Deuterium Exchange Rates Research Articles

Электронное влияние ароматического кольца в положении 2 на H/D обмен метильных протонов в пиримидине

The H/D exchange of hydrogen atoms of methyl groups in pyrimidines containing a phenyl or substituted pyrazole ring in position 2, namely, 2-phenyl-4-methyl-5-ethoxycarbonyl-, 2-phenyl-4-methyl-5-acetyl and 2-(3,5 dimethylpyrazolyl-1-)-4-methyl-5-acetylpyrimidines was studied. It has been shown that the rate of deuterium exchange depends on the electronic influence of the lateral aromatic ring associated with pyrimidine. When a phenyl group is introduced into position 2 of the pyrimidine ring almost immediately after adding deuterated sodium methoxide (CD3ONa) to the CD3OD solution, the signals from the protons of the ester or acetyl groups disappear.

Ion-neutral clustering alters gas-phase hydrogen-deuterium exchange rates.

The rates and mechanisms of chemical reactions that occur at a phase boundary often differ considerably from chemical behavior in bulk solution, but remain difficult to quantify. Ion-neutral interactions are one such class of chemical reactions whose behavior during the nascent stages of solvation differs from bulk solution while occupying critical roles in aerosol formation, atmospheric chemistry, and gas-phase ion separations. Through a gas-phase ion separation technique utilizing a counter-current flow of deuterated vapor, we quantify the degree of hydrogen-deuterium exchange (HDX) and ion-neutral clustering on a series of model chemical systems (i.e. amino acids). By simultaneously quantifying the degree of vapor association and HDX, the effects of cluster formation on reaction kinetics are realized. These results imply that cluster formation cannot be ignored when modeling complex nucleation processes and biopolymer structural dynamics.

Estimation of the slow hydrogen–deuterium exchange rates for local water confined in 1-butyl-3-methylimidazolium tetrafluoroborate via nuclear magnetic resonance

The unique properties of water in ionic liquid (IL)–water mixtures have attracted considerable attention; however, the association state of water in the IL nanospace requires continuous investigations. Here, we determined the proton exchange rate among water molecules from the half-band width of the proton nuclear magnetic resonance water peaks (H2O and HDO) in mixtures of two typical ILs, namely, 1-butyl-3-methylimidazolium tetrafluoroborate ([BMIM][BF4]) and N,N-diethyl-N-methyl-N-(2-methoxyethyl) ammonium tetrafluoroborate ([DEME][BF4]). At H2O:D2O = 1:1, the effects of water concentration and pH were systematically studied. Based on the data, we discussed very slow hydrogen–deuterium exchange in the IL mixtures with the association state of water molecules.

Ionic liquid-based reverse micelles. Use of hydrogen–deuterium exchange as a tool for surfactant self-assembly characterization

We report the study of the rate for hydrogen–deuterium (H-D) exchange in the cation of the ionic liquid-surfactant 1-butyl-3-methylimidazolium 1,4-bis-2-ethylhexylsulfosuccinate (bmim-AOT) when it forms reverse micelles (RMs). The results were compared with those found for bmim-AOT dissolved in D2O showing an inhibition larger than 500-fold on going from bulk D2O to the reverse micelle. Also, the effect of varying water content and surfactant concentration of the RMs on the deuterium exchange was evaluated by 1H NMR technique. The results demonstrate that the H-D exchange at the C(2) position of bmim+ when it forms RMs significantly reflects the properties of the micellar microenvironment, specifically of the micellar interface due to the localization of bmim+ in the interfacial region, intercalated between the AOT moiety. The rate of deuterium exchange is directly related to the properties of interfacial water, which, in turn, is closely related to the water-surfactant interaction. Thus, the analysis of deuterium exchange in the 2-position of bmim+ is proposed as a useful tool to explore the properties of RMs formed by this cation, with the advantage of being a non-invasive technique since the ionic liquid-surfactant itself acts as the molecular probe.

Hydrogen-Deuterium Addition and Exchange in N-Ethylmaleimide Reaction with Glutathione Detected by NMR Spectroscopy.

Glutathione (GSH) is an important and ubiquitous thiol compound abundantly present in virtually every living cell. It is a powerful antioxidant critically required to protect cells from oxidative damage and free radical injury. Its quantification in ex vivo analysis remains a major challenge because it spontaneously oxidizes to form glutathione disulfide. N-Ethylmaleimide (NEM) is a well-known Michael acceptor, which reacts rapidly and irreversibly with thiol and prevents disulfide bond formation. Based on thiol conjugation to NEM, recently, the concentration of GSH was determined in human blood using NMR spectroscopy [Anal. Chem, 2021, 93(44): 14844–14850]. It was found that hydrogen–deuterium addition and exchange occur during the thiol–maleimide reaction as well as NMR analysis, generating a series of poorly explored diastereomers/isotopomers. Here, we establish a general NMR approach to identify the thiosuccinimide diastereomers/isotopomers derived from the thiol–maleimide reaction. The thiol-Michael addition reaction was conducted for GSH and another thiol compound, cysteine, separately, using D2O and H2O. The conjugates were characterized by 1H/13C 1D/2D NMR under different solvent, buffer, and pH conditions. The Michael addition combined with the H/D exchange formed twelve unique diastereomers/isotopomers. NMR measurements allowed the distinct assignment of all structures in solutions and quantification of H/D addition and exchange. Interestingly, the deuterium exchange rate was dependent on structure, pH, and buffer. The elucidation of the thiol–maleimide reaction and H/D exchange mechanism can potentially impact areas including metabolomics, small molecule synthesis, and bioconjugation chemistry.

Desialylation of O‐glycans activates von Willebrand factor by destabilizing its autoinhibitory module

BackgroundThe binding of the A1 domain of von Willebrand factor (VWF) to platelet receptor glycoprotein (GP)Ibα defines the VWF activity in hemostasis. Recent studies suggest that sequences flanking A1 form cooperatively an autoinhibitory module (AIM) that reduces the accessibility of the GPIbα binding site on A1. Application of a tensile force induces unfolding of the AIM. Desialylation induces spontaneous binding of plasma VWF to platelets. Most O‐glycans in VWF are located around the A1 domain. Removing certain O‐glycans in the flanking sequences by site‐directed mutagenesis enhances A1 binding to GPIbα and produces an effect similar to type 2B von Willebrand disease in animals. ObjectivesTo understand if and how desialylation of O‐glycans in the flanking sequences increases A1 activity. MethodsA recombinant AIM‐A1 fragment encompassing VWF residues 1238–1493 and only O‐glycans was treated with neuraminidase to produce desialylated protein. The glycan structure, dynamics, stability, and function of the desialylated protein was characterized by biochemical and biophysical methods and compared to the sialylated fragment. ResultsAsialo‐AIM‐A1 exhibited increased binding activity and induced more apparent platelet aggregation than its sialylated counterpart. It exhibited a lower melting temperature, and increased hydrogen–deuterium exchange rates at residues near the secondary GPIbα binding site and the N‐terminal flanking sequence. Asialo‐AIM‐A1 is less mechanically stable than sialo‐AIM‐A1, with its unstressed unfolding rate approximately 3‐fold greater than the latter. ConclusionsDesialylation of O‐glycans around A1 increases its activity by destabilizing the AIM.

Hydrogen-Deuterium Exchange Plus Mass Spectrometry (HX-MS) Reveals a Novel Mechanism Underlying the pH-Dependent Regulation of ADAMTS13 Activity

Background: ADAMTS13 is a metalloprotease that is essential for the cleavage of von Willebrand factor (VWF). Deficiency of plasma ADAMTS13 activity causes a potentially fatal blood disorder, thrombotic thrombocytopenic purpura. Previous studies demonstrated a two-fold increase in the proteolytic activity of ADAMTS13 towards the VWF73 substrate by deleting the distal C-terminal domains, decreasing the pH, or binding a monoclonal antibody to the C-terminal domains. This suggests an allosteric interaction between the distal and proximal domains of ADAMTS13 modulated by pH.Objectives: To identify peptide regions within a recombinant full-length ADAMTS13 (rADAMTS13) responsible for the pH-modulated allosteric mechanism.Methods: To identify regions in the rADAMTS13 that undergo conformational changes in response to a change in pH, we performed hydrogen deuterium exchange plus mass spectrometry (HX-MS) at three different pH conditions, pH 6, pH 7, and pH 8. Because the rate of hydrogen and deuterium exchange is dependent upon the pH in a logarithmic fashion, a single time point from each of these pH conditions were compared. For instance, 6000 s was used for pH 6, 600 s for pH 7, and 60 s for pH 8. Any differences in the deuterium incorporation in these conditions in a given peptide should be contributed to conformational changes within the protein effecting accessibility and not simply the rate of hydrogen and deuterium exchange.Results: Purified rADAMTS13 and a variant truncated after the spacer domain (rMDTCS) were incubated with deuterium for various times (0- 6000 s). After quenching the reaction by lowering pH to 2.5 and temperature to 0 °C, the rADAMTS13 and rMDTCS were digested by pepsin and fungal protease-13 and then partially resolved by liquid chromatography before being injected into a LTQ ObitrapXL mass spectrometer for peptide identification. Upon comparing the mass spectra of these three conditions, the peptides with mass spectra shift greater than 1 m/z unit as a result of conformational changes from the pH change were identified in the metalloprotease, cysteine-rich, spacer, and CUB-2 domains. In the metalloprotease, three peptide regions were identified (i.e. residues 110 -131, 181-197, and 217-230) with 19, 6, and 1 overlapping peptides, respectively. Within the cysteine rich domain, there were two identified peptide regions, 468 - 480 and 502 - 503. Within the spacer domain, there was only one identified peptide region 629-630, with 5 overlapping peptides. Finally, In CUB-2, there were two peptide regions, 1327-1340 and 1366-1377, with 2 overlapping peptides each (see Figure). All of these regions, with only the exception of residues 217-230, were more accessible to deuterium at pH 6 than at pH 8.To determine if these results are contingent upon the whole protein being present, and possible intramolecular protein interactions, the HX experiment was repeated using two ADAMTS13 variants (i.e. rMDTCS and rT5C). The rT5C contains the C-terminal TSP1 5-8 repeats and CUB domains. Because of smaller size, more overlapping peptides were recovered, which allows for narrowing down the residues central to the conformational change. Two small areas were narrowed down to several peptides, residues 120-123, with 6 overlapping peptides, and residue 503 within the cysteine rich with 3 overlapping peptides. Curiously, both regions within the CUB-2 domain showed little change with pH, despite 6 overlapping peptides in the residues 1327 -1340 and residues 1366-1377 (see Figure), suggesting the necessity of the proximal domains of the protein to modulate the conformation as a function of pH.Finally, the regions within the rADAMTS13 that exhibit a pH dependent conformation change are similar to the regions we reported showing bimodal behavior previously. Regions within the metalloprotease, the cysteine rich, and CUB-2 are all shared in residues 217-230, 468-480, and 1327-1328, respectively (see Figure).Conclusions: Our results, along with the direct binding experiments using a surface plasmon resonance assay, suggest that the N- and C-terminus of ADAMTS13 interact with each other at multiple discrete sites, which mediates the pH-dependent regulation of ADAMTS13 conformation and function. [Display omitted] DisclosuresZheng:Alexion: Speakers Bureau; Ablynx: Consultancy.

Implications for an imidazol-2-yl carbene intermediate in the rhodanase-catalyzed C-S bond formation reaction of anaerobic ergothioneine biosynthesis.

In the anaerobic ergothioneine biosynthetic pathway, a rhodanese domain containing enzyme (EanB) activates tne hercynine's sp2 ε-C-H Dona ana replaces it with a C-S bond to produce ergothioneine. The key intermediate for this trans-sulfuration reaction is the Cys412 persulfide. Substitution of the EanB-Cys412 persulfide with a Cys412 perselenide does not yield the selenium analog of ergothioneine, selenoneine. However, in deuterated buffer, the perselenide-modified EanB catalyzes the deuterium exchange between hercynine's sp2 ε-C-H bond and D2O. Results from QM/MM calculations suggest that the reaction involves a carbene intermediate and that Tyr353 plays a key role. We hypothesize that modulating the pKa of Tyr353 will affect the deuterium-exchange rate. Indeed, the 3,5-difluoro tyrosine containing EanB catalyzes the deuterium exchange reaction with k ex of ~10-fold greater than the wild-type EanB (EanBWT). With regards to potential mechanisms, these results support the involvement of a carbene intermediate in EanB-catalysis, rendering EanB as one of the few carbene-intermediate involving enzymatic systems.

Cation–Anion and Anion–CO2 Interactions in Triethyl(octyl)phosphonium Ionic Liquids with Aprotic Heterocyclic Anions (AHAs)

Ionic liquids with aprotic heterocyclic anions (AHAs) have been developed for postcombustion CO2 capture applications. The anions of AHA ILs play a significant role in tuning anion-CO2 complexation. In addition, AHAs are able to trigger the abstraction of acidic protons located at the α position of phosphonium cations by forming hydrogen bonds between cations and anions, eventually leading to cation-driven CO2 complexation. Here we investigate the role of the anion in cation-anion hydrogen bonding and ylide formation. Using CO2 uptake measurements, 31P nuclear magnetic resonance (NMR), attenuated total reflection-Fourier transform infrared (ATR-FTIR) deuterium exchange equilibrium and rates, two-dimensional nuclear Overhauser effect spectroscopy (2D NOESY), and density functional theory calculations, we show that the key is the proximity of the negatively charged nitrogen atoms on the anion to the α protons, which is governed not just by anion basicity but by sterics. Thus, we show that triethyl(octyl)phosphonium 3-methyl-5-trifluoromethylpyrazolide is much more effective in hydrogen-bonding with and deprotonating the cation than the equivalent [P2228] ILs with more basic 2-cyanopyrrolide and 3-trifluoromethylpyrazolide anions.

Characterization of Recombinant β2GPI Using Hydrogen-Deuterium Exchange and X-Ray Crystallography

Introduction: Antiphospholipid syndrome (APS) is an autoimmune disorder caused by "antiphospholipid" antibodies (aPL) directed against β2-glycoprotein I (β2GPI). However, there remains significant inconsistency in measurements of anti-β2GPI antibodies, possibly due to different sources of β2GPI. To address this issue and others, we have developed a system in which recombinant β2GPI (rβ2GPI) is expressed in mammalian (HEK293) cells using a lentiviral construct with a CMV promoter, in which the signal peptide of β2GPI has been mutated. In previous work, we demonstrated that patient-derived anti-β2GPI antibodies recognize rβ2GPI similarly to purified plasma β2GPI. Plasma Β2GPI has been crystallized and found to be in a "fishhook" or "J" conformation, however the protein was isolated under harsh conditions that included perchloric acid precipitation. Moreover, electron microscopy and other approaches have suggested that β2GPI may adopt several conformations in addition to the fishhook, including circular and other intermediate forms. Methods: cDNA encoding full length APOH (the gene encoding β2GPI), was cloned into pLenti CMV Puro DEST. Lentivirus was produced using the Lentiviral Gateway Expression kit and stable cell lines developed by transducing HEK293 cells with lentivirus encoding APOH. Cell lines were grown in suspension, and supernatants containing secreted full length β2GPI were purified using a hitrap-heparin column. Anti-β2GPI antibody binding to rβ2GPI was determined using a standard β2GPI ELISA. For hydrogen-deuterium exchange (HDX) mass spectrometry, proteins were in 10 mM phosphate buffer, 99.9% D2O in an autosampler at 20 °C. After 10-10,000 seconds of incubation, the reaction was quenched. Sequences of peptic fragments were identified and mass assignment for each peptide without deuterium exchange was checked manually. The average of relative fractional deuterium uptake for each amide proton residue was calculated using HDsite equations. Crystallization of plasma derived and recombinant β2GPI-WT was performed using a hanging-drop vapor diffusion method at 4°C in 150 mM NaCl and 50 mM Tris-HCl pH 7.3. Results: After transduction with lentiviral β2GPI constructs, HEK-293 cells efficiently secreted rβ2GPI-WT, site-directed mutants and several domains constructs. Hydrogen-deuterium exchange (HDX) analysis of plasma derived and rβ2GPI-WT revealed that both have very similar surface deuterium exchange rates and heat map profiles (Figure 1A, B). In both plasma derived and rβ2GPI, limited digestion of domains D-I, II and III by pepsin was observed, consistent with other sushi domain containing proteins. However, solid coverage of domain IV and V was obtained and the heat maps generated for both plasma derived and rβ2GPI-WT were very similar, suggesting similar structure and conformation. Moreover, although crystallization of plasma derived and rβ2GPI-WT revealed differences in crystal size and appearance, X-ray structural analysis to a resolution of 3.1 Å (Figure 1C, D) demonstrated that both plasma derived and rβ2GPI had the same structure and conformation under the conditions employed. Conclusion: These studies demonstrate that recombinant β2GPI produced in mammalian cells following lentiviral transduction has a similar structure and conformation as plasma-derived β2GPI. This methodology may be used for additional mutagenesis studies to better define the underlying structural features regulating the conformation of β2GPI as well as its interactions with other proteins, diagnostic utility and functions. Disclosures McCrae: Sanofi Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Rigel Pharmaceutical: Membership on an entity's Board of Directors or advisory committees; Pfizer Pharmaceutical: Membership on an entity's Board of Directors or advisory committees; Dova Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees.

Hydrogen transfer reactions relevant to Guerbet coupling of alcohols over hydroxyapatite and magnesium oxide catalysts

The rates of double bond hydrogenation, deuterium exchange, and benzyl alcohol dehydrogenation were compared to those of ethanol coupling.

Kinetic isotope effects for fast deuterium and proton exchange rates.

By monitoring the effect of deuterium decoupling on the decay of transverse (15)N magnetization in D-(15)N spin pairs during multiple-refocusing echo sequences, we have determined fast D-D exchange rates kD and compared them with fast H-H exchange rates kH in tryptophan to determine the kinetic isotope effect as a function of pH and temperature.

Properties of water confined in ionic liquids.

The varying states of water confined in the nano-domain structures of typical room temperature ionic liquids (ILs) were investigated by 1H NMR and by measurements of self-diffusion coefficients while systematically varying the IL cations and anions. The NMR peaks for water in BF4-based ILs were clearly split, indicating the presence of two discrete states of confined water (H2O and HOD). Proton and/or deuterium exchange rate among the water molecules was very slowly in the water-pocket. Notably, no significant changes were observed in the chemical shifts of the ILs. Self-diffusion coefficient results showed that water molecules exhibit a similar degree of mobility, although their diffusion rate is one order of magnitude faster than that of the IL cations and anions. These findings provide information on a completely new type of confinement, that of liquid water in soft matter.

Reactivity and H–D Exchange Rate of the α-Methylene of Deoxyvasicinone and its Homologs

The deuterium exchange rate of the α-methylene of deoxyvasicinone (1) and mackinazolinone (2) and their seven- (3) and eight-membered (4) homologs was studied in neutral, acidic, and basic media. It was found that the exchange rate depended on the number of methylenes in the polymethylene ring and the medium. The deuterium exchange rate decreased in the order 2→1→3→4 and was slowest in media close to neutral. The results agreed with those for the reactions of 1–4 with bromine in acidic medium, condensation with substituted aromatic and heterocyclic aldehydes, and acylation by aromatic acid chlorides. Several trends in the reactivity of the tricyclic quinazolin-4-ones were discovered.

Time-resolved multidimensional NMR with non-uniform sampling

Time-resolved experiments demand high resolution both in spectral dimensions and in time of the studied kinetic process. The latter requirement traditionally prohibits applications of the multidimensional experiments, which, although capable of providing invaluable information about structure and dynamics and almost unlimited spectral resolution, require too lengthy data collection. Our work shows that the problem has a solution in using modern methods of NMR data collection and signal processing. A continuous fast pulsing three-dimensional experiment is acquired using non-uniform sampling during full time of the studied reaction. High sensitivity and time-resolution of a few minutes is achieved by simultaneous processing of the full data set with the multi-dimensional decomposition. The method is verified and illustrated in realistic simulations and by measuring deuterium exchange rates of amide protons in ubiquitin. We applied the method for characterizing kinetics of in vitro phosphorylation of two tyrosine residues in an intrinsically disordered cytosolic domain of the B cell receptor protein CD79b. Signals of many residues including tyrosines in both phosphorylated and unmodified forms of CD79b are found in a heavily crowded region of 2D 1H–15N correlation spectrum and the significantly enhanced spectral resolution provided by the 3D time-resolved approach was essential for the quantitative site-specific analysis.Electronic supplementary materialThe online version of this article (doi:10.1007/s10858-013-9811-1) contains supplementary material, which is available to authorized users.

Familial Alzheimer’s mutations within APPTM increase Aβ42 production by enhancing accessibility of ε-cleavage site

The high Aβ42/Aβ40 production ratio is a hallmark of familial Alzheimer's disease, which can be caused by mutations in the amyloid precursor protein (APP). The C-terminus of Aβ is generated by γ-secretase cleavage within the transmembrane domain of APP (APPTM), a process that is primed by an initial ε-cleavage at either T48 or L49, resulting in subsequent production of Aβ42 or Aβ40, respectively. Here we solve the dimer structures of wild-type APPTM (AAPTM WT) and mutant APPTM (FAD mutants V44M) with solution NMR. The right-handed APPTM helical dimer is mediated by GXXXA motif. From the NMR structural and dynamic data, we show that the V44M and V44A mutations can selectively expose the T48 site by weakening helical hydrogen bonds and increasing hydrogen-deuterium exchange rate (kex). We propose a structural model in which FAD mutations (V44M and V44A) can open the T48 site γ-secretase for the initial ε-cleavage, and consequently shift cleavage preference towards Aβ42.

Use of H/D isotope effects to gather information about hydrogen bonding and hydrogen exchange rates

Polar side-chains in proteins play important roles in forming and maintaining three-dimensional structures, and thus participate in various biological functions. Until recently, most protein NMR studies have focused on the non-exchangeable protons of amino acid residues. The exchangeable protons attached to polar groups, such as hydroxyl (OH), sulfhydryl (SH), and amino (NH2) groups, have mostly been ignored, because in many cases these hydrogen atoms exchange too quickly with water protons, making NMR observations impractical. However, in certain environments, such as deep within the hydrophobic interior of a protein, or in a strong hydrogen bond to other polar groups or interacting ligands, the protons attached to polar groups may exhibit slow hydrogen exchange rates and thus become NMR accessible. To explore the structural and biological implications of the interactions involving polar side-chains, we have developed versatile NMR methods to detect such cases by observing the line shapes of 13C NMR signals near the polar groups, which are affected by deuterium-proton isotope shifts in a mixture of H2O and D2O. These methods allow the detection of polar side-chains with slow hydrogen–deuterium exchange rates, and therefore provide opportunities to retrieve information about the polar side-chains, which might otherwise be overlooked by conventional NMR experiments. Future prospects of applications using deuterium-proton isotope shifts to retrieve missing structural and dynamic information of proteins are discussed.

Experimental investigation of rates and mechanisms of isotope exchange (O, H) between volcanic ash and isotopically-labeled water

The hydrogen and oxygen isotope ratios in hydrous minerals and volcanic glass are routinely used as paleo-proxies to infer the isotopic values of meteoric waters and thus paleo-climatic conditions. We report a series of long-term exposure experiments of distal 7700BP Mt. Mazama ash (−149‰ δ2H, +7‰ δ18O, 3.8 wt.% H2O) with isotopically-labeled water (+650‰ δ2H, +56‰ δ18O). Experiments were done at 70, 40 and 20°C, and ranged in duration from 1 to 14454h (∼20months), to evaluate the rates of deuterium and 18O exchange, and investigate the relative role of exchange and diffusion. We also investigate the effect of drying on H2Otot and δ2H in native and reacted ash that can be used in defining the protocols for natural sample preparation. We employ Thermal Conversion Elemental Analyzer (TCEA) mass spectrometry, thermogravimetric analysis and a KBr pellet technique with infrared spectroscopy to measure the evolution of δ2H, total water, and OH water peaks in the course of exposure experiments, and in varying lengths of vacuum drying.Time series experiments aided by infrared measurements demonstrate the following new results: (i) It wasobserved that from 5 to >100‰ δ2H increases with time, with faster deuterium exchange at higher temperatures. Times at 15% of theoretical “full δ2H exchange” are: 15.8years at 20°C, 5.2years at 40°C, and 0.4years at 70°C. (ii) Even at extended exposure durations experiments show no net increase in water weight percent nor in δ18O in ash; water released from ash rapidly by thermal decomposition is not enriched in δ18O. This observation clearly suggests that it is hydrogen exchange, and not water addition or oxygen exchange that characterizes the process. (iii) Our time series drying, Fourier transform infrared (FTIR)-KBr and Thermogravimetric Analyzer (TGA) analyses collectively suggest a simple mechanistic view that there are three kinds of “water” in ash: water (mostly H2O) that is less strongly bonded on the surface of ash particles that are getting lost with 24–48h of drying to up to 200–300°C, bound water in glass in a range of combining proportions of SiOH to H2O, and a small reservoir of residual, tightly held water.Experimentation with vacuum drying at 130–220°C, and with TGA up to 1000°C provides a set of simple relationships and recommendations for users. Ash loses 1–1.2wt.% water with weight loss stabilizing after 48h of vacuum drying at 130°C. This ash drying removes molecular water over the hydroxyl group in a proportion of ∼80:20% resulting in relatively constant δ2H values of the remaining total water in native ash.This study demonstrates that δ2H in ash can be rapidly changed by minor diagenesis even at relatively low temperatures of 20°C. A diagenetic history of ash is needed to interpret the D/H ratio, but the δ18O values of water in ash are more robust.

Structural Comparability Between Recombinant FVIII-Fc and Its Isolated FVIII and Fc Constituents

AbstractAbstract 1135rFVIIIFc is a recombinant fusion protein comprising human B domain-deleted (BDD) coagulation factor VIII covalently attached to the Fc domain of human immunoglobulin G1 (IgG1). rFVIIIFc is currently in a phase 3 clinical study as a novel long-lasting FVIII molecule designed to potentially reduce dosing frequency and provide prolonged protection from bleeds for patients with hemophilia A receiving prophylaxis or on-demand treatment, or undergoing surgery. In this study, we evaluated the structural comparability of BDD rFVIII and rFVIIIFc using small angle X-ray scattering (SAXS) and hydrogen/deuterium exchange (HDX) mass spectrometry. SAXS analysis of BDD rFVIII and rFVIIIFc, performed at the Advanced Light Source Synchrotron, provided the electron pair radius distribution of each molecule and accurate but inherently low resolution structural information. Both BDD rFVIII and rFVIIIFc were monomeric in solution. BDD rFVIII had a radius of gyration (Rg) of 39.7 Å, compared with a Rg of 38 Å calculated from its published atomic coordinates (2R7E.pdb). rFVIIIFc had a larger Rg of 51 Å, which was consistent with a simple computational model of BDD rFVIII tethered to Fc that predicted an Rg of 50 Å. HDX mass spectrometric analysis yielded 94% coverage of the BDD rFVIII component and 87% coverage of the Fc component. Rates of deuterium exchange within peptides derived from each of these components in the context of the rFVIIIFc fusion protein were very similar to those of the isolated BDD rFVIII and Fc constituents. Taken together, the results of SAXS and HDX analyses support the conclusion that fusion of the Fc moiety to BDD rFVIII does not alter the higher order structure of either component. Disclosures:Kulman:Biogen Idec: Employment. Bou-Assaf:Biogen Idec: Employment. Berkowitz:Biogen Idec: Employment. Light:biogenidec: Employment. Mezo:Biogen Idec: Employment. Wang:Biogen Idec: Employment. Whitlow:Colabrativ Inc.: Employment; Biogen Idec: Consultancy. Culyba:Biogen Idec: Employment. Peters:Biogen Idec: Employment.

Effect Of Benzyl Alcohol on Recombinant Human Interleukin-1 Receptor Antagonist Structure and Hydrogen–Deuterium Exchange

Benzyl alcohol, a preservative commonly added to multidose therapeutic protein formulations, can accelerate aggregation of recombinant human interleukin-1 receptor antagonist (rhIL-1ra). To investigate the interactions between benzyl alcohol and rhIL-1ra, we used nuclear magnetic resonance to observe the effect of benzyl alcohol on the chemical shifts of amide resonances of rhIL-1ra and to measure hydrogen–deuterium exchange rates of individual rhIL-1ra residues. Additionof 0.9% benzyl alcohol caused significant chemical shifts of amide resonances for residues 90–97, suggesting that these solvent-exposed residues participate in the binding of benzyl alcohol. In contrast, little perturbation of exchange rates was observed in the presence of either sucrose or benzyl alcohol. © 2011 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 100:4215–4224, 2011