Identification and characterization of deamidation sites in the conserved regions of human immunoglobulin gamma antibodies

Deamidation of asparagine residues of biological pharmaceuticals is a major cause of chemical degradation if the compounds are not formulated and stored appropriately. The mechanism of this nonenzymatic chemical reaction has been studied in great detail; however, the identification of deamidation sites in a given protein remains a challenge. In this study, we identified and characterized all deamidation sites in the conserved region of a recombinant monoclonal antibody. The conserved region of this antibody is shared by all human IgGs with the exception of minor differences in the hinge region. Our high-performance liquid chromatography method could separate the succinimide, isoaspartic, and aspartic acid isoforms of peptide fragments generated using trypsin. Each of the isoforms was unambiguously identified using tandem mass spectrometry. Deamidation at the identified four sites was slow for the intact, folded antibody at accelerated degradation conditions (pH 7.5 and 37 degrees C). Deamidation was enhanced after reduction, alkylation, and tryptic digestion, indicating that the three-dimensional structure of the antibody reduced deamidation. Furthermore, after the reduction, alkylation, and tryptic digestion, only 4 of a possible 25 asparagine residues showed deamidation, demonstrating the effect of the primary amino acid sequence, especially the -1 and +1 amino acids flanking the deamidation site. For instance, the amino acid motifs SNG, ENN, LNG, and LNN were found to be more prone to deamidation, whereas the motifs GNT, TNY, YNP, WNS, SNF, CNV, SNT, WNS, FNW, HNA, FNS, SNK, GNV, HNH, SNY, LNW, SNL, NNF, DNA, GNS, and FNR showed no deamidation. Our findings should help predict deamidation sites in proteins and peptides and help develop deamidation-resistant biological therapeutics.     

超滤膜除谷氨酸发酵液中菌体对等电提取收率的影响

采用截留分子量（ＭＷＣＯ）为６万和１万的卷式超滤膜,去除谷氨酸发酵液中的菌体,同时将发酵液中的固含量浓缩１０倍,再利用等电法提取谷氨酸。经超滤后的发酵液等电收率可达到９０．９６％,在提高等电收率的同时,菌体的去除还降低了污水的处理负荷。     

Formation of lamellar structure in gamma titanium aluminide

Abstracts are not published in this journal This revised version was published online in November 2006 with corrections to the Cover Date.     

Differentiating N-terminal aspartic and isoaspartic acid residues in peptides

Formation of isoaspartic acid (isoAsp) is a common modification of aspartic acid (Asp) or asparagine (Asn) residue in proteins. Differentiation of isoAsp and Asp residues is a challenging task owing to their similar properties and identical molecular mass. It was recently shown that they can be differentiated using ion-electron or ion-ion interaction fragmentation methods (ExD) because these methods provide diagnostic fragments c + 57 and z(?) - 57 specific to the isoAsp residue. To date, however, the presence of such fragments has not been explored on peptides with an N-terminal isoAsp residue. To address this question, several N-terminal isoAsp-containing peptides were analyzed using ExD methods alone or combined with chromatography. A diagnostic fragment [M + 2H - 74](+?) was observed for the doubly charged precursor ions with N-terminal isoAsp residues. For some peptides, identification of the N-terminal isoAsp residue was challenging because of the low diagnostic ion peak intensity and the presence of interfering peaks. Supplemental activation was used to improve diagnostic ion detection. Further, N-terminal acetylation was offered as a means to overcome the interference problem by shifting the diagnostic fragment peak to [M + 2H - 116](+?).     

Intramolecular synergistic effect of glutamic acid, cysteine and glycine against copper corrosion in hydrochloric acid solution

The corrosion protection of copper by glutamic acid, cysteine, glycine and their derivative (glutathione) in 0.5 M hydrochloric acid solution has been studied by the electrochemical impedance spectroscopy and cyclic voltammetry. The inhibition efficiency of the organic inhibitors on copper corrosion increases in the order: glutathione > cysteine > cysteine + glutamic acid + glycine > glutamic acid > glycine. Maximum inhibition efficiency for cysteine reaches about 92.9% at 15 mM concentration level. The glutathione can give 96.4% inhibition efficiency at a concentration of 10 mM. The molecular structure parameters were obtained by PM3 (Parametric Method 3) semi-empirical calculation. The intramolecular synergistic effect of glutamic acid, cysteine and glycine moieties in glutathione is attributed to the lower energy of the lowest unoccupied molecular orbital (E_LUMO) level and to the excess hetero-atom adsorption centers and the bigger coverage on the copper surface.     

Studies on in vitro release of CPM from semi-interpenetrating polymer network (IPN) composed of chitosan and glutamic acid

Interpenetrating polymer network (IPN) beads consisting of chitosan-glutamic acid were prepared for in vitro study of controlled release of chlorpheniramine maleate (CPM). A viscous solution of chitosan-glutamic acid was prepared in 2% acetic acid solution, extruded as droplets through a syringe to alkalimethanol solution and the precipitated beads were crosslinked using glutaraldehyde solution. Swelling and drug release studies were carried out. Transport of release medium through the semi-IPN depended upon its pH and extent of crosslinking. The structural and morphological studies of beads were carried out by using a scanning electron microscope (SEM). The larger surface area of beads as well as their ease of handling makes them ideal agents of controlled release.     

Formation of formic acid,acetic acid and lactic acid from decomposition of citric acid by coal ash particles at room temperature

It was found for the first time that citric acid was decomposed to formic acid, acetic acid and lactic acid in the presence of coal ash particles at pH 3 at 20 °C, while it was not decomposed at more than pH 5. The yield of organic acid at stirring time of 60 min is in the order of formic acid > acetic acid > lactic acid. Since citric acid dissociates to citric anion at more than pH 5, it was suggested that citric anion and negatively charged coal ash particles repelled electrically each other at more than pH 5, resulting in that citric acid could not be adsorbed and not be decomposed on coal ash. Based on the obtained results, the decomposition of citric acid at pH 3 was suggested to be due to catalytic effects of coal ash. Since formic acid and acetic acid can be used as a material of hydrogen fermentation, coal ash could be used as a catalyst to synthesize the important material for hydrogen fermentation from wastewater of citric acid.     

Hydrothermal synthesis of large-sized hydroxyapatite whiskers regulated by glutamic acid in solutions with low supersaturation of precipitation

Large-sized hydroxyapatite (HA) whiskers with length of 50–100 μm and width of 0.5 μm were synthesized by a facile hydrothermal method. Dilute reaction solutions containing glutamic acid were used in order to achieve low degree of supersaturation with respective to HA precipitation. The supersaturation values at different hydrothermal temperatures were calculated theoretically from ion concentrations with considerations of all association–dissociation balances between various ions. Experimentally, it was found that HA whiskers with smaller sizes were obtained when raising the additive amount of NaOH or glutamic acid. The regulation mechanism of crystal growth along c-axis was proposed according to the degree of supersaturation, the adsorption/desorption behaviors of glutamate occurred on HA’s (1 0 0) and (0 0 1) planes, as well as the concentration effect of glutamate’s selective adsorption.     

八肋游仆虫一种富含谷氨酸的蛋白GARP的表达与纯化

为研究游仆虫中含有三核苷酸重复序列的GARP基因编码的GARP蛋白在细胞中的功能,利用定点突变技术,将GARP基因中的TGA突变为通用半胱氨酸密码子TGT.突变后的GARP基因构建于原核表达载体pRSETc中,得到重组质粒pRSETc-GARP,将pRSETc-GARP质粒转化至大肠杆菌BL21(DE3)中,经IPTG诱导,带有纯化标签的重组蛋白His6-GARP获得可溶表达,表达产物与抗His抗体在约26 kDa处有很强的交叉反应.His6-GARP蛋白在不同pH条件下通过两次阴离子交换层析和一次凝胶层析三步纯化达到电泳纯.     

Formation of silver nanoparticles in poly(perfluorosulfonic) acid membrane

The formation of silver nanoparticles by chemical reduction of Ag+-loaded Nafion-117 membrane with NaBH4 was studied using radioactivity tagged ions. The counterion-exchange method (Ag(m)+ <--> Na(s)+) was used to obtain a membrane sample with a varying proportion of Ag+ ions. The X-ray elemental mapping across the thickness of the membrane by energy-dispersive X-ray spectrometer attached to the environmental scanning electron microscope (ESEM/EDAX) indicated that Na+ and Ag+ were uniformly distributed in the membrane samples before reduction. The average size of nanoparticles formed after reduction was found to be 15 +/- 3 nm, irrespective of the concentration of silver ions present in the membrane before reduction. Energy-dispersive X-ray fluorescence (EDXRF) analyses of the membrane samples, carried out before and after reduction, indicated that the Ag concentration on the membrane surface was considerably increased after reduction. EDXRF measurements of the membrane samples, obtained from reduction carried out in a dead end cell, indicated that Ag nanoparticles were formed only on the membrane surface exposed to NaBH4 solution. Reduction carried out with NaBH4 tagged with 22Na showed that the formation of Ag nanoparticles involved exchange of Ag+ ions from ion-exchange sites in the interior of the membrane with Na+ ions, followed by reduction of Ag+ ions with BH4- ions at the surface of membrane. The study of self-diffusion of water, Na+, and Cs+ ions in the membrane loaded with Ag nanoparticles indicated that formation of Ag nanoparticles did not affect the diffusional transport properties of the membrane. The ion-exchange capacity and water uptake capacity were also not affected by the formation of Ag nanoparticles in the membrane. The spatial distribution of Ag nanoparticles across the thickness of the membrane obtained by ESEM/EDAX showed that Ag nanoparticles were confined to a few-micrometer surface layer of the membrane. Based on these observations, an attempt has been made to explain the mechanism of the formation of Ag nanoparticles in the membrane.     

Specific racemization of heavy-chain cysteine-220 in the hinge region of immunoglobulin gamma 1 as a possible cause of degradation during storage

Therapeutic antibodies often suffer from degradation due to various modifications during storage. We detected a degradation of immunoglobulin gamma 1 (IgG1) stored for 6 month at 40 °C, and identified the modification as the racemization of Cys220 in the hinge sequence S(219)CDKTHT(225) of the heavy chain by tryptic peptide mapping and tandem mass spectrometry. The rate of racemization at Cys220 was enhanced deliberately by incubating the protein at 50 °C and pH 9.0, while all the other cysteine residues were not affected. The racemization of Cys220 was confirmed by mass spectrometry in conjunction with extracted ion chromatography of the tryptic digest of IgG1 forced to degrade in D(2)O and a series of synthetic hinge fragments containing D-amino acid, as well as the detection of D-cysteine in the acid hydrolysate. To rationalize the possible relationship between the racemization of Cys220 and isopeptide formation at the neighboring residue of Asp221, we suggest a new reaction mechanism that assumes a base catalyst to initiate these reactions by activating the amide nitrogen of Lys222. Due to the highly flexible nature of the hinge region, Lys222 can participate in either the formation of a cyclic imidazoline intermediate involving the α-carbonyl carbon of Cys220 to facilitate the racemization of Cys220 or that of a succinimide structure leading to the isomerization of Asp221.     

Observation of gamma rays from fusion reactions in a tokamak plasma

Characteristic 5.5 MeV gamma rays from the reaction D(p,γ)³He have been detected during neutral beam heated discharges at Doublet-III. The observed rate is in agreement with a production model based on beam-plasma interactions. Applications of this diagnostic for tokamak and mirror experiments are discussed.     

Formation of ZnS nanorods entrapped in polyacrylic acid (PAA) film

A simplistic approach for synthesis of zinc sulphide (ZnS) nanorods is reported. The synthesis of ZnS nanoparticles involved mixing of zinc acetate, sodium sulphide and acrylic acid in appropriate ratio at proper conditions, which formed the core. These nanoparticles were trapped in PAA by in-situ polymerization of acrylic acid and carefully casted into the film which resulted into entrapped nanorods in the polymer matrix. The nanoparticles as well as nanorods entrapped in PAA were characterized using high resolution scanning electron microscopy (SEM) for morphological investigations; energy dispersive X-ray analysis (EDAX) for composition and its crystalinity was checked using X-ray diffraction (XRD). The length of nanorods was in the range of 2–4 μm and thickness between 50–200 nm.     

Assessment of gamma dose rates from terrestrial exposure in Serbia and Montenegro

The gamma dose rates due to naturally occuring terrestrial radionuclides ((226)Ra, (232)Th and (40)K) were calculated based on their activities in soil samples, determined by gamma-ray spectrometry. A total of 140 soil samples from 21 different regions of Serbia and Montenegro were collected. The gamma dose rates ranged from 7.40 to 29.7 nGy h(-1) for (226)Ra, from 12.9 to 46.5 nGy h(-1) for (232)Th and from 12.5 to 37.1 nGy h(-1) for (40)K. The total absorbed gamma dose rate due to these radionuclides varied from 34.5 to 97.6 nGy h(-1) with mean of 66.8 nGy h(-1). Assuming a 20% occupancy factor, the corresponding annual effective dose varied from 4.23 x 10(-5) to 11.9 x 10(-5) Sv with mean of 8.19 x 10(-5) Sv, i.e. the dose was lower than world wide average value. According to the values of external hazard index (mean: 0.39) obtained in this study, the radiation hazard was found to be insignificant for population living in the investigated area.     

Detection of SNM by delayed gamma rays from induced fission

The Pulsed Neutron Interrogation Test Assembly (PUNITA) is an experimental device for research in NDA methods and field applicable instrumentation for nuclear safeguards and security applications. PUNITA incorporates a standard 14-MeV (D-T) pulsed neutron generator inside a large graphite mantle. The generator target is surrounded by a thick tungsten filter with the purpose to increase the neutron output and to tailor the neutron energy spectrum. In this configuration a sample may be exposed to a relatively high average thermal neutron flux of about (2.2±0.1)×10³ s⁻¹ cm⁻² at only 10% of the maximum target neutron emission. The sample cavity is large enough to allow variation of the experimental setup including the fissile sample, neutron and gamma detectors, and shielding materials.The response from SNM samples of different fissile material content was investigated with various field-applicable scintillation gamma detectors such as the 3×2 in. LaBr₃ detector. Shielding in the form of tungsten and cadmium was applied to the detector to improve the signal to background ratio. Gamma and neutron shields surrounding the samples were also tested for the purpose of simulating clandestine conduct. The energy spectra of delayed gamma rays were recorded in the range 100 keV-9 MeV. In addition time spectra of delayed gamma rays in the range 3.3-8 MeV were recorded in the time period of 10 ms-120 s after the 14-MeV neutron burst. The goal of the experiment was to optimize the sample/detector configuration including the energy range and time period for SNM detection. The results show, for example, that a 170 g sample of depleted uranium can be detected with the given setup in less than 3 min of investigation. Samples of higher enrichment or higher mass are detected in much shorter time.     

Precision energy determinations of gamma transitions from 56Co decay

The energies of 16 γ-rays from ⁵⁶Co decay were determined with a Ge(Li) Compton suppression spectrometer. The mixed source technique was used with γ-ray energy standards provided by ^110mAg, ¹²⁴Sb, ¹⁸²Ta, and ¹⁴⁴Ce β-decay. The main motive for these measurements was to obtain more precise energies for transitions with energies in the 2500–3500 keV range. A least-squares adjustment was made to the present and previous γ-ray energy measurements, resulting in a set of recommended γ-ray calibration lines.     

Formation of haloacetamides during chlorination of dissolved organic nitrogen aspartic acid

The stability of haloacetamides (HAcAms) such as dichloroacetamide (DCAcAm) and trichloroacetamide (TCAcAm) was studied under different experimental conditions. The yield of HAcAms during aspartic acid (Asp) chlorination was measured at different molar ratio of chlorine atom to nitrogen atom (Cl/N), pH and dissolved organic carbon (DOC) mainly consisted of humic acid (HA) mixture. Ascorbic acid showed a better capacity to prevent the decay of DCAcAm and TCAcAm than the other two dechlorinating agents, thiosulfate and sodium sulfite. Lower Cl/N favored the DCAcAm formation, implying that breakpoint chlorination might minimize its generation. The pH decrease could lower the concentration of DCAcAm but favored dichloroacetonitrile (DCAN) formation. DCAcAm yield was sensitive to the DOC due to higher chlorine consumption caused by HA mixture. Two possible pathways of DCAcAm formation during Asp chlorination were proposed. Asp was an important precursor of DCAN, DCAcAm and dichloroacetic acid (DCAA), and thus removal of Asp before disinfection may be a method to prevent the formation of DCAcAm, DCAN and DCAA.     

Determination of the efficiency of gamma detectors from particle-gamma angular correlations

Particle-γ angular correlation technique can be used to determine the efficiency of γ-detectors. This method is demonstrated for 4.44 MeV γ-rays from the reaction ¹²C(α, α′γ).     

Formation of SiC whiskers from silicon nitride

Attempts have been made to produce SiC whiskers through vacuum pyrolysis of Si₃N₄ without any addition of extraneous carbon. Vacuum pyrolysis of Si₃N₄ granules and powder compacts, has been carried out at 1550 and 1700°C using a graphite resistance furnace. The products of pyrolysis have been identified through XRD and SEM as SiC whiskers and particles. Small amounts of elemental silicon at 1550°C and free carbon at 1700°C have been detected through X-ray diffraction. Detection of elemental silicon through X-ray diffraction and solidified silicon droplets at the whisker tips in the SEM provide important clues regarding the mechanism of SiC_w formation, as the one involving the reaction 2Si(l) + CO(g) SiC(s) + SiO(g) Silicon carbide whiskers, 3–4 mm long, have been grown from Si₃N₄ compacts at 1550°C over a short period of 0.5 h. It has been shown in the present study that Si₃N₄ can be completely converted to SiC_w, when a loose bed of Si₃N₄ in the form of granules is pyrolysed in the presence of CO at about 1550°C.