Reactivity toward deamidation of asparagine residues in beta-turn structures.

Mimetics of beta-turn structures in proteins have been used to calibrate the relative reactivities toward deamidation of asparagine residues in the two central positions of a beta-turn and in a random coil. N-Acetyl-Asn-Gly-6-aminocaproic acid, an acyclic analog of a beta-turn mimic undergoes deamidation of the asparaginyl residue through a succinimide intermediate to generate N-acetyl-Asp-N-Gly-6-aminocaproic acid (6-aminocaproic acid, hereafter Aca) and N-acetyl-L-iso-aspartyl (isoAsp)-Gly-Aca (pH 8.8, 37 degrees C) approximately 3-fold faster than does the cyclic beta-turn mimic cyclo-[L-Asn-Gly-Aca] with asparagine at position 2 of the beta-turn. The latter compound, in turn, undergoes deamidation approximately 30-fold faster than its positional isomer cyclo-[Gly-Asn-Aca] with asparagine at position 3 of the beta-turn. Both cyclic peptides assume predominantly beta-turn structures in solution, as demonstrated by NMR and circular dichroism characterization. The open-chain compound and its isomer N-acetyl-Gly-Asn-Aca assume predominantly random coil structures. The latter isomer undergoes deamidation 2-fold slower than the former. Thus the order of reactivity toward deamidation is: asparagine in a random coil approximately 3x(asparagine) in position 2 of a beta-turn approximately 30x (asparagine) in position 3 of a beta-turn.     

Deamidation of calmodulin at neutral and alkaline pH: quantitative relationships between ammonia loss and the susceptibility of calmodulin to modification by protein carboxyl methyltransferase

Measurements of ammonia release provide the first direct evidence that calmodulin becomes extensively deamidated during incubations at 37 degrees C, pH 7.4 or pH 11. A stoichiometry of 0.5 mol of NH3 released/mol of calmodulin is observed after 2 h at pH 11 or after 8-9 days at pH 7.4. These treatments also increase the ability of calmodulin to serve as a substrate for the isoaspartate-specific protein carboxyl methyltransferase from bovine brain. The stoichiometries of methylation are highly correlated with the stoichiometries of ammonia release. Deamidation and increased methyl-accepting capacity also occur in parallel for seven other proteins (aldolase, bovine serum albumin, cytochrome c, lysozyme, ovalbumin, ribonuclease A, and triosephosphate isomerase) upon incubation at pH 11. However, in comparison to calmodulin, these other proteins show very little deamidation and increased methylation capacity following incubation at pH 7.4. Deamidation of calmodulin at pH 7.4 is unaffected by the addition of 10(-7) M Ca2+; however, at 4 X 10(-6) M Ca2+, the rate of deamidation is inhibited by approximately 70%. The Ca2+-protection effect is consistent with the suggestion (B. A. Johnson, N. E. Freitag, and D. W. Aswad, (1985) J. Biol. Chem. 260, 10913-10916) that deamidation occurs preferentially at Asn-60 and/or Asn-97, each of which resides in a distinct Ca2+-binding domain.     

Detection, evaluation and minimization of nonenzymatic deamidation in proteomic sample preparation

Identification of deamidated sites in proteins is commonly used for assignment of N-glycosylation sites. It is also important for assessing the role of deamidation in vivo. However, nonenzymatic deamidation occurs easily in peptides under conditions commonly used in treatment with trypsin and PNGase F. The impact on proteomic sample preparation has not yet been evaluated systematically. In addition, the (13)C peaks of amidated peptides can be misassigned as monoisotopic peaks of the corresponding deamidated ones in database searches. The 19.34 mDa mass difference between them is proposed as a means for eliminating the resulting false positive identifications in large-scale proteomic analysis. We evaluated five groups of proteomic data, obtained mainly through an electrostatic repulsion-hydrophilic interaction chromatography (ERLIC)-reverse phase (RP) chromatography sequence, and ascertained that nonenzymatic asparagine deamidation occurred to some extent on 4-9% of the peptides, resulting in the false positive identification of many N-glycosylation sites. A comprehensive investigation indicated that the chief causative factors were the mildly alkaline pH and prolonged incubations at 37 °C during proteomic sample preparation. An improved protocol is proposed featuring tryptic digestion at pH 6 and deglycosylation at pH 5, resulting in a significant decrease in nonenzymatic deamidation while conserving adequate digestion efficiency. The number of identified deamidation sites was improved significantly by increasing the sample loading amount in liquid chromatography-tandem MS. This permitted the identification of a significant number of glutamine deamidation sites, which featured sequence motifs largely different from those for asparagine deamidation: -Q-V-, -Q-L- and -Q-G- and, to a lesser extent, -Q-A- and -Q-E-.     

Deamidation of labile asparagine residues in the autoregulatory sequence of human phenylalanine hydroxylase.

Two dimensional electrophoresis has revealed a microheterogeneity in the recombinant human phenylalanine hydroxylase (hPAH) protomer, that is the result of spontaneous nonenzymatic deamidations of labile asparagine (Asn) residues [Solstad, T. and Flatmark, T. (2000) Eur. J. Biochem.267, 6302-6310]. Using of a computer algorithm, the relative deamidation rates of all Asn residues in hPAH have been predicted, and we here verify that Asn32, followed by a glycine residue, as well as Asn28 and Asn30 in a loop region of the N-terminal autoregulatory sequence (residues 19-33) of wt-hPAH, are among the susceptible residues. First, on MALDI-TOF mass spectrometry of the 24 h expressed enzyme, the E. coli 28-residue peptide, L15-K42 (containing three Asn residues), was recovered with four monoisotopic mass numbers (i.e., m/z of 3106.455, 3107.470, 3108.474 and 3109.476, of decreasing intensity) that differed by 1 Da. Secondly, by reverse-phase chromatography, isoaspartyl (isoAsp) was demonstrated in this 28-residue peptide by its methylation by protein-l-isoaspartic acid O-methyltransferase (PIMT; EC 2.1.1.77). Thirdly, on incubation at pH 7.0 and 37 degrees C of the phosphorylated form (at Ser16) of this 28-residue peptide, a time-dependent mobility shift from tR approximately 34 min to approximately 31 min (i.e., to a more hydrophilic position) was observed on reverse-phase chromatography, and the recovery of the tR approximately 34 min species decreased with a biphasic time-course with t0.5-values of 1.9 and 6.2 days. The fastest rate is compatible with the rate determined for the sequence-controlled deamidation of Asn32 (in a pentapeptide without 3D structural interference), i.e., a deamidation half-time of approximately 1.5 days in 150 mm Tris/HCl, pH 7.0 at 37 degrees C. Asn32 is located in a cluster of three Asn residues (Asn28, Asn30 and Asn32) of a loop structure stabilized by a hydrogen-bond network. Deamidation of Asn32 introduces a negative charge and a partial beta-isomerization (isoAsp), which is predicted to result in a change in the backbone conformation of the loop structure and a repositioning of the autoregulatory sequence and thus affect its regulatory properties. The functional implications of this deamidation was further studied by site-directed mutagenesis, and the mutant form (Asn32-->Asp) revealed a 1.7-fold increase in the catalytic efficiency, an increased affinity and positive cooperativity of L-Phe binding as well as substrate inhibition.     

Deamidation and succinimide formation by gamma-N-methylasparagine: potential pitfalls of amino acid analysis

The biological function of the post-translationally methylated amino acid gamma-N-methylasparagine (gamma-NMA) in proteins is unknown. We are examining the premise that amide methylation protects against deamidation. The free amino acids Asn, gamma-NMA, Gln, and delta-N-methylglutamine (delta-NMG) were incubated at elevated temperature and a variety of pH conditions to assay for deamidation. Gln disappears 12- to 14-fold more rapidly than delta-NMG, and Asn hydrolyzes to Asp and NH3 as expected. However, the gamma-NMA deamidation rate is severely overestimated by simply measuring the disappearance of starting material because gamma-NMA undergoes a cyclization reaction in preference to deamidation. At pH 1 the predominant gamma-NMA reaction is formation of stable 3-amino-N-methylsuccinimide (NMS) and this occurs greater than 10-fold faster than Asn deamidation. At pH 4.0, 7.4, and 9.0 NMS is readily formed but it is unstable and partitions between the parent compound, gamma-NMA, and a second species, alpha-N-methylasparagine. At pH 7.4 and 9.0 gamma-NMA disappears 4-fold slower than Asn but the methyl amide hydrolysis rate is diminished by as much as 13-fold. The Asn incubations over the pH range 1-9 yield scant evidence of a succinimide intermediate. It is concluded that the amide methylation provides a unique reaction pathway and stabilization for the N-methylsuccinimide species. Amino acid analysis by o-phthalaldehyde postcolumn reaction fails to detect isoasparagine, alpha-N-methylasparagine, and NMS. Amino acid analysis by precolumn derivatization with phenyl isothiocyanate destroys NMS and therefore cannot quantitate this compound. The ninhydrin postcolumn derivatization method is able to detect and quantitate all of these amino acid species.     

Identification and characterization of oxidation and deamidation sites in monoclonal rat/mouse hybrid antibodies

Oxidation of methionine residues and deamidation of asparagine residues are the major causes of chemical degradation of biological pharmaceuticals. The mechanism of these non-enzymatic chemical reactions has been studied in great detail. However, the identification and quantification of oxidation and deamidation sites in a given protein still remains a challenge. In this study, we identified and characterized several oxidation and deamidation sites in a rat/mouse hybrid antibody. We evaluated the effects of the sample preparation on oxidation and deamidation levels and optimized the peptide mapping method to minimize oxidation and deamidation artifacts. Out of a total number of 18 methionine residues, we identified six methionine residues most susceptible to oxidation. We determined the oxidation rate of the six methionine residues using 0.05% H₂O₂ at different temperatures. Methionine residue 256 of the mouse heavy chain showed the fastest rate of oxidation under those conditions with a half life of approximately 200 min at 4 °C and 27 min at 37 °C. We identified five asparagine residues prone to deamidation under accelerated conditions of pH 8.6 at 37 °C. Kinetic characterization of the deamidation sites showed that asparagine residue 218 of the rat heavy chain exhibited the fastest rate of deamidation with a half live of 1.5 days at pH 8.6 and 37 °C. Analysis of antibody isoforms using free flow electrophoresis showed that deamidation is the major cause of the charged variants of this rat/mouse hybrid antibody.     

Deamidated active intermediates in the irreversible acid denaturation of ribonuclease-A.

A study has been made on the changes in the enzymatic activity of Ribonuclease-A**-(RNase-A) exposed to highly acidic (pH less than 1) acqueous environment. Irreversible alterations of activity were observed when the protein was exposed to an acidic medium for a long period (20 to 60 h). Even prior to these changes in activity RNase-A was found to form intermediates which had very nearly the same activity as the native protein. The primary process in the acid denaturation of RNase-A was observed to be deamidation of the protein leading to the formation of active chromotographically distinct derivatives. The initial product of deamidation, a monodeamidated derivative, has been isolated by chromatography on Amberlite XE-64. This initial deamidation reaction proceeded with very high specificity. The subsequent deamidation reaction is comparatively slower, so that nearly 50% of the native protein could be converted to this derivative before any subsequent deamidation took place. This monodeamidated derivative has been designated RNase-Aa1. The conversion of RNase-A to RNase-Aa1 was not accompanied by any changes in the primary structure other than the observed deamidation. Apart from the differences in chromatographic and electrophoretic mobilities, RNase-Aa1 was found to have very nearly the same activity and physicochemical properties as the native enzyme. Significance of this specific and faster deamidation of RNase-A in this denaturing medium as well as the biological significance of such deamidation reactions of proteins are discussed.     

Identification of succinimide sites in proteins by N-terminal sequence analysis after alkaline hydroxylamine cleavage.

Under favorable conditions, Asp or Asn residues can undergo rearrangement to a succinimide (cyclic imide), which may also serve as an intermediate for deamidation and/or isoaspartate formation. Direct identification of such succinimides by peptide mapping is hampered by their lability at neutral and alkaline pH. We determined that incubation in 2 M hydroxylamine, 0.2 M Tris buffer, pH 9, for 2 h at 45 degrees C will specifically cleave on the C-terminal side of succinimides without cleavage at Asn-Gly bonds; yields are typically approximately 50%. N-terminal sequence analysis can then be used to identify an internal sequence generated by cleavage of the succinimide, hence identifying the succinimide site.     

Isolation of a Spore Germination Inhibitor from a Cellular Slime Mold Dictyostelium discoideum

The functional properties of gluten obtained by treating with chymotrypsin at alkali pH were investigated. The gluten was treated by chymotrypsin at pH 10.0 and 20°C, and was found to be deamidated to a state that was scarcely subject to proteolysis by chymotrypsin. The degree of deamidation of the gluten reached about 25% by this treatment for 2 hr. The functional properties of the gluten thus obtained were investigated in regard to deamidation. The enzymatically deamidated gluten greatly improved such functional properties as solubility and emulsifying ability. In particular, the solubility of the treated gluten was remarkably high in the pH range of 5 to 8, in which native gluten is insoluble. It was apparent that the improvement in functional properties of gluten was mainly due to the deamidation induced by treating with chymotrypsin at pH 10.0 and 20°C.     

The effect of hydrocarbon addition on the deamidation rate in immune whey proteins

The influence of some hydrocarbons that are often used at different stages of immunobiological preparation's production as stabilizers of biological activity on the dynamics of nonenzymatic deamidation in proteins of immune whey against conditionally pathogenic microorganisms obtained by means of membrane ultrafiltration technology is investigated. Preparations of whey were incubated in 10 per cent solutions of glucose, fructose and sorbitol at the conditions similar to physiological ones (0.9% NaCl, pH 5.5) and temperature of about +4 degrees C and +35 degrees C for 7, 14 and 28 days. A sample dissolved in 0.9% NaCl (pH 5.5) without addition of hydrocarbons was used as a "control preparate". All explored substances brought about the suppressive effect on deamidation rate of asparaginyl residues whereas that of glutaminyl residues, on the contrary, was obviously increased. The possible reasons for these observations are discussed.     

Effects of amino acid sequence, buffers, and ionic strength on the rate and mechanism of deamidation of asparagine residues in small peptides

The nonenzymatic rates of deamidation of Asn residues in a series of pentapeptides with the sequences VSNXV and VXNSV, where X is one of 10 different amino acids, were determined at neutral, alkaline, and acid pH values. The results demonstrate that in neutral and alkaline solutions the amino acid residue on the amino side of the Asn had little or no effect on the rate of deamidation regardless of its charge or size. The group on the carboxyl side of Asn affected the rate of deamidation significantly. Increasing size and branching in the side chain of this residue decreased the rate of deamidation by as much as 70-fold compared to glycine in the N-G sequence, which had the greatest rate of deamidation. In acidic solution, the rate of deamidation of the Asn residue was not affected by the amino acid sequence of the peptide. The products for each deamidation reaction were tested for the formation of isoAsp residues. In neutral and alkaline solutions, all products showed that the isoAsp:Asp peptide products were formed in about a 3:1 ratio. In acidic solution, the Asp peptide was the only deamidation product formed. All peptides in which a Ser residue follows the Asn residue were found to undergo a peptide cleavage reaction in neutral and alkaline solutions, yielding a tripeptide and a dipeptide. The rate of the cleavage reaction was about 10% of the rate of the deamidation pathway at neutral and alkaline pH values. The rates of deamidation of Asn residues in the peptides studied were not affected by ionic strength, and were not specific base catalyzed. General base catalysis was observed for small bases like ammonia. A model for the deamidation reaction is proposed to account for the observed effects.     

Control of process-induced asparaginyl deamidation during manufacture of Erwinia chrysanthemi l-asparaginase

During the manufacture of the chemotherapeutic enzyme Erwinia chrysanthemi l-asparaginase, a small proportion (approximately 5–15%) of acidic variants, including deamidated species, are observed. Although the deamidated forms appear to have similar specific activity and quaternary structure to the unmodified enzyme, monitoring and control of these forms is important from a regulatory perspective. The extent of Asn to Asp deamidation directly correlates with the time taken to thaw the Erwinia cells. Erwinia l-asparaginase is a tetrameric enzyme containing one site, Asn₂₈₁, theoretically very labile to deamidation due to the sequence Asn-Gly. Structurally, this part of the protein sequence is completely buried inside the tetramer, but solvent-exposed upon tetramer dissociation. During the cell thawing and alkaline lysis sequence of the process, lengthening the cell thaw times by up to 24 h allowed tetramer to reassociate, protected Asn₂₈₁ from deamidation and reduced the acidic species content of the l-asparaginase from approximately 17% to 9% as measured by weak cation-exchange (WCX) HPLC. The correlation of cell thaw time with acidic species content was also confirmed using capillary zone electrophoresis (CZE) and peptide mapping. These studies demonstrate that cell thaw time is an important, if unexpected, control variable for l-asparaginase deamidation.     

Analysis of deamidation of small, acid-soluble spore proteins from Bacillus subtilis in vitro and in vivo.

Deamidation of one specific asparagine residue in an alpha/beta-type small, acid-soluble spore protein (SASP) of Bacillus subtilis took place readily in vitro (time for 50% deamidation [t(1/2)], approximately 1 h at 70 degrees C), and the deamidated SASP no longer bound to DNA effectively. However, DNA binding protected against this deamidation in vitro. A mutant alpha/beta-type SASP in which the reactive asparagine was changed to aspartate also failed to bind to DNA in vitro, and this protein did not restore UV radiation and heat resistance to spores lacking the majority of their alpha/beta-type SASP. When expressed in Escherichia coli, where it is bound to DNA, the alpha/beta-type SASP deamidated with a t(1/2) of 2 to 3 h at 95 degrees C. However, the alpha/beta-type SASP was extremely resistant to deamidation within spores (t(1/2), >50 h at 95 degrees C). A gamma-type SASP of B. subtilis also deamidated readily in vitro (t(1/2) for one net deamidation, approximately 1 h at 70 degrees C), but this protein (which is not associated with DNA) deamidated fairly readily in spores (t(1/2), approximately 1 h at 95 degrees C). Total spore core protein also deamidated in vivo, although the rate was two- to threefold slower than that of deamidation of total protein in heated vegetative cells. These data indicate that protein deamidation is slowed significantly in spores, presumably due to the spore's environment. However, alpha/beta-type SASP are even more strongly protected against deamidation in vivo, presumably by their binding to spore DNA. Thus, not only do alpha/beta-type SASP protect spore DNA from damage; DNA also protects alpha/beta-type SASP.     

Deamidation of glutaminyl residues: dependence on pH, temperature, and ionic strength

We have shown the dependence of the deamidation half-times of the peptides, GlyLeuGlnAlaGly and GlyArgGlnAlaGly upon pH, temperature, and ionic strength. Increase in temperature or ionic strength, variation of pH to pH′s higher or lower than pH 6, and the use of phosphate buffer rather than Tris buffer at high pH all decrease the half-time of dcamidation. Temperature increase of 20°C or pH change of 2 pH units decreases the half-time about fivefold, while increase of one ionic strength unit decreases the half-time about twofold. In pH 7.4, I = 0.2, 37.0°C phosphate buffer, the deamidation half-times are 663 ± 74 and 389 ± 56 days respectively for the two peptides, GlyLeuGlnAlaGly and GlyArgGlnAlaGly.These experiments should serve as a warning to peptide and protein experimenters that even the more stable glutaminyl residues are unstable with respect to deamidation in certain solvent conditions. These experiments also provide, along with previously reported experiments on asparaginyl peptides (7), some quantitative data to help with the extrapolation of in vitro deamidation experiments to in vivo deamidation conditions.     

Glutamine deamidation destabilizes human gammaD-crystallin and lowers the kinetic barrier to unfolding

Human eye lens transparency requires life long stability and solubility of the crystallin proteins. Aged crystallins have high levels of covalent damage, including glutamine deamidation. Human gammaD-crystallin (HgammaD-Crys) is a two-domain beta-sheet protein of the lens nucleus. The two domains interact through interdomain side chain contacts, including Gln-54 and Gln-143, which are critical for stability and folding of the N-terminal domain of HgammaD-Crys. To test the effects of interface deamidation on stability and folding, single and double glutamine to glutamate substitutions were constructed. Equilibrium unfolding/refolding experiments of the proteins were performed in guanidine hydrochloride at pH 7.0, 37 degrees C, or urea at pH 3.0, 20 degrees C. Compared with wild type, the deamidation mutants were destabilized at pH 7.0. The proteins populated a partially unfolded intermediate that likely had a structured C-terminal domain and unstructured N-terminal domain. However, at pH 3.0, equilibrium unfolding transitions of wild type and the deamidation mutants were indistinguishable. In contrast, the double alanine mutant Q54A/Q143A was destabilized at both pH 7.0 and 3.0. Thermal stabilities of the deamidation mutants were also reduced at pH 7.0. Similarly, the deamidation mutants lowered the kinetic barrier to unfolding of the N-terminal domain. These data indicate that interface deamidation decreases the thermodynamic stability of HgammaD-Crys and lowers the kinetic barrier to unfolding due to introduction of a negative charge into the domain interface. Such effects may be significant for cataract formation by inducing protein aggregation or insolubility.     

The propensity for deamidation and transamidation of peptides by transglutaminase 2 is dependent on substrate affinity and reaction conditions

Transglutaminase 2 (TG2) catalyzes cross-linking or deamidation of glutamine residues in peptides and proteins. The in vivo deamidation of gliadin peptides plays an important role in the immunopathogenesis of celiac disease (CD). Although deamidation is considered to be a side-reaction occurring in the absence of suitable amines or at a low pH, a recent paper reported the selective deamidation of the small heat shock protein 20 (Hsp20), suggesting that deamidation could be a substrate dependent event. Here we have measured peptide deamidation and transamidation in the same reaction to reveal factors that affect the relative propensity for the two possible products. We report that the propensity for deamidation by TG2 is both substrate dependent and influenced by the reaction conditions. Direct deamidation is favored for poor substrates and at low concentrations of active TG2, while indirect deamidation (i.e. hydrolysis of transamidated product) can significantly contribute to the deamidation of good peptide substrates at higher enzyme concentrations. Further, we report for the first time that TG2 can hydrolyze iso-peptide bonds between two peptide substrates. This was observed also for gliadin peptides introducing a novel route for the generation of deamidated T cell epitopes in celiac disease.     

Asparagine deamidation: pH-dependent mechanism from density functional theory

Asparagine deamidation is a decisive event in chemotherapy-induced apoptosis and a major obstacle in the formulation of monoclonal antibodies. Despite the importance of deamidation, little is known about the elementary reactions involved. B3LYP/6-31+G(d,p)/COSMO-RS calculations were used to obtain stable structures and transition states for a network of reactions. Calculated rate constants were incorporated into a kinetic model of the pH dependence and compared to a pseudo-steady-state model. At low pH, the calculations show that deamidation occurs by direct acid-catalyzed hydrolysis to aspartate. At neutral to basic pH, deamidation proceeds by the initial formation of a tetrahedral intermediate. The intermediate can be converted to succinimide by two pathways and three rate-determining steps that shift in relative importance with pH. The calculated pH-dependent rate constant qualitatively agrees with the experimental pH dependence. The rate-determining transition state structures may help to understand chemotherapy-induced apoptosis and improve protein formulations.     

Why does ribonuclease irreversibly inactivate at high temperatures?

The mechanism of irreversible thermoinactivation of bovine pancreatic ribonuclease A in the pH range relevant to enzymatic catalysis has been elucidated. At 90 degrees C and pH 4, the enzyme inactivation is caused by hydrolysis of peptide bonds at aspartic acid residues (the main process) and deamidation of asparagine and/or glutamine residues. At 90 degrees C and neutral pH (pH 6 and 8), the enzyme inactivation is caused by a combination of disulfide interchange (the main process), beta-elimination of cystine residues, and deamidation of asparagine and/or glutamine residues. These four processes appear to demarcate the upper limit of thermostability of enzymes.     

Bcl-xL deamidation is regulated by multiple ion transporters and is intramolecularly catalyzed

In susceptible tumor cells, DNA-damaging antineoplastic agents induce an increase in intracellular pH during the premitochondrial stage of apoptosis. The rate of nonenzymatic deamidation of two asparagines in the anti-apoptotic protein Bcl-x_L is accelerated by this increase in pH. Deamidation of these asparagines is a signal for the degradation of Bcl-x_L, which is a component of the apoptotic response to DNA damage. It has previously been shown that the increase in pH is mediated by the ion transporter Na⁺/H⁺ exchanger 1 in some cells. Here we demonstrate that one or more additional ion transporters also have a role in the regulation of Bcl-x_L deamidation in at least some tumor cell lines and fibroblasts. As a second, independent finding, we report that there are histidines in close proximity to the Bcl-x_L deamidation sites that are highly conserved in land-dwelling species and we present evidence that deamidation of human Bcl-x_L is intramolecularly catalyzed in a manner that is dependent upon these histidines. Further, we present evidence that these histidines act as a pH-sensitive switch that enhances the effect of the increase in pH on the rate of Bcl-x_L deamidation. The conservation of such histidines implies that human Bcl-x_L is in essence “designed” to be deamidated, which provides further evidence that deamidation serves as a bona fide regulatory post-translational modification of Bcl-x_L.     

Effect of polyols on the conformational stability and biological activity of a model protein lysozyme

The purpose of this study was to investigate the stabilizing action of polyols against various protein degradation mechanisms (eg, aggregation, deamidation, oxidation), using a model protein lysozyme. Differential scanning calorimeter (DSC) was used to measure the thermodynamic parameters, mid point transition temperature and calorimetric enthalpy, in order to evaluate conformational stability. Enzyme activity assay was used to corroborate the DSC results. Mannitol, sucrose, lactose, glycerol, and propylene glycol were used as polyols to stabilize lysozyme against aggregation, deamidation, and oxidation. Mannitol was found to stabilize lysozyme against aggregation, sucrose against deamidation both at neutral pH and at acidic pH, and lactose against oxidation. Stabilizers that provided greater conformational stability of lysozyme against various degradation mechanisms also protected specific enzyme activity to a greater extent. It was concluded that DSC and bioassay could be valuable tools for screening stabilizers in protein formulations.