Assessment of antibody fragmentation by reversed-phase liquid chromatography and mass spectrometry

Antibody fragmentation in the hinge region and other regions, and the impact of pH on the level and pattern of antibody fragmentation were investigated by reversed-phase (RP) liquid chromatography and mass spectrometry (LC-MS). Extensive fragmentation was observed in the hinge and in regions other than the hinge of a recombinant monoclonal antibody that was incubated in buffers of various pH at 40 degrees C for 10 weeks. Peptide bonds that were susceptible to hydrolysis were located mainly around the domain-domain interfaces close to or in the loop structures. The sites as well as the level of peptide bond hydrolysis were affected by the buffer pH. In agreement with previous findings when only the hinge region fragmentation was monitored, pH 6 was optimal for slowing down antibody fragmentation in regions other than the hinge. It also demonstrated that analysis by RPLC-MS provided a better assessment of the susceptible regions of recombinant monoclonal antibodies than size-exclusion chromatography (SEC) followed by fraction collection and mass spectrometry identification.     

Non-enzymatic hinge region fragmentation of antibodies in solution

Liquid formulations of monoclonal antibodies (MAbs) typically undergo fragmentation near the papain cleavage site in the hinge region, resulting in Fab and Fab+Fc forms. The purpose of this study was to investigate whether this fragmentation is due to proteases. Four closely-related MAbs were exchanged into a pH 5.2 acetate buffer with NaCl and stored at -20 degrees C, 5 degrees C, 30 degrees C, or 40 degrees C for 1 month. Fragmentation generated size-exclusion chromatography (SEC) peak fractions that were analyzed by electrospray mass spectrometry to identify the cleavage sites. The effects of protein inhibitors or host cell proteins on fragmentation were also studied. The extent of fragmentation was equivalent for all four antibodies, occurring in the heavy chain hinge region Ser-Cys-Asp-Lys-Thr-His-Thr sequence. The fragment due to cleavage of the Asp-Lys bond showed two forms that differ by 18 Da. A synthetic peptide with the hinge region sequence terminating with Asp did not show fragmentation or the loss of 18 Da after incubation. Protease inhibitors did not affect rates of cleavage or modify sites of fragmentation. Degradation was not affected by host cell protein content. Fragmentation appears to be a kinetic process that is not caused by low levels of host cell proteases.     

An enzyme-probe study of motile domains in the subfragment-2 region of myosin

The temperature-dependence of local melting within the subfragment-2 region of rabbit skeletal muscle myosin has been investigated using an enzyme-probe technique. Rate constants of fragmentation of two long subfragment-2 particles (61,000 Mr and 53,000 Mr per polypeptide chain) and a short subfragment-2 particle (34,000 Mr per polypeptide chain) by three different enzymes (alpha-chymotrypsin, trypsin and papain) have been determined over the temperature range 5 to 40 degrees C. We followed the time-course of digestion at specific sites at high (I = 0.50, pH 7.3) and low (physiological, I = 0.15, pH 7.3) ionic strengths by electrophoresis of the digestion products on sodium dodecyl sulfate-containing gels. All rate constants were corrected for the intrinsic temperature-dependence of the enzymes by comparison with model substrates. Normalized rate constant versus temperature profiles for the three enzyme-probes are similar in showing that local melting in long subfragment-2 (61,000 Mr) occurs in two distinct stages as was observed earlier for the intact myosin rod. Over the temperature range 5 to 25 degrees C a restricted region at Mr = 53,000 to 50,000 from the N terminus of the rod (the light meromyosin/heavy meromyosin junction) shows the highest susceptibility to proteolytic cleavage. At temperatures above 25 degrees C local melting was detected by all three enzymes at several specific sites within the hinge domain (Mr = 53,000 to 34,000). Activation energies for cleavage at the susceptible sites were similar for the three enzyme probes. They suggest that this region of the myosin rod has significantly lower thermal stability than the flanking light meromyosin and short subfragment-2 segments. These results, together with other physico-chemical studies, point to the hinge domain of the myosin cross-bridge as an important functional element in the mechanism of force generation in muscle.     

Temperature-dependence of local melting in the myosin subfragment-2 region of the rigor cross-bridge

We have used alpha-chymotrypsin as an enzyme-probe to detect local melting in the subfragment-2 region of the cross-bridges of rigor myofibrils and glycerinated psoas fibers. The kinetics of proteolysis and the sites of cleavage were determined at various temperatures over the range 5 to 40 degrees C by following the decay of the myosin heavy chain and the rates of appearance of light meromyosin fragments, using electrophoresis on sodium dodecyl sulfate-containing polyacrylamide gels. Cleavage occurs primarily at the 72,000 Mr and 64,000 Mr (per polypeptide chain from the C terminus of myosin) sites within the light meromyosin-heavy meromyosin hinge domain of the subfragment-2 region, under all experimental conditions. At pH 8.2 to 8.3 and at low divalent metal ion (0.1 mM), where the actin-bound cross-bridges are thought to be released from the thick filament surface, the intrinsic cleavage rate constant (k) increases markedly as the temperature is raised. This suggests substantial thermal destabilization of the released cross-bridge in the intact contractile apparatus. Addition of divalent metal ion (10 mM) lowers the cleavage rate and shifts the k versus temperature profile to higher temperatures. Normalized rate constants for chymotryptic cleavage within the subfragment-2 hinge region of released cross-bridges (pH 8.2, low divalent metal) of rigor fibers were markedly lower than activated fibers at all temperatures investigated (5 to 40 degrees C). Results show that conformational melting within the subfragment-2 hinge region is amplified on activation and is well above that observed when the actin-attached rigor bridge is passively released from the thick filament surface.     

Melting of myosin rod as revealed by electron microscopy. II. Effects of temperature and pH on length and stability of myosin rod and its fragments

Effects of temperature and pH on intact rabbit and chicken myosin, isolated myosin rods, rabbit subfragment-2 (61 kDa, 53 kDa, and 34 kDa) and chicken light meromyosin (LMM) fragments were tested to induce a phase transition from alpha-helix to coil conformation, within the hinge region. The influence of temperature and pH were studied directly with length determination by electron microscopy. An increase of temperature to 50 degrees C yielded a shortening of 16 nm, 8 to 9 nm and 7 to 11 nm for intact myosin, isolated rods and long S-2 fragments, respectively. The length of the 34 kDa short S-2 and LMM fragments were unchanged. An increase of pH from neutral to pH 8.0 yielded values that were somewhat smaller, e.g. 12 nm, 6 nm and 6 to 8 nm for intact myosin, isolated rods and long S-2 fragments, respectively, whereas the 34 kDa short S-2 LMM fragments were also unaffected. Thus, melting and subsequent shortening is confined to the region between LMM and short S-2 segment, that is the hinge region. Alteration of temperature had a stronger shortening effect than alteration of pH, and shortening of long S-2 was more pronounced under physiological salt conditions as compared with high (0.3 M) salt. The shortening of rods in intact myosin amounted to twice the value observed with isolated rods. The amount of contraction was somewhat smaller in rods than in the 61 kDa and 53 kDa long S-2 fragments.     

Temperature and pH dependence of immunoglobulin G conformation

Previous indirect observations have indicated that IgG may change its conformation at low or high pH and at a temperature of about 35 degrees C. By means of small angle neutron scattering a change in the value of the gyration radius of two different native IgG's was observed above 44 degrees C. No similar change was detected when the sample was previously dissolved in an acidic buffer. The acidic pretreatment caused a significant decrease in the gyration radius (Rg) value measured at 20 degrees C which was partially recovered by increasing the temperature. These observations led to the assumption that the main conformational change observed appears either in the hinge region of the molecular or in the interdomain areas separating the constant and the variable domains of the Fab parts.     

An enzyme-probe method to detect structural changes in the myosin rod

The temperature-dependence of local melting within the alpha-helical, coiled-coil structure of rabbit myosin rod has been investigated by following changes in the rate constants of proteolytic digestion. The kinetics of fragmentation of the rod by three different enzymes (alpha-chymotrypsin, trypsin and papain) over the temperature range 5 to 40 degrees C (pH 7, I = 0.5) has been monitored by electrophoresis of the digestion products on sodium dodecyl sulfate/polyacrylamide gels. All rate constants were corrected for the intrinsic temperature-dependence of the enzyme by comparison with model substrates. Results from the three enzyme-probes are similar in showing that local melting within the rod occurs in two distinct stages. At temperatures between 5 and 25 degrees C, melting is confined to a restricted segment of the rod structure near the light meromyosin/heavy meromyosin junction. At temperatures between 25 and 40 degrees C, a wider segment of the rod lysing between the junction and the short subfragment-2 segment (the hinge domain) appears to be melting, judging from the broad spectrum of cleavage sites observed in this region. Results are compared with those from other physicochemical methods that measure the hinging or opening of the coiled-coil structure of the rod.     

Structural effect of a recombinant monoclonal antibody on hinge region peptide bond hydrolysis

IgG hinge region peptide bonds are susceptible to degradation by hydrolysis. To study the effect of Fab and Fc on hinge region peptide bond hydrolysis, a recombinant humanized monoclonal IgG1 antibody, its F(ab')2 fragment, and a model peptide with amino acid sequence corresponding to the hinge region were incubated at 40 degrees C in formulation buffer including complete protease inhibitor and EDTA for 0, 2, 4, 6 and 8 weeks. Two major cleavage sites were identified in the hinge region of the intact recombinant humanized monoclonal antibody and its F(ab')2 fragment, but only one major cleavage site of the model peptide was identified. Hinge region peptide bond hydrolysis of the intact antibody and its F(ab')2 fragment degraded at comparable rates, while the model peptide degraded much faster. It was concluded that Fab region of the IgG, but not Fc portion had significant effect on preventing peptide bond cleavage by direct hydrolysis. Hydrolysis of hinge region peptide bonds was accelerated under both acidic and basic conditions.     

Evolutionary hypervariability in the hinge region of the immunoglobulin alpha gene

The hinge region of the immunoglobulin molecule is responsible for antigen-binding and cross-linking reactions, varying the distance between the two antigen-binding sites. As the amino acid sequence of the hinge region is identical among immunoglobulin molecules of the same (sub)class, it has been regarded as a constant region. By comparison of the nucleotide sequences among primate C alpha genes, it is clear that there is a wide variety of length among the hinge regions of hominoid C alpha genes, which basically consist of tandem repeats of a 15 base-pair sequence. This reiterated structure probably facilitates rapid evolutionary changes in the length of the hinge region. The hinge region of the Old World monkey C alpha gene has a non-reiterated structure whose nucleotide sequence is quite different from those of the hominoid C alpha genes, although its surrounding region is conserved during evolution. This unusual hypervariability reveals that the hinge region has evolved as a semi-variable region in contrast to its constant character from an ontogenic viewpoint.     

Self-association of a high molecular weight subfragment-2 of myosin induced by divalent metal ions

The effect of divalent cations on the self-association of high molecular weight subfragment-2 (long S-2) and low molecular weight subfragment-2 (short S-2) of rabbit skeletal muscle myosin has been investigated. In the presence of millimolar concentrations of Ca2+ or Mg2+ long S-2 associates at neutral pH to form ordered, high molecular weight aggregates whereas short S-2 does not associate. The association process is co-operative and results from binding two to four divalent cations within the light meromyosin-heavy meromyosin (LMM-HMM) hinge region of long S-2. Optical diffraction of electron micrographs of the long S-2 aggregates revealed several periodicities including reflections near 143 A. High molecular weight HMM showed a similar divalent metal induced self-association. Chymotryptic digestion studies of rod filaments reveal that cleavage within the LMM-HMM hinge is also strongly dependent on the presence of divalent cations. At pH 8, in the absence of divalent cations, the S-2 region appears to be displaced away from the filament backbone resulting in rapid proteolysis in the hinge domain. At high cation concentrations (greater than 10 mM) proteolytic cleavage is suppressed. A similar depression of the (substantially lower) hinge cleavage rate was also observed at neutral pH following addition of these divalent metal ions. Results suggest that binding of Mg2+ within the hinge domain under physiological conditions may act to lock the cross-bridge onto the thick filament surface in its resting-state orientation.     

Enzymatic characterization of the streptococcal endopeptidase, IdeS, reveals that it is a cysteine protease with strict specificity for IgG cleavage due to exosite binding

Streptococcus pyogenes, an important pathogen in humans, secretes an IgG specific endopeptidase named IdeS. To elucidate the mechanism that is responsible for this specificity, we have here characterized the activity of IdeS in detail. Both gamma chains of human IgG or its Fc fragment were cleaved in the hinge region after Gly236 by IdeS, but other proteins or synthetic peptides containing sequences such as the P(4)-P(1) segment in the IgG cleavage site, or long peptides resembling the IgG hinge, were not hydrolyzed at all. This is likely due to a second binding site interacting with the Fc part of IgG. The lack of IdeS activity on peptide substrates necessitated the development of an assay with IgG as the substrate for kinetic studies. IdeS showed a sigmoidal velocity curve at physiological IgG concentrations, and a declining enzyme rate at higher IgG concentrations. This atypical velocity curve suggests product inhibition and/or allosteric control, which again indicates the presence of an exosite involved in substrate binding. The pseudoequilibrium constant for IdeS hydrolysis of IgG was 90 microM. The enzyme exhibited activity in the pH range of 5.1-7.6, with an optimum at pH 6.6. IdeS was stable above pH 10 but not at acidic pH. It exhibited an activity maximum around 37 degrees C and a decreased thermal stability at 42 degrees C. Iodoacetate and iodoacetamide inhibited IdeS, as expected for a cysteine protease, and biochemical evidence verified this classification. E-64 and chicken cystatin, specific inhibitors of family C1 and C13 cysteine proteases, were without effect on enzyme activity, as were class specific serine, aspartic, and metallo protease inhibitors. No significant similarities were found in protein sequence comparisons with known enzyme families, suggesting that IdeS represents a novel family of cysteine proteases.     

A study of the hinge-bending mechanism of yeast 3-phosphoglycerate kinase.

The hinge-bending mechanism proposed as part of the catalytic mechanism for phosphoglycerate kinase (PGK) has been investigated using yeast PGK and the site-directed mutant [H388Q]PGK, where His388 is replaced by Gln. The emission and quenching of fluorescence, supported by the aromatic CD band, show that the mutation in the waist region affects the tryptophan environment in the C-terminal domain. The mutant is also less stable to guanidine denaturation and less cooperative in its unfolding. The effect of substrates on the conformation of PGK was studied using 8-anilino-1-naphthalenesulphonic acid (ANS), a competitive inhibitor of ATP binding to the C-terminal domain, and 8-(2-[(iodoacetyl)ethyl]amino)naphthalene (I-AEDANS), attached to Cys197 on the N-terminal domain. Under the influence of substrates the novel anisotropy decay curves for ANS indicate a 1-5 degrees change in the orientation of the probe, interpreted as a small reorientation of the domains about the waist region. The experimental data are interpreted as a small swivelling of the domains about the waist region under the influence of substrate. The results with AEDANS anisotropy decay are consistent with those for ANS. The enzyme activity of PGK shows a break in the Arrhenius plot at 20 degrees C mirrored by a break in the temperature dependence of tryptophan ellipticity. This is interpreted as a change in protein dynamics associated with destabilisation of the waist region. This destabilisation is shown to have already taken place in the mutant enzyme and in the wild type at pH 5.6, both of which exhibit linear Arrhenius plots. NMR titration curves show that the pH effect must be due to a group other than histidine. The results give further support to the permissive model of hinge bending previously proposed by one of the authors, in which binding of substrate destabilises the waist region. This loosens the hinge which can then swing slightly to bring the domains closer together to make favourable interactions between the domains and the substrates, with the exclusion of water.     

Low and high pH form of cadmium carbonic anhydrase determined by nuclear quadrupole interaction.

The pH dependence of the nuclear quadrupole interaction between the excited 247-keV state in 111Cd bound to the active site in human carbonic anhydrase B and the nearest protein surroundings has been studied by means of the nuclear spectroscopic technique of perturbed angular correlation of gamma rays. The enzyme has been studied in the pH region 5.6-11.0 at 22 and -196 degrees C. The results show that the Cd enzyme changes from one form at low pH to another form at high pH both at 22 and -196 degrees C. The pK of the transition is 8.9 +/- 0.2 at -196 degrees C and close to 9 at 22 degrees C. Parallel to this transformation, the esterase activity of the Cd enzyme for the hydration of p-nitrophenyl acetate exhibits a pH dependency with a pH of 9.1 +/- 0.2. The sulfonamide inhibitor acetazolamide completely inhibits this activity of the Cd enzyme. The quadrupole interaction parameters for the Cd enzyme are not significantly different at -196 degrees C from those obtained at 22 degrees C. A measurement at 0 degrees C pH 5.7 shows, however, a form different from those at 22 degrees C pH 5.6 and -196 degrees C pH 5.7. The change in the quadrupole interaction with pH is, in a simple model, consistent with an ionization of a metal-bound water molecule.     

Streptococcus gordonii soluble inorganic pyrophosphatase: an important role for the interdomain region in enzyme activity

Streptococcus gordonii DL1(Challis) soluble inorganic pyrophosphatase was shown to be a homo dimer with a subunit molecular mass of 33407. In solution, in the presence of Mn(2+), the protein is ellipsoidal with an axial ratio of 3.37 and molecular mass of 67000. In the absence of the divalent cation, the molecular mass is unchanged but the axial ratio increases to 3.94. The enzyme, in the presence of 5 mM Mg(2+), at 25 degrees Celsius and pH 9.0, has K(m) and k(cat) values of 62 microM and 6290 s(-1), respectively. The free N- and C-terminal domains of Streptococcus gordonii PPase did not interact productively when mixed together. Replacing the interdomain region with that from Bacillus subtilis decreased the catalytic efficiency of the enzyme whereas inserting the same region from the Archaeglobus fulgidus thermophilic enzyme yielded an inactive protein. Substitution, deletion and insertion of amino acid residues in the interdomain region were found to affect the monomer dimer equilibrium in the absence of Mn(2+) ions. In the presence of these ions however the variant proteins were dimers. Proteins with altered interdomain regions also displayed a 2- to 625-fold decrease in catalytic efficiency. These data together with that of computer analysis show that the interdomain region has characteristics of a mechanical hinge. Modelling mutant proteins onto the wild type shows that the active site regions are not significantly perturbed. These results show that, although distant from the active site, the interdomain region plays a role in enzyme activity and both its length and composition are important. This supports the hypothesis that catalytic activity requires the N- and C terminal domains of the enzyme to open and close using the interdomain region as a hinge.     

Conformational stability of the myosin rod

Chymotryptic cleavage patterns of myosin rods from pig stomach, chicken gizzard, and rabbit skeletal muscle indicate that short (approximately 45 nm) heavy meromyosin subfragment 2 (SF2) is a consistent product of all three rods, whereas long (approximately 60 nm) SF2 is derived only from skeletal muscle myosin. Differential scanning calorimetry was used to follow the thermally induced melting transition of the rods and certain of their subfragments. In 0.12 M KCl, sodium phosphate buffer, pH 6.2-7.6, the light meromyosin (LMM) and SF2 domains of each rod had essentially identical conformational stabilities. Temperature midpoints for the melting transitions were 54-56 degrees C for the two smooth muscle myosin rods and 50-53 degrees C for the skeletal muscle myosin rod. In 0.6 M K Cl buffer, melting transitions for the smooth muscle myosin rods were essentially unchanged, but skeletal muscle myosin rods showed multiphase melting, with major transitions at 43 degrees C and 52 degrees C. The first of these was tentatively attributed to LMM, and the second to SF2. In 0.12 M K Cl buffer, the LMM transition was stabilised so that it superimposed on that of SF2. No melting was observed in any of the rods at physiological temperature. These results indicate that, excluding a possible but only narrow hinge region, the entire myosin rod has essentially uniform conformational stability at physiological pH and ionic strength, and thus that the contractile and elastic properties of the cross-bridge exist in the heavy meromyosin subfragment 1 (SF1) domains of the molecule.     

Insights into the role of the (alpha+beta) insertion in the TIM-barrel catalytic domain, regarding the stability and the enzymatic activity of chitinase A from Serratia marcescens

Chitinase A (ChiA) from Serratia marcescens is a mesophilic enzyme with high catalytic activity and high stability. The crystal structure of ChiA has revealed a TIM-barrel fold of the catalytic domain, an (alpha+beta) insertion between the B7 beta-strand and A7 alpha-helix of the TIM-barrel, an FnIII domain at the N-terminus of the molecule and a hinge region that connects the latter to the catalytic domain. In this study, the role of the (alpha+beta) domain on the stability, catalytic activity and specificity of the enzyme was investigated by deleting this domain and studying the enzymatic and structural properties of the resulting truncated enzyme. The obtained data clearly show that by removing the (alpha+beta) domain, the thermal stability of the enzyme is substantially reduced, with an apparent T(m) of 42.0+/-1.0 degrees C, compared to the apparent T(m) of 58.1+/-1.0 degrees C of ChiA at pH 9.0. The specific activity of ChiADelta(alpha+beta) was substantially decreased, the pH optimum was shifted from 6.5 to 5.0 and the substrate and product specificities were altered.     

Local melting in the subfragment-2 region of myosin in activated muscle and its correlation with contractile force

Local melting within the subfragment-2 region of activated rabbit skeletal glycerinated muscle fibers has been investigated over the temperature range 5 to 37 degrees C, using an enzyme (chymotrypsin)-probe method. The cleavage rates were determined from the time-course of formation of digestion products by electrophoresis on sodium dodecyl sulfate-containing polyacrylamide gels. We found the cleavage sites to be localized in a restricted region Mr = 64,000 to 90,000/polypeptide chain, measured from the C terminus of the myosin rod (the subfragment-2 hinge domain). The cleavage rate constant for activated muscle fibers in the presence of an ATP-regenerating system was about 100 times larger at each temperature than that for rigor or for relaxed muscle fibers and showed a marked increase in magnitude with increasing temperature. Comparative plots of the apparent rate-constant for cleavage within the subfragment-2 hinge domain and the isometric force generated by active fibers versus MgATP concentration gave closely similar profiles suggesting a strong positive correlation. Thus, there appears to be a close coupling between the conformational transition within the subfragment-2 hinge domain and contractile force when the cross-bridges undergo cycling.     

Oligomeric state and mode of self-association of Thermotoga maritima ribosomal stalk protein L12 in solution

The "stalk" of the prokaryotic 50S ribosomal subunit is comprised of four copies of the protein L7/L12. In Escherichia coli, L7/L12 is a dimeric protein at micromolar concentrations, which is able to undergo rapid subunit exchange. A recent structural study indicated a tetrameric arrangement of the L12 proteins isolated from Thermotoga maritima, in which the proteins engaged in two different dimerization modes. In one mode, the two monomers of L12 form a tight symmetric and parallel dimer held together by a four-helix bundle, which encompasses the hinge region between the N- and C-terminal domains. In the other mode, the two monomers bind through their N-terminal region in an antiparallel configuration, in which one monomer comprises an alpha-helical hinge and the other monomer adopts an elongated shape with an unfolded hinge region. Presently, it is unclear which dimer contact prevails in solution and on the ribosome. Using cysteine mutants of T. maritima labeled with fluorescent probes, we investigated the mode of interactions between L12 subunits. Data from Forster resonance energy transfer experiments support a dimerization of L12 in solution, in which two monomers bind through their N-terminal region in an antiparallel configuration. We also demonstrate that the rate of subunit exchange in T. maritima L12 is significantly slower at 25 degrees C than that in the E. coli system. The exchange rate increases with increasing temperature and approaches the one observed for the E. coli system at 50 degrees C. Possible factors responsible for this difference are discussed.     

On the flexibility of myosin in solution.

Rabbit skeletal muscle myosin from the same rabbit was prepared by two different methods, and then purified by either Sephadex or hydroxylapatite chromatography. The resulting myosin samples were analyzed in 2-10 mM sodium pyrophosphate solutions at pH 9 using transient electric birefringence. The birefringence decay signals were fitted using a Fortran program called DISCRETE and two relaxation times, 49.7 +/- 5.6 and 11.2 +/- 2.5 microseconds, were determined. These relaxation times were independent of the method of myosin preparation, the method of myosin purification, the concentration of sodium pyrophosphate between 2 and 10 mM, the concentration of myosin between 0.08 and 1.59 mg/mL, and the temperature between 4.0 and 20.0 degrees C, after correction to 20.0 degrees C. The longer relaxation time is consistent with a rigid, linear myosin molecule. The shorter relaxation time is consistent with myosin that has a completely flexible hinge region in the myosin tail. Both relaxation times are inconsistent with the previously reported single relaxation time of myosin obtained by fitting the birefringence decay data to only 90% of the decay signal. By forcing some of the birefringence decay data in the presence work to fit 90% of the decay signal with a single relaxation time, approximately the same relaxation time as previously reported was obtained.