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.     

Fluoroalcohols as structure modifiers in peptides and proteins: hexafluoroacetone hydrate stabilizes a helical conformation of melittin at low pH

Abstract: The effect of hexafluoroacetone hydrate (HFA) on the structure of the honey bee venom peptide melittin has been investigated. In aqueous solution at low pH melittin is predominantly unstructured. Addition of HFA at pH ≈ 2.0 induces a structural transition from the unstructured state to a predominantly helical conformation as suggested by intense diagnostic far UV CD bands. The structural transition is highly cooperative and complete at 3.6 m (50% v/v) HFA. A similar structural transition is also observed in 2,2,2 trifluoroethanol which is complete only at a cosolvent concentration of ≈ 8 m . Temperature dependent CD experiments support a ‘cold denaturation’ of melittin at low concentrations of HFA, suggesting that selective solvation of peptide by HFA is mediated by hydrophobic interactions. NMR studies in 3.6 m HFA establish a well-defined helical structure of melittin at low pH, as suggested by the presence of strong NH_i/NH_i+1 NOEs throughout the sequence, along with many medium range helical NOEs. Structure calculations using NOE-driven distance constraints reveal a well-ordered helical fold with a relatively flexible segment around residues T10–G11–T12. The helical structure of melittin obtained at 3.6 m HFA at low pH is similar to those determined in methanolic solution and perdeuterated dodecylphosphocholine micelles. HFA as a cosolvent facilitates helix formation even in the highly charged C-terminal segment.     

Spectroscopic studies on proteinase K and subtilisin DY

Circular dichroic spectroscopy has been used to study the effect of pH, guanidinium hydrochloride concentration and temperature on the conformation of the fungal subtilisin-like proteinase K and the bacterial DY. The ellipticity of the bands in the far ultraviolet region remains almost unchanged in the pH range 3.0-11.0 (PMS-proteinase K) and 5.0-10.0 (PMS-subtilisin DY). The same ranges of pH stability were determined from the pH dependence of the near ultraviolet dichroic spectra. Hence the changes in the tertiary and secondary structure occur in parallel. Proteinase K is considerably more stable at acidic and somewhat more stable at alkaline pH than subtilisin DY. At neutral pH proteinase K is more resistant to denaturation by guanidinium hydrochloride than is subtilisin DY. The midpoints of the denaturation curves were 6.2 M and 3.2 M guanidinium, respectively. The thermal unfolding of proteinase K occurred at a higher temperature than for subtilisin DY, the transition midpoints being 65° and 48°, respectively. Thus proteinase K is overall a much more robust molecule than subtilisin DY, showing greater resistance to all three forms of denaturation. The differences in the stability of the two proteinases can be partly explained by differences in their calcium binding sites.     

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.     

Application of Second-Derivative Fluorescence Spectroscopy to Monitor Subtle Changes in a Monoclonal Antibody Structure

In this study, the tertiary structure of a monoclonal antibody was analyzed under thermal and chemical stresses using second‐derivative fluorescence spectroscopy. The effect of polyols, sucrose, and ethylene glycol on the tertiary structure of monoclonal antibody‐U (mAb‐U) (pH 7.0) was studied under thermal stress (25°C–75°C). The tertiary structure of mAb‐U was also analyzed upon chemical denaturation using urea (2.0–8.0 M). The second derivative of mAb‐U showed three bands corresponding to the three spectral classes of tryptophan, class I (330 nm), class II (340 nm), and class III (350 nm). Class II was higher in intensity in the presence of polyols compared with the solution without any polyol. Thermally denatured structure of mAb‐U in sucrose and ethylene glycol was distinctly different than that in buffer. Addition of urea resulted in a decrease in intensity of class I and II, and an increase in intensity of class III implying unfolding. This study showed that second‐derivative fluorescence spectroscopy is an effective tool to monitor subtle alterations in the tertiary structure of proteins. The unfolding of a protein is reflected as an increase in the intensity of the polar class III accompanied with a decrease in the intensity of class I.     

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.     

Effects of pH, temperature, and sucrose on benzyl alcohol-induced aggregation of recombinant human granulocyte colony stimulating factor

Antimicrobial preservatives (e.g., benzyl alcohol), which are required in multidose formulations, can induce protein aggregation. In this study, the mechanism of benzyl alcohol-induced aggregation of recombinant human granulocyte colony-stimulating factor (rhGCSF) was investigated by determining the effects of temperature, pH, and sucrose on this process. rhGCSF was incubated at 25 and 37 degrees C and at pH 7.0 (phosphate-buffered saline, PBS) and pH 3.5 (HCl). Benzyl alcohol (0.9% w/v) accelerated aggregation of rhGCSF at pH 7.0, an effect that was much greater at 37 degrees C than at 25 degrees C and partially counteracted by 1.0 M sucrose. At pH 3.5, benzyl alcohol did not induce aggregation of rhGCSF. Spectroscopic studies showed that 0.9% benzyl alcohol altered the tertiary structure of rhGCSF at both pH, without detectably altering secondary structure. Structural perturbation was greater at 37 degrees C than at 25 degrees C. At both pH 7.0 and 3.5, the hydrogen-deuterium (H-D) exchange rate for rhGCSF was increased by 0.9% benzyl alcohol. Sucrose (1.0 M) partially counteracted the benzyl alcohol-induced perturbation of tertiary structure and the increase in H-D exchange rate. Thus, benzyl alcohol accelerates aggregation of rhGCSF at pH 7.0, because it favors partially unfolded aggregation-prone conformations of the protein. Sucrose partially counteracts benzyl alcohol-induced rhGCSF aggregation by shifting the molecular population away from these species and towards more compact conformations. We postulate that the absence of aggregation at pH 3.5, even with benzyl alcohol-induced structural perturbation, is due to the unfavorable energetics of intermolecular interactions (i.e., colloidal stability) between rhGCSF molecules at this pH.     

Elucidation of two major aggregation pathways in an IgG2 antibody

Two major aggregation pathways observed in an IgG2 molecule are described. Different aggregate species generated by long-term incubation of the antibody at 37 degrees C were collected by a semi-preparative size exclusion chromatography method. These purified species were analyzed extensively by denaturing size-exclusion chromatography methods. The major aggregation pathway at low pH (4.0) resulted in the formation of both dimers and high molecular weight (HMW) aggregates. It was found that these dimers and HMW aggregates contain antibody molecules that have a peptide bond cleavage between an aspartic acid and proline residue in the CH2 domain. Evidence that unfolding of the CH2 domain may be driving the aggregation at low pH is presented. At higher pH (pH - 6.0), formation of a dimer having approximately 75% covalent character was the major aggregation pathway while formation of higher molecular weight aggregates were largely suppressed. The covalent dimer consisted of both disulfide linked antibody molecules and another species (approximately 26%) that was formed due to nondisulfide covalent bonds between two heavy chains. At pH - 5.0, both dimer and higher molecular weight aggregates were formed and the aggregation pathway was a combination of the major pathways observed at pH - 4.0 and 6.0. The dimer species formed at pH - 5.0 had a larger contribution from covalent species-both disulfide and nondisulfide linked, while the HMW aggregate contained a higher percentage of molecules that had the peptide bond cleavage in the CH2 domain. The dimer formed at pH - 6.0 was found to have identical secondary and tertiary structure as the intact antibody molecule. However, the dimer and higher molecular weight aggregate formed at pH - 4.0 have altered secondary and tertiary structure.     

Biophysical characterisation of thermal-induced precipitates of recombinant anthrax protective antigen: Evidence for kinetically trapped unfolding domains in solid-state

Insoluble aggregation or precipitation is one of the most common degradation pathways observed for biotherapeutics; despite this, the structural mechanisms by which this occurs remain poorly understood due to difficulties associated with biophysical characterisation of protein particulates. To address this knowledge gap, we developed a solid-state circular dichroism (CD) technique, which allows in situ measurements of the secondary and tertiary structural changes associated with the formation of visible therapeutic protein aggregates. We demonstrate how solid-state CD, in conjunction with other biophysical and computational methods can aid in gaining valuable insights into the mechanisms and pathways of thermal-induced precipitation of Bacillus anthracis recombinant protective antigen (rPA), the primary immunogen of anthrax subunit vaccine. Using these methods, we show the domains d3 and d4 are the most labile of the four structurally distinct domains of rPA and play the critical role in nucleating the cascade of unfolding and aggregation. During the assembly process, the domains d1 and d2 become kinetically trapped within the insoluble aggregate and reveal previously intractable distinct tertiary structural elements of the rPA native structure. These findings reveal a uniquely detailed insight into the role of rPA domains on protein stability and provide a mechanistic framework for thermal-induced unfolding and precipitation. It also shows that solid-state CD provides a novel approach in characterising protein precipitation that may facilitate rational improvements to the stability of biopharmaceuticals.     

The application of capillary electrophoresis for monitoring effects of excipients on protein conformation

Studies were conducted to assess the utility of free solution capillary electrophoresis (CE) for monitoring the effects of selected excipients on the thermal denaturation of a model protein (Ribonuclease A, RNase A) at low pH. Thermal denaturation/unfolding experiments were conducted via temperature-controlled CE using a run buffer of 20 mM citric acid in the pH range of 2.3–3.1, with a marker peptide incorporated to correct for temperature-induced changes in endoosmotic flow. The effects of selected excipients on the thermal unfolding of RNase A were then evaluated by adding either sorbitol, sucrose, polyethylene glycol 400 (PEG 400) or 2-methyl-2,4-pentanediol (MPD) to the electrophoretic run buffer (pH 2.3). Confirmatory denaturation experiments were conducted under the same solution conditions using circular dichroism (CD) spectropolarimetry. Using temperature-controlled CE, an increase in solution pH from 2.3 to 2.7 and 3.1 resulted in an increase in transition temperatures of RNase A by approximately 8 and 13°C, respectively. Similar shifts in transition temperatures were observed when thermal denaturation transitions were monitored by far-UV CD. Sorbitol (0.55–1.1 M) and sucrose (0.55 M) each shifted the denaturation transition temperatures of RNase A to higher values, whereas PEG 400 and MPD had minimal effect on the unfolding transition midpoint at the concentrations evaluated (0.55 M for each). The observed changes in the transition temperatures for RNase A as a function of pH and selected excipients were similar when measured by either CE or far-UV CD. These results support the utility of CE for monitoring the effects of neutral excipients on the thermal denaturation of a model protein under selected conditions. The widespread utility of the technique may be limited by the narrow temperature range of most commercial CE instruments and the need to use extreme pH conditions to monitor the complete denaturation transition.     

Solid state stability of proteins III: calorimetric (DSC) and spectroscopic (FTIR) characterization of thermal denaturation in freeze dried human growth hormone (hGH)

This research is a study of the changes in secondary structure (Fourier transform infrared spectroscopy, FTIR), aggregation, and loss of the magnitude of the heat of denaturation upon scanning to and partially through the temperature range of the thermal denaturation peak of a model protein, human growth hormone (hGH). We study two formulations, a system of essentially pure protein (with a trace of phosphate buffer) and a system formulated with trehalose in a 3:1 trehalose:hGH weight ratio. The extent of denaturation is measured by loss of secondary structure by FTIR, the loss of heat of denaturation by differential scanning calorimetry (DSC), and the fraction of protein aggregated by HPLC. We examine loss of structure on heating to the DSC onset of thermal denaturation and restoration of structure by cooling below the denaturation temperature and holding to (nominally) allow time for refolding, and we also examine restoration of structure upon dissolving and refreeze drying samples heated to selected temperatures in the denaturation range. We find that denaturation occurs only above the glass transition temperature, is highly cooperative, and is only reversible by redissolving the "denatured" formulated (trehalose) solid. Further, all measures of the extent of denaturation are in essential agreement.     

Spectroscopic study on the structure and stability of beef liver arginase

The secondary and tertiary structure of the oligomeric arginase (EC 3.5.3.1) from beef liver was investigated by circular dichroism (CD) and fluorescence measurements. The far-ultraviolet CD spectrum of the enzyme at neutral pH is indicative of high helical content. The intrinsic fluorescence emission of the protein is due to tryptophan, the contribution of tyrsoine being small. Upon excitation at 295 nm, the maximum of emission occurs at 330 nm, implying that the trytophan residues are rather buried in a hydrophobic interior of the protein. Ethylenediaminetetraacetic acid (EDTA), which inactivates the enzyme by removing the functional Mn²⁺-ion from the enzyme, does not dissociate the enzyme into subunits, nor affect noticeably its secondary and tertiary structure. Inactivation occurs in the acid pH range, being complete at pH below 4. However, acidification up to pH 1.5 produced only limited changes in the far-ultraviolet CD spectrum and intrinsic fluorescence emission properties. The enzyme shows noteworthy thermal stability, as shown by measuring the residual activity after heating and by evaluating the temperature dependence of the CD signal at 220 nm and the intensity of emission fluorescence. A temperature of half inactivation (Tm) of 77° was determined upon heating the enzyme at pH 7.5 in the presence of Mn²⁺-ions for 10 min; in the presence of EDTA, Tm is shifted to 55°. Taken together, these observations indicate that the structural stability of beef liver arginase arises from a clustering of hydrophobic amino acids and from Mn²⁺-ion binding.     

Effects of PEG size on structure, function and stability of PEGylated BSA

The effects of PEGylation on the structural, thermal and functional stability of bovine serum albumin (BSA) were investigated using BSA and 6 linear mono-PEGylated BSA compounds. The secondary and tertiary structure of BSA measured by circular dichroism (CD) was independent of PEGylation. In contrast, the thermal stability of BSA was affected by PEGylation. The apparent unfolding temperature T_max measured by differential scanning calorimetry (DSC) decreased with PEGylation, whereas the temperature of aggregation, T_agg, measured by dynamic light scattering (DLS) increased with PEGylation. The unfolding temperature and the temperature of aggregation were both independent of the molecular weight of the PEG chain. Possible functional changes of BSA after PEGylation were measured by Isothermal Titration Calorimetry (ITC), where the binding of sodium dodecyl sulphate (SDS) to BSA and PEGylated BSA was analysed. At 25 °C, two distinct classes of binding sites (high affinity and low affinity) for BSA and one class of binding site (low affinity) for PEGylated BSA were identified. The binding isotherm was modelled assuming independence and thermodynamic equivalence of the sites within each class. From the present biophysical characterisation, it is concluded that after PEGylation BSA appears to be unaffected structurally (secondary and tertiary structure), slightly destabilised thermally (unfolding temperature), stabilised kinetically (temperature of aggregation) and has an altered functionality (binding profile). These biophysical characteristics are all independent of the molecular weight of the attached polymer chain.     

Non-native Intermediate Conformational States of Human Growth Hormone in the Presence of Organic Solvents

Purpose. Manufacturing processes expose protein pharmaceuticals to organic solvents that may perturb the native folded state, increasing the potential for irreversible aggregation or surface adsorption. The aim of this study was to characterize the conformational states of human growth hormone (hGH) in aqueous ethanolic solutions.Methods. The higher order structure of hGH was investigated using far- and near-UV circular dichroism (CD) and fluorescence spectroscopy as orthogonal techniques, and the hydrodynamic size was monitored using dynamic light scattering.Results. CD data suggested that the secondary structure of hGH remained unchanged up to 50\% (v/v) ethanol, but the tertiary structure was perturbed at ã20% ethanol. Fluorescence anisotropy, however, showed that the mobility of the buried Trp residue was restricted even at 30% ethanol, suggesting a differently packed structural core in 30% ethanol relative to the native structure. Consistent with this result, thermal unfolding of hGH in 30% ethanol was more facile compared to that in 0% and 20% ethanol. At >40% ethanol, fluorescence data were consistent with increased solvent exposure of the tryptophan.Conclusions. The results point to progressive unfolding of hGH that increases solvent exposure of the hydrophobic core as a function of ethanol concentration and suggest that non-native intermediate states are populated in 30–60% ethanol.     

Interdomain Interactions in the Chimeric Protein Toxin sCD4(178)-PE40: A Differential Scanning Calorimetry (DSC) Study

The thermal denaturation of the chimeric protein toxin known as sCD4(178)-PE40 (sCD4-PE40) was studied using differential scanning calorimetry (DSC). sCD4-PE40 consists of HIV-binding domains of the T-cell membrane protein known as CD4 and the cytotoxic domains of Pseudomonas exotoxin A (PE40). sCD4-PE40 undergoes two DSC transitions. An endothermic transition associated with unfolding of the CD4 and PE40 components occurs at approximately 46°C in buffered saline at pH 6.5. An exothermic transition associated with precipitation of unfolded protein occurs at higher temperatures. Both transitions are irreversible. DSC studies of solutions of pH 5.0 to 9.5 indicate that sCD4-PE40 shows maximal thermal stability at around pH 6.5. Variable pH experiments are also presented on solutions of sCD4(183) and PE40 revealing how these components denature as independent structural entities. sCD4(183) denaturation occurs at significantly higher temperatures than does the CD4 component of sCD4-PE40. PE40 denaturation occurs at the same temperatures as sCD4-PE40. These results suggest that the native CD4 and PE40 components are independent and non-interacting entities in the chimeric sCD4-PE40 molecule and that unfolding of the less-stable PE40 component induces unfolding of the CD4 component. These destabilizing interdomain interactions of sCD4-PE40 are in contrast to the stabilizing interactions which apparently exist in wild-type Pseudomonas exotoxin A between its PE40 domains and the cell binding domain of the native toxin (analogous to the CD4 component in sCD4-PE40). Reasons are discussed why the type of interdomain interactions observed for sCD4-PE40 might be the norm for chimeric proteins.Brian E. Collins: B.E.C.'s contribution was made while he was a summer intern at The Upjohn Company.     

CONFORMATIONAL STABILITY OF FRUCTOSE-1,6-BIPHOSPHATE ALDOLASE FROM CERATITIS CAPITATA

Fructose 1,6-biphosphate aldolase from Ceratitis capitata is a tetramer of identical subunits with 34% α-helix, 22% β structure and 44% of aperiodic order. Increase of urea concentration up to 4.0 M results in non-cooperative reversible dissociation of the enzyme. Sodium dodecylsulphate 0.06% (w/v) dissociates the tetramer cooperatively with retention of the helical content. Thermal denaturation was a non-reversible cooperative process with a midpoint for the transition at 55°. Cysteine residues are involved in this process and 2-mercapto-ethanol preserves partially the enzyme activity. The acidic dissociation of the enzyme is a non-reversible process in contrast to the reversible basic dissociation. Increase of ionic strength results in a more ordered secondary structure for the monomer after acidic dissociation.     

Preformulation Study of Highly Purified Inactivated Polio Vaccine,Serotype 3

To improve the effectiveness of the polio vaccination campaign, improvements in the Thermal Stability of IPV3 at pH 7 as the vaccine are being investigated. Here, inactivated polio vaccine, serotype 3 (IPV3) was characterized via a number of biophysical techniques. The size was characterized by transmission electronic microscopy and light scattering. The capsid protein conformation was evaluated by intrinsic fluorescence and circular dichroism (CD), and the D-antigen content by enzyme-linked immunosorbent assay (ELISA). The pH thermal stability of IPV3 (pH 3.0–8.0; 10°C–87.5°C) was evaluated by fluorescence, CD, and static light scattering. The transition temperatures reflect the responses, respectively, of tertiary structure, secondary structure, and size to applied thermal stress. The data were summarized as empirical phase diagrams, and the most stable conditions were found to be pH 7.0 with temperature lower than 40°C. CD detected a higher transition temperature for capsid protein than that for RNA. The effects of certain excipients on IPV3 thermal stability and antigen content were evaluated. The results of their effects, based on intrinsic fluorescence and ELISA, were in good agreement, suggesting the feasibility of applying intrinsic fluorescence as a high-throughput tool for formulation development. The study improves the understanding of IPV3 thermal stability, and provides a starting point for future formulation development of IPV3 and other serotypes. © 2013 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 103:140–151, 2014     

Biophysical characterisation of GlycoPEGylated recombinant human factor VIIa

The effects of GlycoPEGylation on the structural, kinetic and thermal stability of recombinant human FVIIa were investigated using rFVIIa and linear 10 kDa and branched 40 kDa GlycoPEGylated(?) recombinant human FVIIa derivatives. The secondary and tertiary structure of rFVIIa measured by circular dichroism (CD) was maintained upon PEGylation. In contrast, the thermal and kinetic stability of rFVIIa was affected by GlycoPEGylation, as the apparent unfolding temperature T(m) measured by differential scanning calorimetry (DSC) and the temperature of aggregation, T(agg), measured by light scattering (LS) both increased with GlycoPEGylation. Both T(m) and T(agg) were independent of the molecular weight and the shape of the PEG chain. From the present biophysical characterisation it is concluded that after GlycoPEGylation, rFVIIa appears to be unaffected structurally (secondary and tertiary structure), slightly stabilised thermally (unfolding temperature) and stabilised kinetically (temperature of aggregation).     

Trypsin-pancreatic secretory isoinhibitor A from bovine pancreas (Kazal type)

The secondary and tertiary structure of isoinhibitor A from bovine pancreas secretion (Kazal inhibitor) was investigated by circular dichroism (CD) and fluorescence measurements. The protein shows noteworthy thermal stability as seen by the temperature dependence of the CD spectra and the intensity of emission fluorescence at different pH values.     

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.