Effects of Solution Conditions on Methionine Oxidation in Albinterferon Alfa-2b and the Role of Oxidation in its Conformation and Aggregation

Physical and chemical degradation of therapeutic proteins can occur simultaneously. In this study, our first objective was to investigate how solution conditions that impact conformational stability of albinterferon alfa-2b, a recombinant fusion protein, modulate rates of methionine (Met) oxidation. Another objective of this work was to determine whether oxidation affects conformation and rate of aggregation of the protein. The protein was subjected to oxidation in solutions of varying pH, ionic strength, and excipients by the addition of 0.02% tertiary-butyl hydroperoxide (TBHP). The rate of formation of Met-sulfoxide species was monitored by reversed-phase high-performance liquid chromatography and compared across solution conditions. Albinterferon alfa-2b exhibited susceptibility to Met oxidation during exposure to TBHP that was highly dependent on solution parameters, but there was not a clear correlation between oxidation rate and protein conformational stability. Met oxidation resulted in significant perturbation of both secondary and tertiary structure of albinterferon alfa-2b as shown by both far-ultraviolet (UV) and near-UV circular dichroism. Moreover, oxidation of the protein caused a noticeable reduction in the protein's resistance to thermal denaturation. Surprisingly, despite its negative effect on solution structure and conformational stability, oxidation actually reduced the protein's aggregation rate during agitation at room temperature as well as during quiescent incubation at 40°C. Oxidation of the protein resulted in improved colloidal stability of the protein, which is manifested by a more positive B₂₂ value in the oxidized protein. Thus, the reduced aggregation rate after oxidation suggests that increased colloidal stability of oxidized albinterferon alfa-2b counteracted oxidation-induced decreases in conformational stability.     

Spectral properties of Bitis gabonica venom phospholipase A2 in the presence of divalent metal ion, substrate and hydrolysis products.

Bitis gabonica phospholipase A₂ binds 2 moles of Ca²⁺ per mole of dimer, giving rise to spectral perturbations of tryptophan chromophores in the enzyme. The spectral changes are taken to originate from both solvent and charge effects. A pH dependent perturbation is noted both in the presence and absence of Ca²⁺; however, the character of the perturbation in absence of metal ions is different from the perturbation observed in the presence of Ca²⁺. The data suggest that Ca²⁺ induces a conformational change in the enzyme, allowing the productive binding of substrate. A change in tryptophan absorption is also noted in the presence of dipalmitoyllecithin, lysolecithin and palmitic acid. However, perturbation by palmitic acid causes a blue shift of the spectrum as opposed to a red shift caused by lysolecithin and dipalmitoyllecithin. Fine structure in the 270–300 nm range due to tryptophan perturbation, is abolished when lysolecithin binds to the enzyme in the presence of Ca²⁺. This is interpreted to mean that the perturbable tryptophan is involved in substrate binding.     

Effect of Phosphate Ion on the Structure of Lumazine Synthase,an Antigen Presentation System From Bacillus anthracis

Lumazine synthase (LS) is an oligomeric enzyme involved in the biosynthesis of riboflavin in microorganisms, fungi, and plants. LS has become of significant interest to biomedical science because of its critical biological role and attractive structural properties for antigen presentation in vaccines. LS derived from Bacillus anthracis (BaLS) consists of 60 identical subunits forming an icosahedron. Its crystal structure has been solved, but its dynamic conformational properties have not yet been studied. We investigated the conformation of BaLS in response to different stress conditions (e.g., chemical denaturants, pH, and temperature) using a variety of biophysical techniques. The physical basis for these thermal transitions was studied, indicating that a molten globular state was present during chemical unfolding by guanidine HCl. In addition, BaLS showed 2 distinct thermal transitions in phosphate-containing buffers. The first transition was due to the dissociation of phosphate ions from BaLS and the second one came from the dissociation and conformational alteration of its icosahedral structure. A small conformational alteration was induced by the binding/dissociation of phosphate ions to BaLS. This work provides a closer view of the conformational behavior of BaLS and provides important information for the formulation of vaccines which use this protein.     

Preparation of clear solutions of reconstituted acetylcholinesterase Effect of ionic strength and lipid/protein ratio

The detergent-soluble globular dimer of acetylcholinesterase from Torpedo californica was reconstituted through dialysis into preformed egg phosphatidylcholine vesicles. The formation of the enzyme-lipid complexes depended on the ionic strength of the dialysis buffer as well as the molar lipid/protein ratio (R). The enzyme was unstable at I < 0.05; increasing the ionic strength increased the size of the complex. A too low R value (e.g. 1000) would promote self-aggregation of the enzyme and produce heterogeneous complexes, especially at high I values. On the other hand, a too high R value (e.g. > 5000) favored the formation of large enzyme-lipid complexes; their solutions were too turbid for optical studies. The enzyme reconstituted at I = 0.07 and R = 4000 gave a clear solution and showed no artifacts due to light scattering. The conformation based on circular dichroism and enzymatic activity of the detergent-soluble enzyme were unchanged upon reconstitution. The reconstituted enzyme in lipid vesicles seemed to be slightly more stable against thermal denaturation than the protein in sodium cholate solution.     

THE DENATURATION OF COVALENTLY INHIBITED SWINE PEPSIN

Studies are reported on the denaturation of freshly prepared, intact swine pepsin, which was inactivated by reaction with diazoacetylglycine ethyl ester, to prevent autolysis. Denaturation about pH 6 was found to involve a small expansion of the molecular domain with some loss of organized secondary structure. On the other hand, increasing concentrations of guanidine hydrochloride induced cooperative transitions in both the native and alkali denatured forms to give a cross-linked random coil. No conditions could be found in which these reactions were reversible. Removal of denaturing conditions usually resulted in aggregation and precipitation of protein. From these studies, it would seem that the active conformation is largely predetermined in the zymogen.     

Mucopolysaccharides in aqueous solutions: effect of ionic strength on titration curves

We study the changes taking place in hyaluronic acid, chondroitin 4-sulfate (C4-S) and condroitin 6-sulfate (C6-S), at ionic strengths of 0.10, 0.15, and 0.20 in NaCl, in a neutralization process in aqueous solution. We apply the equation of Henderson Hasselbalch modified for polyelectrolytes and evaluate the changes in the electrostatic free energy starting from the pK curves as a function of the dissociation degree. For a dissociation degree next to 0.4 corresponding to the -COOH group of the hyaluronic acid, we observed a change in the conformation of the three glycosaminoglycans studied. This conformational change takes place as a consequence of the break of intramolecular links and the beginning of the ionization process. The macromolecules in solution show a structure of random coil sufficiently expanded so that the interaction among the close ionizable groups is negligible.     

Denaturation of concanavalin A by urea at acid pH

The denaturation of dimeric concanavalin A induced by urea at pH 3 has been studied using optical activity and sedimentation velocity. Under the conditions employed Mn⁺² and Ca⁺² are dissociated from the protein, but the basic structural elements are little changed from those prevailing in the functional lectin at pH 5.5 [H.E. Auer and T. Schilz, preceding paper in this issue]. The protein passes through three stages as the urea concentration is varied from 0 to 10 M. Below 4M urea the only effect observed is the loss of optical activity of the aromatic amino acid residues. At 4 M, a conformational change occurs producing extensive aggregation, which persists to 7 M. At 8–10 M urea a disordered monomeric protein molecule prevails. The protein could be reactivated provided that dilution to native conditions was very rapid and the protein concentration remained very low. Kinetics of denaturation were monitored by optical activity at 218, 225 and 283 nm, Transients with one, two or three components were observed, which were resolved by nonlinear regression according to sequential first-order decay laws. First order character was confirmed by independence of the kinetic parameters from protein concentration over a two- to four-fold range. Enthalpies and entropies of activation for the various steps were also determined. The transients at the three wavelengths monitor changes in β structure, β turns and aromatic groups, respectively. The urea dependence of the rate constants is unique in most cases. It is concluded that different structural elements of the concanavalin A molecule unfold independently from one another.     

Interplay between molecular conformation and intermolecular interactions in conformational polymorphism: a molecular perspective from electronic calculations of tolfenamic acid

Tolfenamic acid exhibits conformational polymorphism. The molecules in its two commonly occurred crystal structures form similar hydrogen-bonded dimers but differ in conformation. The conformational variance was analyzed by electronic calculation methods with the aim to unravel intrinsic connection between the conformational flexibility and intermolecular interactions in the polymorphs. The study was conducted mainly by conceptual density functional theory (DFT) and natural bond orbital (NBO) analysis. It is found that the conformational polymorphism is resulted from the energy competition between intramolecular π-conjugation and intermolecular hydrogen bonding. By adapting conformation that departs from being the most energetically stable, tolfenamic acid molecules can strengthen the intermolecular hydrogen-bonding interactions in the crystals. The study illustrates how the molecule's electronic properties are influenced by conformational variation and, inherently, how the intermolecular interactions become regulated. Moreover, understanding molecular interaction and crystal packing necessitates electronic structure calculation and analysis, which can be further facilitated by utilizing DFT and NBO concepts.     

Hydrogels of N-isopropylacrylamide copolymers with controlled release of a model protein

Temperature- and pH-sensitive hydrogels, based on N-isopropylacrylamide (NiPAAm) and itaconic acid (IA), were synthesized by free radical crosslinking copolymerization in the presence of lipase from Candida rugosa. The samples were characterized for their sensitivity to the changes of external conditions and the ability to control the release of a hydrophilic model protein, lipase. These hydrogels were highly responsive to temperature and pH, at constant ionic strength. Parameters, such as the crosslinking degree and non-ionic/ionic (NiPAAm/IA) ratio, were found to impact the hydrogel structure, mechanical properties, morphology and swelling kinetics at different pH and temperatures. The hydrogels demonstrated protein loading efficiency as high as 95 wt%. Release studies of a hydrophilic model protein at a physiological temperature of 37 °C were performed at different pH values. High dependence of lipase release kinetics on hydrogel structure and the environmental pH was found, showing generally low release rates, lower in acidic media (pH 2.20) and higher at higher pHs (6.80). Lipase activity was retained even after treatment conditions that would provoke denaturation of the enzyme if it was not protected in the gel. The obtained hydrogels were found suitable for releasing therapeutic proteins in a controlled manner at specific sites in gastrointestinal tract.     

Simultaneous differential scanning calorimetry,X-ray diffraction and FTIR spectrometry in studies of ovalbumin denaturation

The new application of differential scanning calorimetry (DSC) and the susceptibility of ovalbumin to α-chymotrypsin gave a quantitative estimation of protein denaturation in solid ovalbumin. Solid ovalbumin in granules with 11% of water was heated at 100 °C in closed and nonclosed ampules. In order to compare effects of size and crystal structure, two proteins (bovine albumin and γ-globulin) were examined at similar conditions for the extent of denaturation. Ovalbumin and bovine albumin showed similar extents of denaturation, but γ-globulin, with a very different molecular mass, showed the maximal conformational changes. The enthalpy of denaturation was measured to elucidate the conformational changes in solid proteins. Its value was used for calculation of the degree of denaturation. The thermodynamic data associated with transition were calculated and the number of bonds broken during denaturation was determined. Intrinsic fluorescence was utilized in order to compare these two methods. Moreover, X-ray diffraction and FTIR spectrometry were applied to native and denatured proteins.     

Simulations of conformers of tuftsin and a cyclic tuftsin analog

The conformational properties of the configurational isomers of tuftsin, a linear tetrapeptide with the sequence Thr-Lys-Pro-Arg, were investigated with six 1 ns molecular dynamics simulations in explicit water and in a 1.0 M NaCl solution. The average conformation of the cis isomer is a type VI β-turn. Our results indicate that water-peptide hydrogen bonding, in addition to intramolecular hydrogen bonds, stabilizes the cis conformer. The trans isomer is neither a β- nor a γ-turn. Results are compared with parallel studies on a cyclic analog of tuftsin, cyclo(Thr-Lys-Pro-Arg-Gly). The addition of salt does not influence the backbone conformation of the peptide. Differences between the structures are confined to the side-chain orientations of the Lys and Arg residues. © Munksgaard 1995.     

STRUCTURAL STUDIES ON THE SUCCINYLATED BOVINE SERUM ALBUMIN

An analysis of succinylated bovine serum albumin showed that all of the 62 α-amino groups of lysine residues, 13 hydroxyamino acid residues and 12 tyrosin residues were succinylated, but the succinylated tyrosines were later deacylated. Structural properties of the modified protein have been studied with circular dichroism and sedimentation velocity. At neutral pH (pH 7.60) and in a salt-free aqueous solution the modified protein is in an expanded form and its helical content is only 30% of that of unmodified protein. The increase of ionic strength restores the original conformation of the protein, whereas the increase of pH further disorganizes the structure of the protein. The results suggest that the electrostatic force alone is responsible for the compact structure of the protein molecule. The same mechanism is believed to underlie the effect of H₃O+ and the effect of succinylation of side chain groups on the conformation of bovine serum albumin.     

Predicted secondary structure of bovine prothrombin fragment 1 and related proteins in different environments by circular dichroism spectroscopy

Circular dichroism spectroscopy was used to investigate the structure of bovine prothrombin fragment 1 (BF1) and related proteins in several environments. The conformational change induced in BF1 by the addition of Mg[II] ions was found to be different from that induced by Ca[II] or Sr[II]. The Ca[II] and Sr[II] conformations appear to differ only slightly from the apo-metal conformation. The conformation of the 1–45 fragment of prothrombin, however, is markedly different than the conformation of the same fragment in the presence of either Ca[II] of Mg[II]; both of the latter structures differ substantially from one another. The presence of phospholipids has almost no effect on the structure of either BF1 or the 1–45 fragment; in the presence of both phospholipids and Ca[II] a structural change is seen for the 1–45 fragment but not BF1 (relative to the protein alone). The addition of phospholipids to the Mg[II]/BFl structure did not induce a CD-detectable conformational change, while the addition of phospholipids to the Ca[II]/BFl or Sr[II]/BFl structures induced a change to a conformation similar in secondary structure composition to the relative apometal structures.     

THEORY OF REVERSIBLE DENATURATION OF GLOBULAR PROTEINS

A theoretical method is developed by which the character of the process of protein denaturation (e.g., whether or not it is of the all-or-none type) can be discussed in terms of conformation of native proteins and the forces stabilizing it. An important role is played by a quantity S(H): entropy of a protein molecule in solution in the conformational states with a given value of enthalpy H. It is demonstrated that the all-or-none type denaturation of proteins is a rather direct consequence of the globularity and specificity of the native conformations. Denaturations with significant intermediate states are discussed. Denaturations induced by added denaturants are also discussed.     

Elucidation of different binding modes of purine nucleosides to human deoxycytidine kinase

Purine nucleoside analogues of medicinal importance, such as cladribine, require phosphorylation by deoxycytidine kinase (dCK) for pharmacological activity. Structural studies of ternary complexes of human dCK show that the enzyme conformation adjusts to the different hydrogen-bonding properties between dA and dG and to the presence of substituent at the 2-position present in dG and cladribine. Specifically, the carbonyl group in dG elicits a previously unseen conformational adjustment of the active site residues Arg104 and Asp133. In addition, dG and cladribine adopt the anti conformation, in contrast to the syn conformation observed with dA. Kinetic analysis reveals that cladribine is phosphorylated at the highest efficiency with UTP as donor. We attribute this to the ability of cladribine to combine advantageous properties from dA (favorable hydrogen-bonding pattern) and dG (propensity to bind to the enzyme in its anti conformation), suggesting that dA analogues with a substituent at the 2-position are likely to be better activated by human dCK.     

Utilization of Zwitterion-Based Solutions to Dissect the Relative Effects of Solution pH and Ionic Strength on the Aggregation Behavior and Conformational Stability of a Fusion Protein

Solution pH and ionic strength (I) have complex effects on protein stability. We developed an experimental approach based on exploitation of the zwitterionic characteristic of amino acid molecules to probe the relative contribution from each. A variety of types of amino acid solutions were adopted to investigate the effects of pH and I in a manner that allows independent evaluation of each factor. The same effect could not be achieved using conventional buffer solutions. Size-exclusion chromatography, capillary differential scanning calorimetry, and fluorescence spectroscopy were utilized to probe the protein aggregation and conformation. The results suggested that, in addition to pH, solution ionic strength as a function of ionization state of the amino acid molecules and the ions introduced by pH adjustment played an important role in the aggregation and conformation of the protein studied. This experimental approach offers a useful tool to aid fundamental understanding of the relative effects of solution pH and ionic strength on protein stability.     

Improved stability of proteins immobilized in microparticles prepared by a modified emulsion polymerization technique.

Proteins can be immobilized in spherical microparticles of polyacrylamide gel (having a diameter of about 1 microgram) by an emulsion--polymerization technique. Higher cross-linked gels have a structure consisting of relatively large pores. This structure is advantageous when dealing with biologically active proteins acting on molecules dissolved in the surrounding medium. A rapid equilibrium is established between the interior of the particles and the medium, and rate-limiting diffusion is not observed. A suspension of carbonic anhydrase immobilized in microparticles will thus have kinetic properties very much like the free enzyme. In addition to the entrapment of the protein molecules in the three-dimensional network formed by the polyacrylamide threads, protein molecules are also fixed in the cross-linked threads of polyacrylamide. This fixation is probably responsible for the improved stability of the protein molecules against heat denaturation. Not even autoclaving at 110 degrees for 30 min denatured the immobilized enzyme completely (more than 25% of the enzyme activity was left). The higher resistance of molecules in microparticles against proteolytic degradation also is documented.     

Role of a novel excipient poly(ethylene glycol)-b-poly(L-histidine) in retention of physical stability of insulin at aqueous/organic interface

The aim of this study was to investigate whether a cationic polyelectrolyte, poly(ethylene glycol)-b-poly(L-histidine) diblock copolymer (PEG-polyHis), can stabilize insulin, at the aqueous/methylene chloride interface formed during the microencapsulation process. Insulin aggregation at this interface was monitored spectrophotometrically at 276 nm. The effects of protein concentration, pH of the aqueous medium, and the presence of poly(lactic-co-glycolic acid) (PLGA) in methylene chloride (MC) on insulin aggregation were observed. For the 2.0 mg/mL insulin solutions in phosphate buffer (PB), the effect of addition of Pluronic F-127 as a positive control and addition of PEG-polyHis as a novel excipient in PB was also evaluated at various insulin/polymeric excipient weight ratios. The conformation of insulin protected by PEG-polyHis and recovered after interfacial exposure was evaluated via circular dichroism (CD) spectroscopy. Greater loss in soluble insulin was observed with increasing insulin concentrations. pH 6.0 was selected for optimal ionic interactions between insulin and PEG-polyHis based on zeta potential and particle size studies. pH 4.5 and 7.4 (no ionic complexation between insulin and PEG-polyHis) were selected as controls to compare the stabilization effect of PEG-polyHis with that at pH 6.0. Incubation of PEG-polyHis with insulin at pH 6.0 drastically reduced protein aggregation, even in the presence of PLGA. PEG-polyHis and F-127 reduced insulin aggregation in noncomplexing pH conditions pointing to the role played by PEG in modulation of insulin adsorption at the interface. Far-UV (205-250 nm) CD study revealed negligible qualitative effects on soluble insulin's secondary structure after interfacial exposure. RP-HPLC and size-exclusion HPLC showed no deamidation of insulin or formation of soluble high molecular weight transformation products respectively. MALDI-TOF mass spectrometry confirmed the results from chromatographic procedures. Radioimmunoassay carried out on select samples showed that recovered soluble insulin had retained its immunoreactivity. An experimental method to simulate interfacial denaturation of proteins was designed for assessment of protein stability at the interface and screening for novel protein stabilizers. Understanding and manipulation of such polyelectrolyte-insulin complexation will likely play a role in insulin controlled delivery via microsphere formulation(s).     

Physicochemical studies on the stability of influenza haemagglutinin in vaccine bulk material.

The relative unknown conformational stability of monovalent bulks of influenza virus haemagglutinin (HA) from three different strains (B/Guangdong, A/New Caledonia and A/Panama) was investigated with fluorescence and circular dichroism (CD) spectroscopy. Various stress conditions (concentration of denaturant, freeze-thawing, pH and temperature) affected the spectroscopic properties of the haemagglutinin proteins differently. Unfolding experiments revealed a poor stability of Guangdong haemagglutinin (GD-HA) in comparison with New Caledonia (NC-HA) and Panama haemagglutinin (P-HA). Freeze-thawing altered the secondary and tertiary structure of Guangdong haemagglutinin and only the tertiary structure of Panama haemagglutinin. From pH 4.6-9.2 the tertiary structures of Guangdong, New Caledonia and Panama haemagglutinin were all affected to a different extent. The secondary structure was only altered at low pH. Incubation of haemagglutinin at 60 degrees C resulted in denaturation of the protein and a dramatic change of the fluorescence spectrum, indicative of oxidised tryptophan (Trp). In conclusion, fluorescence and circular dichroism spectroscopy are highly suitable techniques to monitor the stability of haemagglutinin in a straightforward and fast way.     

Dissociation,unfolding and inactivation of creatine kinase in urea solutions

It was previously reported that, during unfolding of creatine kinase in guanidinium chloride or urea solutions, inactivation occured before noticeable conformational change could be detected, suggesting that the conformation at the active site is more easily perturbed and, hence, more flexible than the molecule as a whole [Tsou (1986) Trends Biochem. Sci. 11 , 417–429]. In the present paper, the urea-gradient electrophoresis and the isoenzyme hybrid of creatine kinase has been studied. The results show that at low urea concentrations, creatine kinase is still in the dimeric state or only slightly dissociated. The dissociation and inactivation of creatine kinase during denaturation by urea are also compared. It was found that the enzyme was nearly inactivated in low urea concentrations before noticeable dissociation was detected. It therefore appears that in low urea concentrations, inactivation of creatine kinase is not due to the dissociation of the active dimer. The present result supports the hypothesis of the conformational flexibility of the active site in this enzyme. © Munksgaard 1996.