Secondary structure of interleukin-2(Alal25) in unfolded state*

Secondary structure of interleukin-2(Alal25) in unfolded state was examined by circular dichroism (CD). Unfolding of tertiary structure of the protein, as determined by CD, was observed when the solvent pH was decreased below 3.0 or the disulfide bond was reduced. Consistent with the CD results, a stronger fluorescence enhancement of 1-anilinonaphthalene-8-sulfonic acid was observed on acidification or reduction of interleukin-2(Alal25) relative to that of the native protein, indicating a larger hydrophobic surface exposed to solvent. However, the secondary structure was fully retained in 5% acetic acid or aqueous HCl, pH 3.0. It seemed that α-helical content of the protein is even greater at pH 2.0. Reduced protein showed a far u.v. CD spectrum indistinguishable from the oxidized one at pH 4.0. These results suggest that the secondary structure of interleukin-2(Alal25) does not require tertiary structure.     

Thiol-Disulfide Exchange in Human Growth Hormone

Purpose

Thiol-disulfide exchange was monitored in recombinant human growth hormone (hGH) and in model tryptic peptides derived from hGH to investigate the effects of higher-order structure on the reaction.

Methods

Different free thiol-containing peptides, varying in length and amino acid sequence, were used to initiate the reaction at pH 7.0 and 37°C in hGH. Protein samples were digested with trypsin and analyzed for native disulfides, scrambled disulfides and free thiols using LC/MS. The loss of native disulfide and disulfide exchange was compared with model peptides derived from hGH.

Results

Loss of native disulfide in cyclic (cT20-T21) and linear peptides (T20-T21_pep) derived from the C-terminal hGH disulfide during the first 60 min of reaction was greater than loss of the C-terminal disulfide in hGH itself. Of the thiols tested, glutathione (GSH) was the most reactive, forming the highest percentage of mixed disulfides in intact hGH and in the model peptides. At longer reaction times (>240 min), native disulfides in both hGH and cT20-T21 were regenerated. The fastest rates of regeneration were observed for Cys and the di- or tripeptide containing an Arg residue adjacent to Cys, suggesting that they may be useful in refolding.

Conclusions

Thiol-disulfide exchange reactions in hGH and related model peptides were influenced by higher order structure, by the size of the thiol reactant and by an Arg residue adjacent to Cys in the thiol reactant. Reduction of disulfide bonds in hGH did not affect higher order structure as measured by CD and HDX-MS.

     

Thermal denaturation of tryptophan synthase α-subunit

Thermal denaturation for the wild-type of tryptophan synthase α-subunit from E. coli and one of its mutant proteins was followed by CD measurements at various pHs in the alkaline region and the results from van't Hoff analyses of the thermal denaturation curves were compared with those from calorimetry. Although the far-u.v. CD spectra of the thermally denatured proteins differed from those of the completely denatured states in 3.2 M guanidine hydrochloride, the titration curves by denaturants at higher temperatures were not sigmoidal but straight lines, indicating that the cooperative structure of the proteins has been completely destroyed by heating. The ratio of calorimetric enthalpy change to van't Hoff enthalpy change obtained from calorimetric study was unity, indicating that the thermal denaturation of the proteins was a two-state system. The unfolding heat capacity change (ΔCp) of the wild-type protein from van't Hoff analysis of the thermal denaturation curves by CD measurement was estimated to be 2.45 kcal/mol ± deg, which was similar to that from calorimetry. The values of unfolding enthalpy change at denaturation temperatures were lower by about 15 kcal/mol compared to those from calorimetry.     

Probing the structure of human growth hormone by limited proteolysis

Digestion of human growth hormone (hGH) with the Glu-specific protease from Staphylococcus aureus V8 was performed at 20-22°C or 37°C at a 1:20 ratio (by weight) at pH 7.8 with or without 0.2% SDS. There are 14 Glu-residues evenly distributed along the polypeptide chain of hGH as possible sites of proteolytic cleavage of V8-protease. The pattern of fragmentation of hGH was analyzed by electrophoresis and reversed-phase HPLC, and the identity of the proteolytic fragments isolated to homogeneity was established by their partial sequencing and amino acid analysis after acid hydrolysis. Kinetic analysis of the proteolytic digestion process allowed to establish that initial nicking of the protein occurs at Glu³³ and subsequently at Glu⁵⁶ and Glu⁶⁶. Much slower cleavages occur at G1u³⁰ and Glu¹⁸⁶. These cleavage sites are located at chain loops in the hGH molecule, and in particular outside the helical segments of the four-helix bundle of the crystal structure of hGH. Fragments 1-33 and 67-191 comprising entirely the N-terminal helix and the three C-terminal helices of hGH, respectively, were isolated to homogeneity in amounts useful for subsequent conformational and functional studies. The results of this study and of previous ones [Li, C.H. (1982) Mol. Cell. Biochem. 46 , 31-41] describing limited proteolysis of hGH by various proteases have been interpreted on the basis of the three-dimensional structure and dynamics of hGH. Overall, it is shown that proteolytic enzymes preferentially cleave hGH at exposed and flexible loops only, thus emphasizing the fact that proteases can be used as reliable probes of protein structure and dynamics. © Munksgaard 1995.     

Enthalpy and Entropy Contributions to the Solubility of Sulphamethoxypyridazine in Solvent Mixtures Showing Two Solubility Maxima

The solubility of sulphamethoxypyridazine was measured at several temperatures in mixtures of water: ethanol and ethanol: ethyl acetate. Sulphamethoxypyridazine was chosen as a model drug to compare the solvation effects of proton donor-proton acceptor (water and ethanol) and proton acceptor (ethyl acetate) solvents and mixtures of these solvents because this drug contains functional groups capable of Lewis acid-base interaction. A plot of the mole fraction solubility against the solubility parameter (δ₁) of these solvent mixtures showed two solubility maxima, one at δ₁ = 30·87 MPa^1/2 (20:80 v/v water: ethanol) and another at δ₁ = 20·88 MPa^1/2 (30:70 v/v ethanol: ethyl acetate) at all the temperatures under study. The enthalpies and entropies of mixing as well as the enthalpies and entropies of transfer of sulphamethoxypyridazine from ethanol to water:ethanol and ethanol:ethyl acetate mixtures were calculated to compare solvation characteristics of the solvent mixtures toward the drug. As ethanol is added to water, the entropy increases and the structure of the solvent mixture became less ordered, favouring the interaction of the drug with the solvent mixture. On the other hand, in the case of the ethanol: ethyl acetate mixture, solubility is favoured by the more negative enthalpy values. This way, the same result, i.e. a solubility maximum, is obtained by different routes. In the ethanol: water mixtures, the dissolution process is entropy-controlled while enthalpy is the driving force in the case of ethanol: ethyl acetate mixtures. The two solvent systems show enthalpy-entropy compensation. Water deviates from the linear relationship due possibly to its hydrophobic effect.     

Between-subject and within-subject variations in the pharmacokinetics of ethanol.

1. Twelve healthy men drank 0.80 g ethanol kg-1 body weight on four occasions spread over several weeks. Ethanol was given as 96% v/v solvent which was diluted with orange juice to make a cocktail (20-25% v/v). This drink was ingested in exactly 30 min at 08.00 h after an overnight (10 h) fast. 2. Samples of venous blood were obtained at exactly timed intervals of 0, 10, 20, 30, 45, 60, 90, 120, 150, 180, 240, 300, and 360 min after the start of drinking. The concentrations of ethanol in whole blood were determined by headspace gas chromatography. 3. Summary measures were used to evaluate the concentration-time profiles of ethanol for each subject. The between-subject and within-subject components of variation for the pharmacokinetics of ethanol were derived by one-way analysis of variance (ANOVA). 4. The variation between different subjects dominated the total variance for all of the pharmacokinetic parameters studied except the rate of disappearance of ethanol from blood (ko). For this latter parameter, 42% and 58% of the total variation arose from variations between- and within-subjects respectively. These results might be important to consider when experiments on the clinical pharmacokinetics of ethanol are being planned.     

Chromatographic Methods for Quantitative Analysis of Native,Denatured, and Aggregated Basic Fibroblast Growth Factor in Solution Formulations

High-performance liquid chromatography (HPLC) methods were developed for evaluating stability of human recombinant basic fibroblast growth factor (bFGF) against denaturation and aggregation in solution formulations. Re versed-phase chromatography (RP-HPLC)-insensitive to bFGF tertiary structure-was used to measure total soluble protein; heparin affinity chromatography (HepTSK) provided quantitative analysis of native bFGF species. The folding state of bFGF was determined by fluorescence spectroscopy: Tryptophan emission, which was quenched in native protein, increased upon unfolding. Slow unfolding/refolding kinetics of bFGF in 2 M guanidine hydrochloride made possible the separation of native from denatured species by size exclusion chromatography (SEC). Although the tertiary structure affected bFGF retention times, it did not change the sample recovery by SEC. These chromatographic techniques, which quantitatively measure physical and chemical changes taking place in solution formulations, can be used in future investigations of bFGF stability.     

Voluntary ethanol intake in rats following exposure to ethanol on various schedules

Voluntary intake of 20% (v/v) ethanol solutions was assesed in groups of male Wistar rats following various forms of ethanol exposure. Some animals were first exposed to gradually increasing weak solutions of ethanol (acclimation); while others were given 20% solutions from the start. Some were given ethanol every day (continuous schedule); others were given ethanol every other day (intermittent schedule). Some were given ethanol solutions with plain water also available (free-choice); others were given ethanol solutions as the only fluid available (forced-choice). The animals on intermittent schedules for a 30 day period developed a slight preference for 20% ethanol solutions; they came to drink an average of over 9 g/kg/day of absolute ethanol when tested in free choice conditions. Previous acclimation did not add significantly to this effect. The effect held whether the animals received their ethanol in free- or forced-choice conditions. Forced-choice experience inhibited subsequent free-choice intake in the continuous-exposure group, but forced-choice coupled with intermittent exposure led to the highest intake levels in the shortest total ethanol exposure. The intake levels of these animals are encouraging for those interested in developing animal analogues for human ethanol abuse.Supported by grants from the Licensed Beverages Industries and the Medical Research Council of Canada.     

Effects of ethanol/l-menthol on the dynamics and partitioning of spin-labeled lipids in the stratum corneum.

The interaction of ethanol as well as ethanol/L-menthol mixtures with the uppermost layer of epidermis, the stratum corneum, was investigated by electron paramagnetic resonance (EPR) spectroscopy utilizing spin-labeled analogs of androstanol (ASL), stearic acid (5-DSA) and methyl stearate (5-DMS). The EPR spectra of these spin probes structured in stratum corneum tissue of neonatal rat are characterized by the coexistence of two spectral components indicating the presence of two classes of spin labels with very different states of mobility. Probably, one class of spin labels is H-bonded to the polar surface of the membrane and another class corresponds to spin labels more deeply inserted in the hydrophobic core. EPR results showed that in the ethanol range 0-70% neither fluidity in stratum corneum membranes nor the relative fractions of these two components changes were observed. Instead, ethanol only caused a selective extraction of spin labels. The removal of the steroid ASL began at 30% ethanol, reaching extraction levels over 50% at ethanol concentrations of 60-70%, whereas the more hydrophobic 5-DMS was partially removed only with 70% ethanol. Addition of 5% L-menthol to the solvent containing 20% ethanol increases both the mobility and the fraction of those spin labels situated in the hydrophobic core (more mobile spectral component). Altogether, these findings suggest that the L-menthol stabilizes mainly in the central region of stratum corneum membranes attracting the membrane lipids and causing hydrogen bond ruptures in the polar membrane interface.     

Mixtures of trifluoroethanol or hexafluoroisopropanol and dimethylformamide are not of general applicability for peptide condensations catalyzed by trypsin

Mixtures of a good hydrogen bond donor, 2,2,2-trifluoroethanol (TFE) or 1,1,1,3,3,3-hexafluroisopropanol, and an acceptor, dimethylformamide (DMF) (1:1,v/v), containing 4% buffer have been described as adequate solvent systems for trypsin-catalyzed peptide fragment condensations [Mihara et al. (1993) Int. J. Pept. Protein Res. 41 , 405]. Thus, we decided to study the behaviour of trypsin in such solvent systems. We investigated whether this protease would efficiently catalyze condensations between fragments derived from an analogue of the gp-41 capsid protein of HIV virus or from cholecystokinin-22. None of the reactions carried out yielded the desired condensation products. However, when Fmoc-NLQNLDPSHR-OH and cholecystokinin-12 (H-ISDRDYMGWMDF-NH₂) were used as substrates, the last had its R-D peptide bond hydrolyzed producing cholecystokinin-8. The proteolytic activity of this enzyme measured against a fluorogenic peptide derivative was 50 times lower in DMF/TFE containing 5% of aqueous phase than in buffer. Steady-state fluorescence studies in DMF/TFE buffer were performed to examine the structure of this protease in these media. Steady-state spectra obtained with increasing proportions of these two organic solvents in buffer showed that the emission intensities built up. Quenching studies with iodide revealed that the I_o/I ratio (where I_o and I are the fluorescence emission intensities in the absence and presence of quencher, respectively) changed from 1.2 in aqueous media to 2.2 in DMF/TFE (1:1, v/v) containing 11% 0.2 m Tris-HCI buffer, pH 8.0, for 0.5 m iodide. The complete data indicated a higher exposure of tryptophan residues to the quencher in organic media, probably because of the partial unfolding of the enzyme.     

ENOLASE FROM BACILLUS STEAROTHERMOPHILUS

Enolase from Bacillus stearothermophilus was homogeneous in the ultracentrifuge and had the hydrodynamic properties of a globular protein with a molecular weight of 3.51 times 105 (sedimentation-diffusion) and 3.60 times 105 (Archibald approach to equilibrium). Electrophoretic and gel filtration measurements in presence of dissociating agents indicated that the enzyme consists of eight apparently identical polypeptide chains of molecular weight of 47 000. The denaturation of enolase has been studied as a function of urea concentration. A broad transition curve was observed, the enzyme in 8 M urea being inactive and dissociated into monomers. The far-ultraviolet circular dichroism spectrum of enolase in 8 M urea solution indicates extensive unfolding of the globular structure. Accordingly, fluorescence emission measurements indicate that the tryptophanyl residues of the denatured enolase are largely exposed to the aqueous solvent medium. The enzyme can be reversibly denatured by 8 M urea and the enzymatic activity then recovered by dilution 1:100 of the denatured enzyme into buffer or by removing urea by dialysis. Under suitable conditions and in the presence of Mg2+-ions, 90–100% of original activity was recovered and the renatured enzyme was indistinguishable from the native one, as judged from several enzymological and physicochemical criteria, including molecular weight. The far-ultraviolet circular dichroism spectrum and the fluorescence emission properties of the native enzyme were regained very rapidly (less than 1 min), while enzymatic activity was recovered much more slowly. Octameric enzyme species were obtained on refolding the denatured enzyme at temperatures between 4 and 55.     

The effects of water-soluble cyclodextrins on the histological integrity of the rat nasal mucosa

The aim of this study was to investigate the effect of highly water-soluble cyclodextrins (CDs) on the histological integrity of the nasal mucosa. In order to evaluate their effects, the in vivo single and repeated nasal exposure studies were performed using male Wistar rats. The rat nasal cavity was excised after an application of various CD solutions at different times. The morphological appearances of the rat nasal mucosae were analyzed with the light microscopic and the scanning electron microscopic studies. By utilizing 5-min exposure of each CD solution to the nasal mucosa, no tissue damage was visible for 1.5% w/v beta-CD and 5 and 20% w/v hydroxypropyl beta-CD (HP beta-CD), and the effects were quite similar to controls. However, using 20% w/v randomly methylated beta-CD (RM beta-CD) showed severe damage on the integrity of nasal mucosa. The severity was similar to 1% w/v polyoxyethylene-9-lauryl ether or l% w/v sodium deoxycholate. Meanwhile, 30 or 60 min exposure to 10% w/v HP beta-CD or RM beta-CD resulted in no obvious mucosal damage. In addition, in vivo repeated dosing of RM beta-CD did not show any toxicity up to 20% w/v. These results suggest that at least, less than 10% w/v CD solutions do not induce gross tissue damage and can keep the histological integrity of the nasal mucosa.     

Measurement of the Kinetics of Protein Unfolding in Viscous Systems and Implications for Protein Stability in Freeze-Drying

Purpose The aim of the study is to determine the degree of coupling between protein unfolding rate and system viscosity at low temperatures in systems relevant to freeze-drying.Methods The cold denaturation of both phosphoglycerate kinase (PGK) and β-lactoglobulin were chosen as models for the protein unfolding kinetics study. The system viscosity was enhanced by adding stabilizers (such as sucrose), and denaturant (guanidine hydrochloride or urea) was added to balance the stabilizing effect of sucrose to maintain the cold denaturation temperature roughly constant. The protein unfolding kinetics were studied by both temperature-controlled tryptophan emission fluorescence spectroscopy and isothermal high-sensitivity modulated differential scanning calorimetry (MDSC) (T_zero). Viscometers were used to determine the system viscosity. To verify the predictions of structure based on protein unfolding dynamics, protein formulations were freeze-dried above the glass transition temperatures, and the protein structures in dry products were determined by fluorescence spectroscopy of reconstituted solids by extrapolation of the solution data to the time of reconstitution.Results Empirical equations describing the effect of sucrose and denaturant (urea and guanidine hydrochloride) on protein cold denaturation were developed based on DSC observations [X. C. Tang and M. J. Pikal. The Effects of Stabilizers and Denaturants on the Cold Denaturation Temperature of Proteins and Implications for Freeze-Drying. Pharm. Res. Submitted (2004)]. It was found that protein cold denaturation temperature can be maintained constant in system of increasing sucrose concentration by simultaneous addition of denaturants (urea and guanidine hydrochloride) using the empirical equations as a guide. System viscosities were found to increase dramatically with increasing sucrose concentration and decreasing temperature. The rate constants of protein unfolding (or the half-life of unfolding) below the cold denaturation temperature were determined by fitting the time dependence of either fluorescence spectroscopy peak position shift or DSC heat capacity increase to a first-order reversible kinetic model. The half-life of unfolding did slow considerably as system viscosity increased. The half-life of PGK unfolding, which was only 3.5 min in dilute buffer solution at −10°C, was found to be about 200 min in 37% sucrose at the same temperature. Kinetics of protein unfolding are identical as measured by tryptophan fluorescence emission spectroscopy and by high-sensitivity modulated DSC. The coupling between protein unfolding kinetics and system viscosity for both proteins was significant with a stronger coupling with PGK than with β-lactoglobulin. The half-lives of PGK and β-lactoglobulin unfolding are estimated to be 5.5 × 10¹¹ and 2.2 years, respectively, even when they are freeze-dried in sucrose formulations 20°C above T_g′. Thus, freeze-drying below T_g′ should not be necessary to preserve the native conformation. In support of this conclusion, native PGK was obtained after the freeze-drying of PGK at a temperature more than 60°C above the system T_g′ in a thermodynamically unstable system during freeze-drying.Conclusions Protein unfolding kinetics is highly coupled with system viscosity in high viscosity systems, and the coupling coefficients are protein dependent. Protein unfolding is very slow on the time scale of freeze-drying, even when the system is freeze-dried well above T_g′. Thus, it is not always necessary to freeze-dry protein formulations at temperature below T_g′ to avoid protein unfolding. That is, protein formulations could be freeze-dried at product temperature far above the T_g′, thereby allowing much shorter freeze-drying cycle times, with dry cake structure being maintained by the simultaneous use of a bulking agent and a disaccharide stabilizer.     

Temperature- and pH-Induced Multiple Partially Unfolded States of Recombinant Human Interferon-α2a: Possible Implications in Protein Stability

PURPOSE: To study the effect of solution conditions on the structural conformation of recombinant human interferon-alpha2a (IFNalpha2a) to investigate its tendency to form partially unfolded intermediates. METHODS: The structural properties of IFNalpha2a were studied at various pH values (2.0-7.4) and temperatures (5 degrees C-80 degrees C) using Trp fluorescence emission, fluorescence quenching, near- and far-UV circular dichroism (CD) spectroscopy, and DSC. RESULTS: Fluorescence intensity measurements as a function of temperature indicated the onset of the thermal unfolding of IFNalpha2a, denoted by Td, around 60 degrees C above pH 4.0. Td was not observed at pH 3.5 and below. Acrylamide and iodide quenching studies indicated partial unfolding of protein with decrease in pH and with increase in temperature up to 50 degrees C. Near-UV CD studies indicated a significant loss in the tertiary structure of protein on increase in temperature from 15 degrees C to 50 degrees C at all solution pHs. DSC scans supported results obtained from fluorescence and CD studies at pH 4.0 and below. DSC, however, was insensitive to changes that occurred at moderate temperatures at pH 5.0 and 7.4. CONCLUSIONS: IFNalpha2a has a tendency to acquire multiple partially unfolded states with structural conformations sensitive to solution pH and temperature. These states were formed at moderate temperatures, and it is speculated that these partially unfolded states could play an important role in the aggregation of proteins during the long-term storage of aqueous protein formulations.     

Ethanol and the peripheral benzodiazepine receptor: in vivo and in vitro experiments.

1. The effect of chronic ethanol exposure on rat peripheral benzodiazepine receptors (PBR) was studied. 2. The binding of 3H-RO 5-4864 to PBR was increased (35.4%) in the kidneys of rats treated with 10% (v/v) ethanol for 12 weeks, but not in renal membranes isolated from rats exposed to ethanol 30% (v/v) during the same period. 3. Similarly, short-term administration of ethanol (4 weeks) did not alter the binding of 3H-RO 5-4864 to renal membranes. 4. To examine the possibility of a direct interaction of ethanol with PBR, in vitro experiments were carried out. Only high concentrations of ethanol (> 100 mM) caused a significant inhibition of 3H-RO 5-4864 binding in kidney, testis and cerebral cortex. 5. The results presented indicate that chronic ethanol exposure causes a time and dose-dependent increase in renal PBR.     

Comparative studies on the effects of water, ethanol and water/ethanol mixtures on chemical partitioning into porcine stratum corneum and silastic membrane.

The effects of water and ethanol vehicles on stratum corneum and silastic membrane partitioning of 11 industrial and agricultural compounds were studied to aid in characterizing and assessing risk from skin exposure. Zero percent, 50% and 100% aqueous ethanol solutions were used as solvents for (14)C labeled phenol, 4-nitrophenol, pentachlorophenol, dimethyl parathion, parathion, chloropyrifos, fenthion, triazine, atrazine, simazine and propazine. Compound partitioning between the solvents and porcine stratum corneum/silastic membrane were estimated. Stratum corneum was exposed to aqueous ethanol ranging from 0% to 100% v/v ethanol in 20% increments and Fourier transform infrared spectroscopy (FT-IR) was used to obtain an index of lipid disorder. Gravimetry and FT-IR were used to demonstrate lipid extraction in aqueous ethanol solutions. Partitioning patterns in silastic membranes resembled those in stratum corneum and were correlated with octanol/water partitioning. Partitioning was highest in water and was higher from 50% ethanol than from 100% ethanol, except for parathion, 4-nitrophenol, atrazine and propazine. Correlation existed between molecular weight and partitioning in water, but not in ethanol and ethanol/water mixtures. Lipid order, as reflected in FT-IR spectra, was not altered. These studies suggest that stratum corneum partitioning of the compounds tested is primarily determined by relative compound solubility between the stratum corneum lipids and the donor solvent. Linear relationships existed between octanol/water partitioning and stratum corneum partitioning. Partitioning was also correlated with molecular weight in water solvent systems, but not in ethanol and ethanol/water mixtures. Ethanol and ethanol/water mixtures altered the stratum corneum through lipid extraction, rather than through disruption of lipid order.     

Oxide terpenes as human skin penetration enhancers of haloperidol from ethanol and propylene glycol and their modes of action on stratum corneum

In this study, two terpenes with the same functional group; limonene oxide and pinene oxide were used at 5% w/v concentration in 50% v/v ethanol and 100% v/v propylene glycol (PG) to enhance the in vitro permeation of haloperidol (HP) through the human epidermis (or stratum corneum, SC). The enhancement mechanism of terpenes from both solvents was elucidated with HP-SC binding studies, Fourier transform infrared spectroscopy and differential scanning calorimetry. The enhancement activity of these terpenes was higher in 50% v/v ethanol than in 100% v/v PG. These terpenes in 50% v/v ethanol were predicted to provide the required therapeutic plasma concentration and daily-permeated amounts of the drug. Limonene oxide showed higher enhancement in both solvents, which was attributed to its less bulky structure. The terpenes in both solvents did not increase the partition of HP. Instrumental studies showed that these terpenes in 50% v/v ethanol extracted the SC lipids, disrupted the bilayer packing and partially fluidised the lipids. Limonene oxide in 100% v/v PG possibly disrupted the lipid bilayer, whilst leaving the overall bilayer structure intact and pinene oxide in the same vehicle fluidised the lipids within the ordered environment. This study showed that the mode of interactions of terpenes with SC were different in two solvent systems.     

Feasibility Study on Spray-Drying Protein Pharmaceuticals: Recombinant Human Growth Hormone and Tissue-Type Plasminogen Activator

The feasibility of spray-drying solutions of recombinant methionyl human growth hormone (hGH) and tissue-type plasminogen activator (t-PA) was investigated. hGH was formulated in a mannitol phosphate buffer and t-PA was used in an arginine phosphate formulation containing 0.004% (w/v) polysorbate 80. Using filtered air (90 – 150°C) as the drying medium, hGH could be dried to a residual moisture content of 4%. However, approximately 25% of the protein was degraded during the processing. Results of atomization studies suggest that surface denaturation at the air–liquid interface of the droplets in the spray plays a major role in the degradation of the protein. The addition of 0.1% (w/v) polysorbate 20 into the hGH formulation reduced the formation of soluble and insoluble aggregates by approximately 90% during atomization. During spray-drying the addition of 0.1% (w/v) polysorbate 20 reduced the formation of soluble and insoluble aggregates by approximately 70 and 85%, respectively. In contrast, t-PA remained intact upon atomization. Depending on the spray-drying conditions, product powders with a residual moisture content between 5 and 8% were obtained. No oxidation, aggregation, or denaturation occurred in the protein under several operation conditions. Overall, this study demonstrates that it is feasible to spray-dry t-PA in the current marketed formulation.     

Stability of subtilisins and related proteinases (subtilases)

The stability towards thermal and chemical (guanidine hydrochloride, GnHCl) denaturation of six inhibited subtilases (mesentericopeptidase, subtilisins BPN′, Carlsberg and DY, proteinase K and thermitase) has been investigated by kinetic and equilibrium studies. The unfolding processes were monitored by circular dichroic and fluorescence spectroscopy. Experiments in the absence and presence of extraneous calcium in the concentration range 2×10^?3-10^?1 M were performed. The presence of calcium in the weak calcium binding site changes the denaturation drastically. The heat- (or GnHCl-) induced unfolding curves obtained using CD spectroscopy show two independent transitions which seem not to have been resolved before. The presence of Ca²⁺ in the second (third in the case of thermitase) binding site increases the T_m, values by 11-21 °C and the δG_D(H₂O) values obtained from denaturation experiments in GnHCl by 6.7-7.2 kcal/mol when an extraneous Ca²⁺ concentration of 2 × 10^?2 M was used. One interpretation is that the initial step of denaturation in the presence of added calcium is the formation of a partially unfolded intermediate form, retaining a highly ordered structure with 60-85% of the a-helix structure of the native enzyme. This intermediate then unfolds at a temperature considerably higher than that of the same proteinases in the absence of added Ca²⁺. The free energy of stabilization of the intermediates is increased by 1.8-2.8 times in comparison with that for the unfolding reactions of the subtilases with empty Ca2/Ca3 binding sites. A second interpretation is that the two steps in the unfolding curves correspond to enzyme without and with calcium in the weak binding site. Fluorescence experiments confirm the mechanism involving the formation of intermediate states. The results are discussed in relation to the X-ray models of the six subtilases.     

Synthesis and conformational studies of peptides encompassing the carboxy-terminal helix of thermolysin

The 21-residue fragment Tyr-Gly-Ser-Thr-Ser-Gln-Glu-Val-Ala-Ser-Val-Lys-Gln-Ala-Phe-Asp-Ala-Val-Gly-Val-Lys, corresponding to sequence 296–316 of thermolysin and thus encompassing the COOH-termi-nal helical segment 301–312 of the native protein, was synthesized by solid-phase methods and purified to homogeneity by reverse-phase high performance liquid chromatography. The peptide 296–316 was then cleaved with trypsin at Lys³⁰⁷ and Staphylococcus aureus V8 protease at Glu³⁰², producing the additional fragments 296–307, 308–316, 296–302, and 303–316. All these peptides, when dissolved in aqueous solution at neutral pH, are essentially structureless, as determined by circular dichroism (CD) measurements in the far-ultraviolet region. On the other hand, fragment 296–316, as well as some of its proteolytic fragments, acquires significant helical conformation when dissolved in aqueous trifluoroethanol or ethanol. In general, the peptides mostly encompassing the helical segment 301–312 in the native thermolysin show helical conformation in aqueous alcohol. In particular, quantitative analysis of CD data indicated that fragment 296–316 attains in 90% aqueous trifluoroethanol the same percentage (～58%) of helical secondary structure of the corresponding chain segment in native thermolysin. These results indicate that peptide 296–316 and its subfragments are unable to fold into a stable native-like structure in aqueous solution, in agreement with predicted location and stabilities of isolated subdomains of the COOH-terminal domain of thermolysin based on buried surface area calculations of the molecule