of dry, microparticulate (1-5 microns) protein powders. Three proteins were studied: trypsin, lysozyme, and insulin. Amide I band Raman spectra were used to estimate the alpha-helix and beta-sheet structural contents of native and precipitate powders of each protein. Analysis of the Raman spectral revealed minimal (lysozyme), intermediate (trypsin), and appreciable (insulin) changes in secondary structure with respect to the commercial starting materials. The perturbations in secondary structure suggest that the most significant event during supercritical fluid-induced precipitation involved the formation of beta-sheet structures with concomitant decreases of alpha-helix. Amide I band Raman and Fourier-transform infrared (FTIR) spectra indicate that higher operating temperatures and pressures lead to more extensive beta-sheet-mediated intermolecular interactions in the precipitates. Raman and FTIR spectra of redissolved precipitates are similar to those of aqueous commercial proteins, indicating that conformational changes were reversible upon reconstitution. These results suggest that protein precipitation in supercritical fluids can be used to form particles suitable for controlled release, direct aerosol delivery to the lungs, and long-term storage at ambient conditions.

The solution structure and conformational dynamics of tumor necrosis factor-alpha and a (Cys69----Asp; Cys101----Arg) analog as examined by IR spectroscopy and hydrogen exchange.

An analog of human tumor necrosis factor-alpha (TNF-alpha) was created with Cys69 and Cys101 replaced with Asp and Arg respectively. We have undertaken a comparative study of the solution conformation and dynamics of the native and analog molecules using a combination of Fourier transform IR spectroscopy and hydrogen-deuterium (H-D) exchange kinetics. IR spectroscopic results indicate that the analog molecule adopts a gross structure similar to that of the native molecule but significant differences in the conformation of the beta-sheets are observed. Increased bandwidths observed for several of the amide I components also suggest a less rigid structure for the analog molecule. Further, by monitoring the frequency shifts of the individual amide I component bands as a function of hydrogen exchange, we have enhanced our ability to assign these components to individual protein secondary structures, particularly the high frequency beta-strand mode. Hydrogen exchange kinetic studies indicate that the Asp-Arg analog adopts a looser, more flexible solution structure relative to the natural sequence molecule.

Separation of freezing- and drying-induced denaturation of lyophilized proteins using stress-specific stabilization. I. Enzyme activity and calorimetric studies.

Stabilization of labile proteins during lyophilization requires protection of the protein against both freezing and dehydration stresses. Solutions of 1-10% (wt/vol) polyethylene glycol (PEG) fully protected both lactate dehydrogenase and phosphofructokinase during freezing and thawing, but did not stabilize the proteins during freeze-drying. Thus, with this lyophilization system a second compound could be tested for its capacity to stabilize dried proteins, independent of its ability to provide cryopreservation. In the presence of low concentrations of glucose or trehalose (which alone provided minimal protection) and 1% PEG (wt/vol), almost full enzyme activity was recovered after freeze-drying and rehydration. Differential scanning calorimetry indicated that the PEG was crystalline and the sugars were amorphous in the dried samples. Experiments with lactose and mannitol demonstrated that if these compounds also crystallized during freeze-drying, protein stabilization was reduced or abolished. PEG stabilizes the proteins during freezing, due to preferential exclusion of PEG from the protein's surface. The sugars protect the proteins during dehydration by hydrogen bonding to the dried protein, thus serving as water substitutes. This report provides the first example of stabilization of proteins during lyophilization through separate, specific treatments of the fundamentally different stresses of freezing and dehydration.

Separation of freezing- and drying-induced denaturation of lyophilized proteins using stress-specific stabilization. II. Structural studies using infrared spectroscopy.

The conformation of two labile enzymes, lactate dehydrogenase and phosphofructokinase, has been examined in the aqueous and lyophilized states, using infrared spectroscopy. In the preceding paper it was demonstrated that a stress-specific stabilization scheme, which employs a combination of a cryoprotectant (polyethylene glycol) and a compound which protects the dried protein (sugars or mannitol), can be used to optimize recovery of activity of these enzymes upon freeze-drying and rehydration. The purpose of the present study is to determine the effects of these additives on the conformation of these enzymes during lyophilization. Lyophilization in the absence of stabilizers was observed to induce significant conformational changes in both enzymes. Addition of 10 mM mannitol, lactose, or trehalose or 1% polyethylene glycol to the enzyme solutions attenuated the unfolding, but significant spectral differences for the enzymes in the dried state are still observed when compared to the aqueous conformation. Addition of any one of these stabilizers does not improve recovery of activity. However, when a combination of 1% PEG and either 10 mM mannitol, lactose, or trehalose is added, the native structure is preserved during lyophilization and essentially full enzymatic activity is recovered upon reconstitution. The ability of the stabilizers to preserve the native structure during lyophilization correlates directly with the recovery of enzymatic activity upon reconstitution. It appears that for labile proteins, preservation of the native structure during lyophilization is requisite for recovery of activity following rehydration. This study demonstrates that the infrared spectroscopic technique is a rapid and useful method for studying protein conformation in the dried state and can aid in determining the optimal conditions for stabilization of proteins during lyophilization.

Infrared spectroscopic discrimination between alpha- and 3(10)-helices in globular proteins. Reexamination of Amide I infrared bands of alpha-lactalbumin and their assignment to secondary structures.

We have undertaken a new and more detailed Fourier-transform infrared (FTIR) spectroscopic study of alpha-lactalbumin (in D2O solution) aimed at correlating its secondary structures to observed Amide I' infrared bands. The spectra reported here were interpreted in light of the recently determined crystal structure of alpha-lactalbumin and by comparison with the spectra and structure of the homologous protein lysozyme. Of particular importance is the new evidence supporting the assignment of the band at 1639 cm-1 to 3(10)-helices. This assignment is in excellent agreement with one based on theoretical and experimental studies of 3(10)-helical polypeptides. The frequency observed for 3(10)-helices is distinctly different from that at which alpha-helices are typically found (viz., around 1655 cm-1). In the present study, two bands are clearly resolved in the latter region at 1651 and 1659 cm-1. Both are apparently associated with alpha-helices. These results suggest that for D2O solutions of globular proteins. FTIR spectroscopy can be a facile method for detecting the presence of these two different types of helical conformation and distinguishing between them. This provides a distinct advantage over ultraviolet circular dichroism spectroscopy (UV-CD). This work also provides a basis for future studies of alpha-lactalbumin which examine the effects of environment (e.g., pH, temperature) and ligands (e.g., Ca2+, Mn2+) on its conformation.

Comparison of various molecular forms of bovine trypsin: correlation of infrared spectra with X-ray crystal structures.

Fourier-transform infrared spectroscopy is a valuable method for the study of protein conformation in solution primarily because of the sensitivity to conformation of the amide I band (1700-1620 cm-1) which arises from the backbone C = O stretching vibration. Combined with resolution-enhancement techniques such as derivative spectroscopy and self-deconvolution, plus the application of iterative curve-fitting techniques, this method provides a wealth of information concerning protein secondary structure. Further extraction of conformational information from the amide I band is dependent upon discerning the correlations between specific conformational types and component bands in the amide I region. In this paper, we report spectra-structure correlations derived from conformational perturbations in bovine trypsin which arise from autolytic processing, zymogen activation, and active-site inhibition. IR spectra were collected for the single-chain (beta-trypsin) and once-cleaved, double-chain (alpha-trypsin) forms as well as at various times during the course of autolysis and also for zymogen, trypsinogen, and beta-trypsin inhibited with diisopropyl fluorophosphate. Spectral differences among the various molecular forms were interpreted in light of previous biochemical studies of autolysis and the known three-dimensional structures of the zymogen, the active enzyme, and the DIP-inhibited form. Our spectroscopic results from these proteins in D2O imply that certain loop structures may absorb in the region of 1655 cm-1. Previously, amide I' infrared bands near 1655 cm-1 have been interpreted as arising solely from alpha-helices. These new data suggest caution in interpreting this band. We have also proposed that regions of protein molecules which are known from crystallographic experiments to be disordered absorb in the 1645 cm-1 region and that type II beta-turns absorb in the region of 1672-1685 cm-1. Our results also corroborate assignment of the low-frequency component of extended strands to bands below 1636 cm-1. Additionally, the results of multiple measurements have allowed us to estimate the variability present in component band areas calculated by curve fitting the resolution-enhanced IR spectra. We estimate that this approach to data analysis and interpretation is sensitive to changes of 0.01 unit or less in the relative integrated intensities of component bands in spectra whose peaks are well resolved.

Effect of metal ion binding on the secondary structure of bovine alpha-lactalbumin as examined by infrared spectroscopy.

We have examined the influence of monovalent and divalent cations on the secondary structure of bovine alpha-lactalbumin at neutral pH using Fourier-transform infrared spectroscopy. Our present studies are based on previously reported amide I' component band assignments for this protein [Prestrelski, S. J., Byler, D. M., & Thompson, M. P. (1991) Int. J. Pept. Protein Res. 37, 508-512]. The results indicate that upon dissolution, alpha-lactalbumin undergoes a small, but significant, time-dependent conformational change, regardless of the ions present. Additionally, these studies provide the first quantitative measure of the well-known secondary structural change which accompanies calcium binding. Results indicate that removal of Ca2+ from holo alpha-lactalbumin results in local unfolding of the Ca(2+)-binding loop; the spectra indicate that approximately 16% of the backbone chain changes from a rigid coordination complex to an unordered loop. We have also examined the effects of binding of several other metal ions. Our studies have revealed that binding of Mn2+ to apo alpha-lactalbumin (Ca(2+)-free), while inducing a small, but significant, conformational change, does not cause the alpha-lactalbumin backbone conformation to change to that of the holo (Ca(2+)-bound) form as characterized by infrared spectroscopy. Similar changes to those induced by Mn2+ are observed upon binding of Na+ to apo alpha-lactalbumin, and furthermore, even at very high concentrations (0.2 M), Na+ does not stabilize a structure similar to the holo form. Binding of Zn2+ to the apo form of alpha-lactalbumin does not result in significant backbone conformational changes, suggesting a rigid Zn(2+)-binding site.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding of heparin to basic fibroblast growth factor induces a conformational change.

The binding of heparin to basic fibroblast growth factor (bFGF) induces a small but highly reproducible conformational change observable in the amide I region of the protein's infrared spectrum. The observed spectral changes suggest that the conformational change is highly localized most likely in the beta-turn regions of the bFGF molecule. Heparan sulfate, a component of the endothelial extracellular matrix, was also observed to bind to bFGF and induce a similar conformational change to that observed for heparin. Further, sucrose octasulfate, a compound which mimics the effects of heparin biologically, was also observed to induce this same conformational change. This spectroscopically observable change has allowed us to probe the functional determinants necessary for heparin to bind the bFGF and to induce the observed conformational change. We have determined the effects of binding of various monomeric and polymeric, sulfated and nonsulfated glycosaminoglycans and carbohydrate compounds. The results indicate that the binding of heparin involves highly specific interactions. Further, heparin was observed to greatly increase the thermal stability of bFGF, raising the Tm by 25 degrees C. Sucrose octasulfate was also able to enhance the thermal stability of bFGF, but not to the same extent as heparin.

Application of Fourier transform infrared spectroscopy to studies of aqueous protein solutions.

Modern protein Fourier transform infrared (FT-IR) spectroscopy has proven to be a versatile and sensitive technique, applicable to many aspects of protein characterization. The major practical drawback for the FT-IR spectroscopy of proteins is the large absorbance band of water, which overlaps the amide I resonances. D2O is often substituted for H2O in infrared experiments. Removal of water from protein samples can be complicated and tedious and potentially lead to denaturation, aggregation, or sample loss. Solvent removal by dialysis is difficult for suspensions and sols. A new method called the D2O dilution technique (Ddt) is described which simplifies the sample preparation step and improves the solvent subtraction. The effect of the D2O concentration on the IR spectrum of aqueous solutions of several model proteins was studied. Dilution of aqueous samples with D2O yields good quality spectra. The Ddt has been evaluated for quantitative analysis using standard proteins and its applicability to solutions and suspensions of a genetically engineered malaria antigen is demonstrated. Use of resolution-enhancement techniques with spectra in mixed solvents has also been investigated.

Generation of a substructure library for the description and classification of protein secondary structure. I. Overview of the methods and results.

Protein secondary structure has been typically classified into four major classes--alpha-helices, extended strands, reverse turns, and loops. Available methods for secondary structure analysis utilize predefined structure templates to search for structural matches among proteins. By this approach a significant portion of a proteins backbone conformation is assigned to one of a limited number of conformations or, if unassigned, to random coil. To expand our ability to describe protein secondary structure, we have developed an algorithm that operates independently of a predefined structure template. The procedure uses two geometric descriptors, the linear distance and the backbone dihedral angle, to represent the conformation form the alpha-carbon coordinates. The algorithm functions by searching for conformationally equivalent, contiguous fragments without regard to secondary structural classification and is thus independent of the complexity of the backbone fold. The result is a library of conformationally equivalent structure fragments that exhibit some novel characteristics. The library contains features that reproduce the major secondary structure classes as well as defining conformations previously described only as random or undefined conformations. Additionally, the library defines several subclassifications of beta-strands. We present here a validation of this method and a presentation and discussion of the most significant results. In a second study, we report the results of application of this method to spectra-structure correlations in Fourier transform infrared spectroscopy.

Generation of a substructure library for the description and classification of protein secondary structure. II. Application to spectra-structure correlations in Fourier transform infrared spectroscopy.

Fourier transform infrared spectroscopy has become well known as a sensitive and informative tool for studying secondary structure in proteins. Present analysis of the conformation-sensitive amide I region in protein infrared spectra, when combined with band narrowing techniques, provides more information concerning protein secondary structure than can be meaningfully interpreted. This is due in part to limited models for secondary structure. Using the algorithm described in the previous paper of this series, we have generated a library of substructures for several trypsin-like serine proteases. This library was used as a basis for spectra-structure correlations with infrared spectra in the amide I' region, for five homologous proteins for which spectra were collected. Use of the substructure library has allowed correlations not previously possible with template-based methods of protein conformational analysis.

Optimization of lyophilization conditions for recombinant human interleukin-2 by dried-state conformational analysis using Fourier-transform infrared spectroscopy.

PURPOSE: Examination of the dried-state conformation of interleukin-2 (IL-2) was used to determine the pH conditions and stabilizers that provide optimal storage stability for the lyophilized product. METHODS: Fourier-transform infrared spectroscopy and accelerated stability studies which examined solubility, aggregate formation, and covalent cross-linking were used. RESULTS: Varying the pH in the absence of excipients resulted in dramatic differences in the dried state conformation of IL-2. At pH 7, IL-2 unfolds extensively upon lyophilization while at pH below 5 it remains essentially native. Additional unfolding was observed upon incubation at elevated temperatures. A strong direct correlation between the retention of the native (aqueous) structure during freeze-drying and enhanced stability is demonstrated. IL-2 prepared at pH 5 is approximately an order of magnitude more stable than at pH 7 with regard to formation of soluble and insoluble aggregates. A similar pH profile was observed in the presence of excipients, although the excipients alter the overall stability profile. Additional accelerated stability studies examined the stabilizers necessary for optimal stability. CONCLUSIONS: Excipients with the capacity to substitute for water upon dehydration better preserve the native structure resulting in enhanced stability. Those that have high glass transition temperatures provide the highest level of stability during storage, although they do not prevent dehydration induced unfolding.