Dimethyl Adipimidate Research Articles

Nitrite decreases sickle hemoglobin polymerization in vitro independently of methemoglobin formation

The root cause of sickle cell disease (SCD) is the polymerization of sickle hemoglobin (HbS) leading to sickling of red blood cells (RBC). Earlier studies showed that in patients with SCD, high-dose nitrite inhibited sickling, an effect originally attributed to HbS oxidation to methemoglobin-S even though the anti-sickling effect did not correlate with methemoglobin-S levels. Here, we examined the effects of nitrite on HbS polymerization and on methemoglobin formation in a SCD mouse model. In vitro, at concentrations higher than physiologic (>1 μM), nitrite increased the delay time for polymerization of deoxygenated HbS independently of methemoglobin-S formation, which only occurred at much higher concentrations (>300 μM). In vitro, higher nitrite concentrations oxidized 100% of normal hemoglobin A (HbA), but only 70% of HbS. Dimethyl adipimidate, an anti-polymerization agent, increased the fraction of HbS oxidized by nitrite to 82%, suggesting that polymerized HbS partially contributed to the oxidation-resistant fraction of HbS. At low concentrations (10 μM-1 mM), nitrite did not increase the formation of reactive oxygen species but at high concentrations (10 mM) it decreased sickle RBC viability. In SCD mice, 4-week administration of nitrite yielded no significant changes in methemoglobin or nitrite levels in plasma and RBC, however, it further increased leukocytosis. Overall, these data suggest that nitrite at supra-physiologic concentrations has anti-polymerization properties in vitro and that leukocytosis is a potential nitrite toxicity in vivo. Therefore, to determine whether the anti-polymerization effect of nitrite observed in vitro underlies the decreases in sickling observed in patients with SCD, administration of higher nitrite doses is required.

The stability increase of α-amylase enzyme from Aspergillus fumigatus using dimethyladipimidate

Abstract This study’s purpose is to improve the α-amylase enzyme’s stability from Aspergillus fumigatus applying dimethyladipimidate (DMA). It was conducted in different stages, including production, isolation, purification, modification, and the characterization of native and modified enzymes by the DMA addition. The enzyme activity was specified using the Fuwa and Mandels methods, while the protein level was conducted by the Lowry method. The results indicated that the native enzyme contains a specific activity of 7010.42 U/mg, with an increase of 7.8 times than the crude extract, which contains 904.38 U/mg. Meanwhile, the native enzyme contains an optimum pH of 5 at 55 °C, with residual activity of 17.17% after 60 min of incubation at 55 °C and a half-life of 25.86 min. After the DMA addition with 0.5, 1, and 1.5% concentration, the enzymes had 5.5 optimum pH and 65 °C temperature. Meanwhile, after 60 min of incubation at 65 °C, the modified enzymes had 54.17, 46.18, and 34.44% of residual activity, and 85.55 58.25 and 37.46 min of half-lives, respectively. This showed that the addition of DMA to the native α-amylase from A. fumigatus increased the stability of the modified enzymes by 1.5–3.3 times than the native enzyme.

A disposable lab-on-a-chip platform for highly efficient RNA isolation

Despite recent advances in lab-on-a-chip (LOC) technology, RNA-based miniaturized diagnostic platforms have been falling behind due to a lack of on-chip RNA isolation techniques. However, RNA analysis plays an important role in gene expression determination and pathogen detection. To meet the increasing need for the analysis of RNA with point-of-care (POC) testing, a reliable LOC platform for RNA isolation needs to be developed. Here, we present a method of RNA isolation using a LOC platform that provides high-quality RNA from a variety of biological samples. By taking advantage of pH-dependent reversible dimethyl adipimidate (DMA) binding to RNA, we were able to perform RNA isolation on a disposable LOC platform within 30min without the need for chaotropic salts or solvent. Using this LOC platform, gene expression analyses were successfully carried out with RNA isolated from different cell lines (HEK293, Caco-2, MCF-7, and T24) by subsequent real-time reverse transcription PCR. In addition, we validated the utility of this LOC platform with viral RNA isolated from Dengue viruses (DENV-1, −2, −4). We demonstrated a high recovery rate and rapid processing with minimal contamination. Therefore, this novel method could be an ideal RNA isolation technique in gene expression research and for the diagnosis of infectious diseases caused by RNA viruses in POC testing.

Two-stage sample-to-answer system based on nucleic acid amplification approach for detection of malaria parasites

Rapid, early, and accurate diagnosis of malaria is essential for effective disease management and surveillance, and can reduce morbidity and mortality associated with the disease. Although significant advances have been achieved for the diagnosis of malaria, these technologies are still far from ideal, being time consuming, complex and poorly sensitive as well as requiring separate assays for sample processing and detection. Therefore, the development of a fast and sensitive method that can integrate sample processing with detection of malarial infection is desirable. Here, we report a two-stage sample-to-answer system based on nucleic acid amplification approach for detection of malaria parasites. It combines the Dimethyl adipimidate (DMA)/Thin film Sample processing (DTS) technique as a first stage and the Mach-Zehnder Interferometer-Isothermal solid-phase DNA Amplification (MZI-IDA) sensing technique as a second stage. The system can extract DNA from malarial parasites using DTS technique in a closed system, not only reducing sample loss and contamination, but also facilitating the multiplexed malarial DNA detection using the fast and accurate MZI-IDA technique. Here, we demonstrated that this system can deliver results within 60min (including sample processing, amplification and detection) with high sensitivity (<1 parasite μL−1) in a label-free and real-time manner. The developed system would be of great potential for better diagnosis of malaria in low-resource settings.

Solid phase nucleic acid extraction technique in a microfluidic chip using a novel non-chaotropic agent: dimethyl adipimidate

Here, we present a silicon microfluidic system for the purification and extraction of nucleic acids from human body fluid samples utilizing a dimethyl adipimidate (DMA)-based solid-phase extraction method. We propose DMA, which has been used as an amino-reactive cross-linking agent within cells and proteins, as a non-chaotropic reagent for the capture of nucleic acids to overcome the limitations of existing chaotropic and non-chaotropic techniques such as low binding efficiency, PCR inhibition and so on. DMA contains bi-functional imidoesters that form reversible cross-linking structures with DNA therefore providing a high surface-area to volume ratio for capturing DNA without structurally modifying microfluidic channels. In this work, we have first demonstrated highly efficient capture and purification of genomic DNA (T24 cell line) with DMA using a label-free silicon microring resonator sensor device. In addition, we observed the improvement of the DNA amplification efficiency by using the proposed technique for both the genetic (HRAS) and epigenetic (RARβ) analysis of DNA biomarkers. Particularly, we confirmed that the DMA-based solid-phase extraction technique can be applied for the extraction of genomic DNA with higher purity (p < 0.001) using human body fluids (blood and urine) in silicon microfluidic devices compared to other chaotropic methods. Therefore, the proposed technique would be able to harmonize with a micro-total analysis system platform for the analysis of genetic and epigenetic DNA biomarkers related to human diseases in the field of point-of-care (POC) diagnostic applications.

The monomeric state of the proton‐coupled folate transporter represents the functional unit in the plasma membrane

Folic acid is an essential vitamin required for de novo biosynthesis of nucleotides and amino acids. The proton-coupled folate transporter (PCFT; SLC46A1) has been identified as the major contributor for intestinal folate uptake. It is also involved in folate transport across the blood-brain barrier and into solid tumors. PCFT belongs to the major facilitator superfamily. Major facilitator superfamily members can exist in either monomeric or homo-oligomeric form. Here, we utilized blue native polyacrylamide gel electrophoresis (BN/PAGE) and crosslinking with bi-functional chemicals to investigate the quaternary structure of human PCFT after heterologous expression in Xenopus laevis oocytes and CHO cells. PCFT was expressed in the plasma membrane in both expression systems. The functionality of the utilized PCFT construct was confirmed in oocytes by folic acid induced currents at acidic pH. For both the oocyte and CHO expression system [(3)H]folic acid uptake studies indicated that PCFT was functional. To analyze the oligomeric state of PCFT in the plasma membrane, plasma membranes were isolated by polymerization with colloidal silica and polyacrylic acid and subsequent centrifugation. The digitonin-solubilized non-denatured PCFT migrated during BN/PAGE as a monomer, as judged by comparison with a membrane protein (5-HT(3A) receptor) of known pentameric assembly that was used to create a molecular sizing ladder. The chemical crosslinkers glutaraldehyde and dimethyl adipimidate were not able to covalently link potential higher order PCFT structures to form oligomers that were stable following SDS treatment. Together, our results demonstrate that plasma-membrane PCFT functions as a monomeric protein.

Chemical Modification of Saccharomycopsis fibuligera R64 α-Amylase to Improve its Stability Against Thermal, Chelator, and Proteolytic Inactivation

α-Amylase catalyzes hydrolysis of starch to oligosaccharides, which are further degraded to simple sugars. The enzyme has been widely used in food and textile industries and recently, in generation of renewable energy. An α-amylase from yeast Saccharomycopsis fibuligera R64 (Sfamy) is active at 50 °C and capable of degrading raw starch, making it attractive for the aforementioned applications. To improve its characteristics as well as to provide information for structural study ab initio, the enzyme was chemically modified by acid anhydrides (nonpolar groups), glyoxylic acid (GA) (polar group), dimethyl adipimidate (DMA) (cross-linking), and polyethylene glycol (PEG) (hydrophilization). Introduction of nonpolar groups increased enzyme stability up to 18 times, while modification by a cross-linking agent resulted in protection of the calcium ion, which is essential for enzyme activity and integrity. The hydrophilization with PEG resulted in protection against tryptic digestion. The chemical modification of Sfamy by various modifiers has thereby resulted in improvement of its characteristics and provided systematic information beneficial for structural study of the enzyme. An in silico structural study of the enzyme improved the interpretation of the results.

An approach to peptide-based ATP receptors by a combination of random selection, rational design, and molecular imprinting

Random selection, rational design and molecular imprinting were cooperatively utilized to develop peptide-based ATP synthetic receptors. In this fusion strategy, combinatorial chemistry was utilized for screening a precursor peptide useful for construction of ATP receptors, and rational design was employed in modification of the selected precursor peptide for higher affinity and selectivity. Finally, molecular imprinting was used for pre-organizing the conformation of the precursor peptide as complementary to a target molecule ATP. The fusion strategy appeared to have advantage to sole use of the individual strategy: (1) a low hit-rate of combinatorial chemistry will be improved by customizing a higher order structure of a selected peptide by molecular imprinting, (2) combinatorial chemistry allows us to semi-automatically select components of water-compatible synthetic receptors, (3) rational design improves the selected peptide sequence for better molecularly imprinted receptors. A peptide consisting of a randomly selected sequence and a rationally designed sequence (Resin-Lys-Gly-Arg-Gly-Lys-Gly-Gly-Gly-Glu-Lys-Tyr-Leu-Lys-NHAc) was designed and synthesized as a precursor peptide. The rational design was made according to the sequence of the adenine binding site of biotin carboxylase. The on-beads peptide was cross-linked with dimethyl adipimidate in the presence of ATP. In the saturation binding tests, the cross-linked on-beads peptide showed 5.3 times higher affinity compared to the non-cross-linked peptide with the same sequence. Furthermore, the cross-linked peptide showed improved selectivity; the ratios of binding constants, K (ATP)/ K (ADP) and K (ATP)/ K (GTP), were increased from 2.4 to 19, and from 0.8 to 10, respectively. It would be notable that the peptide without the rationally designed sequence showed no discrimination between ATP and GTP ( K (ATP)/ K (GTP) as 0.9), suggesting that the rationally designed site was successfully engaged for recognition of the adenine base.

SAP18 Promotes Krüppel-dependent Transcriptional Repression by Enhancer-specific Histone Deacetylation

Body pattern formation during early embryogenesis of Drosophila melanogaster relies on a zygotic cascade of spatially restricted transcription factor activities. The gap gene Krüppel ranks at the top level of this cascade. It encodes a C2H2 zinc finger protein that interacts directly with cis-acting stripe enhancer elements of pair rule genes, such as even skipped and hairy, at the next level of the gene hierarchy. Krüppel mediates their transcriptional repression by direct association with the corepressor Drosophila C terminus-binding protein (dCtBP). However, for some Krüppel target genes, deletion of the dCtBP-binding sites does not abolish repression, implying a dCtBP-independent mode of repression. We identified Krüppel-binding proteins by mass spectrometry and found that SAP18 can both associate with Krüppel and support Krüppel-dependent repression. Genetic interaction studies combined with pharmacological and biochemical approaches suggest a site-specific mechanism of Krüppel-dependent gene silencing. The results suggest that Krüppel tethers the SAP18 bound histone deacetylase complex 1 at distinct enhancer elements, which causes repression via histone H3 deacetylation.

Effect of Intracellular Magnesium and Oxygen Tension on K+-Cl- Cotransport in Normal and Sickle Human Red Cells

In red cells from normal individuals (HbA cells), theK+-Cl- cotransporter (KCC) is inactivated by low O2tension whilst in those from sickle cell patients (HbScells), it remains fully active. Changes in freeintracellular [Mg2+] have been proposed as amechanism. In HbA cells, KCC activity was stimulatedby Mg2+ depletion and inhibited by Mg2+ loading butthe effect of O2 was independent of Mg2+. At all [Mg2+]is,the transporter was stimulated in oxygenated cells,minimally active in deoxygenated ones. By contrast,the stimulatory effects of O2 was abolished by inhibitorsof protein (de)phosphorylation. HbS cells hadelevated KCC activity, which was of similar magnitudein oxygenated and deoxygenated cells, regardlessof Mg2+ clamping. In deoxygenated cells, theantisickling agent dimethyl adipimidate inhibitedsickling, Psickle and KCC. Results indicate a role forprotein phosphorylation in O2 dependence of KCC,with different activities of the relevant enzymes in HbAand HbS cells, probably dependent on Hb

Preparation of well-defined bovine polyhemoglobin based on dimethyl adipimidate and glutaraldebyde cross-linkage

A well-defined bovine polyhemoglobin was prepared by dimethyl adipimidate (DMA) and glutaraldehyde double cross-linkage method. DMA was used to block some amino groups of hemoglobin, followed by further polymerization with glutaraldehyde. The amino modification degree of hemoglobin was 32% when DMA reacted with hemoglobin at the molar ratio of 200. The bovine polyhemoglobin with narrow molecular weight distribution (mainly 128 kDa) was obtained when glutaraldehyde reacted with DMA-modified hemoglobin. The P 50 and the Hill coefficient for DMA-modified hemoglobin were 19.4 mm Hg and 2.28, respectively, while those for the bovine polyhemoglobin were 15.1 mm Hg and 1.70, respectively. The number of Bohr protons released for DMA-modified hemoglobin and the polyhemoglobin was 0.86 and 0.56 H/tetramer, respectively.

Intramolecular cross-linking experiments on cytochrome c and ribonuclease A using an isotope multiplet method.

Mass spectral analysis of tryptic digests of cross-linked proteins offers considerable promise as a simple technique to probe protein structure and study protein-protein interactions. We describe the use of a 1:1 mixture of isotopically labeled and unlabeled cross-linkers, disuccinimidyladipate (DSA) and dimethyladipimidate (DMA), to enhance visualization of cross-linked peptides in a tryptic digest. Optimized intramolecular reactions of cytochrome c and ribonuclease A (RNase A) with DSA yielded an average of two cross-links per protein molecule. After digestion of the cross-linked cytochrome c with trypsin and analysis by liquid chromatography/mass spectrometry (LC/MS) and matrix-assisted laser desorption/ionization (MALDI), eight modified peptides, five cross-linked and two end-capped, were detected by virtue of their doublet character. An eighth modified peptide's identity remained ambiguous because of its inability to fragment. The lysine-lysine distance constraints obtained are discussed in the context of the known NMR and X-ray structures of cytochrome c. Analysis of cross-linked RNase A by LC/MS and MALDI yielded nine modified peptides, four of which were modified twice, as indicated by the isotopic triplets. Although seven of these peptides contained cross-links, few distance constraints were gained due to the fact that the cross-linked products were variations of modification of the same three lysine residues.

Effect of dimethyl adipimidate on K + transport and shape change in red blood cells from sickle cell patients

Dimethyl adipimidate (DMA) reduces K + loss from, and dehydration of, red cells containing haemoglobin S (HbS cells). Three membrane transporters may contribute to these processes: the deoxygenation-induced cation-selective channel (P sickle), the Ca 2+-activated K + channel (or Gardos channel) and the K +-Cl − cotransporter (KCC). We show that DMA inhibited all three pathways in deoxygenated HbS cells. The Gardos channel could be activated following Ca 2+ loading. Considerable KCC activity was present in oxygenated HbS cells, showing a selective action of DMA on the transporter in deoxygenated cells. Inhibition of sickling correlated strongly with that of P sickle and moderately with that of KCC activity. We conclude that DMA does not inhibit the K + pathways directly, but acts mainly by preventing HbS polymerisation and sickling. These findings are relevant to the development of novel chemotherapeutic agents for amelioration of sickle cell disease.

Nucleotide binding and hydrolysis properties of Neurospora crassa cytosolic molecular chaperones, Hsp70 and Hsp80, heat-inducible members of the eukaryotic stress-70 and stress-90 families.

Formation of a hetero-oligomeric complex between Hsp70 and Hsp80 of Neurospora crassa was observed previously by means of chemical crosslinking and enzyme-linked immunosorbent assays (ELISA). The present study documents the effect of nucleotides on the subunit structure of Hsp70 and Hsp80 by crosslinking with bifunctional reagents: glutaraldehyde, dimethyl adipimidate (DMA), and dimethyl suberimidate (DMS). The inter-protomer crosslinking of Hsp80 with DMA and DMS was suppressed by ATP and to a lesser extent by ADP, CTP, and NAD. Crosslinking of purified Hsp70 by glutaraldehyde yielded dimers and higher order oligomers. Binding of ATP, ADP, CTP, and NAD, but not NADH, led to a marked reduction in the yield of oligomers. Similarly, crosslinking by DMA and DMS was suppressed by ADP, ATP, and CTP. Both Hsp70 and Hsp80 exhibited intrinsic ATPase activity. Interestingly, ATP levels exceeding 25 microM resulted in pronounced inhibition of the ATPase activity of Hsp80 and 0.5 mM and 0.25 mM ATP led to a prolonged lag in the reaction. Addition of NAD resulted in the abolition of the lag period. The binding of 2-p-toluidinylnapthalene-6-sulfonate (TNS) to Hsp70 and its displacement by ATP and other nucleotides demonstrated the hydrophobic nature of the nucleotide-binding region.

Activation of bovine tracheal chloride channels by amino group‐specific reagents

1. The predominant Cl- channel in bovine tracheal epithelial cells has a conductance of approximately 71 pS and accounts for more than 80 % of the total chloride conductance. We examined the effects of protein-modifying reagents on channel function and found that amino groups are critically involved in gating. 2. Patch clamp studies showed that lysine-specific reagents, such as dimethyl adipimidate (DMA), significantly increased the channel open probability, but not its conductance. This suggests that modified residues are involved in the gating mechanism, but are distant from the channel permeation pathway. 3. Kinetic analysis of channel activity showed that histograms of open and closed durations could be well fitted by double exponential distributions, suggesting that the channel has at least two open and two closed states. DMA did not change the number of open or closed states, but increased channel mean open time. 4. Since membrane impermeant reagents were effective only from the extracellular side, we conclude that lysine residues in the extracellular domain of the channel are critically involved in gating. These residues may present an important target for site-directed mutagenesis and pharmacological activation of Cl- channels in epithelial cells.

The quaternary geometry of transcription termination factor rho: assignment by chemical cross-linking

Transcription termination factor rho from Escherichia coli is a ring-shaped homohexamer of 419 amino acid subunits and catalyzes an ATP-dependent release of nascent RNA transcripts. Previous chemical cross-linking studies suggested that the rho hexamer might have D 3 symmetry with three isologous dimers as protomers. However, our recent mutational analysis of rho alongside its putative structural homology to F 1-ATPase rather argued for C 6 symmetry. To resolve this discrepancy, we have re-investigated the pattern of cross-linking of rho using various cross-linkers with different functional groups and spacer lengths. Upon reaction with dimethyl suberimidate followed by SDS-polyacrylamide gel electrophoresis, rho protein generated a series of cross-linked oligomers up to hexamers, of which dimers migrated as distinct doublet bands of approximately equal intensities. However, the lower band became much stronger than the upper one with dimethyl adipimidate and difluorodinitrobenzene, and vice versa with disuccinimidyl glutarate, disuccinimidyl suberate and disulfosuccinimidyl tartarate. Furthermore, the trimeric products also produced doublet bands, whose relative intensities were again variable with cross-linkers, but in an inverse correlation with those of the dimer bands. These results combined with theoretical considerations support a C 6 symmetry model in which cross-linking is assumed to occur stochastically at one of two alternative sites within each subunit interface with variable relative frequencies depending on cross-linkers. The D 3 symmetry is excluded, for the putative trimeric subspecies should always retain mutually equal intensities in that case. Detailed inspections of the cross-linking kinetics further revealed a moderate characteristic of C 3 symmetry for the rho hexamer such that the collective as well as relative rates of cross-linking at the two available sites could fluctuate between alternating interfaces. The final model designated as C 3/6 is also compatible with other functional and structural properties known for rho.

A study of the oligomeric state of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-preferring glutamate receptors in the synaptic junctions of porcine brain.

The number of the subunits in an alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-preferring L-glutamate receptor in the synaptic junctions of porcine brain was investigated in this study. Upon incubation of the synaptic junctions with three cross-linking regents, dimethyl adipimidate (DMA), dimethyl suberimidate (DMS) and N-succinimidyl-(4-azidophenyl)-1,3'-dithiopropionate (SADP), AMPA receptor subunits in higher-molecular-mass aggregates were detected by immunoblotting. These aggregates migrated as proteins of approx. 200, 300 and 400 kDa. The number and identity of the subunits in a solubilized AMPA receptor were also investigated here. Two samples, W1 and W2, enriched in AMPA receptors were prepared from synaptic junctions by a combination of detergent-solubilization, anion-exchange chromatography and wheatgerm agglutinin affinity chromatography. Hydrodynamic behaviour analyses revealed that the majority of the AMPA receptors in either one of these samples were asymmetrical detergent-surrounded particles with a protein mass around 350 kDa. SDS/PAGE analysis revealed that the majority of AMPA receptors in the W1 sample were comprised of dimers of 106 kDa subunits which were covalently linked by disulphide bonds. Cross-linking these receptors with SADP yielded a new band of approx. 400 kDa. The results obtained here, either from the studies of AMPA receptors embedding in synaptic junctions or from those of detergent-solubilized and partially purified receptors, suggest that AMPA receptors contain a basic core structure comprising of four 106 kDa subunits.

Stabilization of penicillin G acylase against pH by chemical cross-linking

The effect of pHs between 2·0 and 10·0 on the inactivation kinetics of penicillin G acylase (PGA) obtained from a mutant of Escherichia coli ATCC 11105 and the stabilization of enzyme against pH by chemical cross-linking with dimethyladipimidate (DMA) were studied. The inactivation mechanisms of native and DMA cross-linked PGA both appeared to obey first-order inactivation kinetics during prolonged incubation of enzyme solutions at 40°C and at different pH values. The lowest inactivation rate constants were obtained with both native and DMA cross-linked PGA at pH 7·0. Inactivation rate constants decreased with an increase in pH from 2·0 to 7·0 and then increased again at pH values above 7·0. Chemical cross-linking of PGA by DMA resulted in the stabilization of enzyme against pH. Highest enhancement of pH stabilization (nearly four-fold) was obtained at pH 7·0 and 8·0.

Production of specific antibodies and development of a non-isotopic immunoassay for carbamazepine by the carbonyl metallo-immunoassay (CMIA) method

As part of our ongoing work to extend the range of applications of the non-isotopic carbonyl metalloimmunoassay (CMIA), previously developed in our laboratory, we describe here the first CMIA study of carbamazepine. The CMIA method uses a metal carbonyl complex as a non-isotopic tracer, and in this case we chose to employ the dicobalt hexacarbonyl moiety (Co 2(CO) 6) attached to an alkyne. Two organometallic tracers, 3 and 7 , were synthesized, differentiated by the nature and length of the spacer arm of the Co 2(CO) 6 moiety. Two different coupling methods were subsequently used to synthesize the immunogens 1 and 2, the first one used a carbodiimide, while the second, employed dimethyl adipimidate as coupling agent. Titer values of the antisera obtained by injection of these immunogens into rabbits, were determined by CMIA, using one of the organometallic complexes, 3 or 7 , as tracer. Both antisera had higher titer values with the long-chain tracer, 7 , than with the short-chain tracer, 3 . However these titer values were very different: low for antiserum 1 and high for antiserum 2. The cross-reactivity of antiserum 2 with other antiepileptic drugs was negligible. For competition curves, these was good sensitivity with the antibody 2 3 pairing, while a broad assay range was obtained with antibody 2 7 pairing. These results demonstrate the viability of CMIA as an immunoassay method for carbamazepine, and open the way to development of a simultaneous multiassay by CMIA of the principal antiepileptic drugs.

Thermostabilization of penicillin G acylase obtained from a mutant of Escherichia coli ATCC 11105 by bisimidoesters as homobifunctional cross-linking agents

We investigated the effects of three different bisimidoesters as homobifunctional cross-linking agents on the thermostabilization of penicillin G acylase (PGA) obtained from a mutant of Escherichia coli ATCC 11105. Cross-linkers were dimethyladipimidate (DMA), dimethylsuberimidate (DMS), and dimethyl-3,3′-dithiobispropionimidate (DTBP). The thermal inactivation mechanisms of the native and cross-linked PGA were both considered to obey first-order inactivation kinetics during prolonged heat treatment, forming fully active, susceptible transient states. The efficacy of the cross-linkers on the thermostabilization of PGA was estimated to be DMA > DMS > DTBP. Optimal concentrations of DMA, DMS, and DTBP for cross-linking of PGA were found to be 0.5, 0.4, and 0.3% (w/v), respectively. The greatest enhancement of the thermostabilities was observed during DMA cross-linking, as a nearly 15-fold increase at temperatures above 50°C. Cross-linking by DMA did not cause much change in the parameters V m, K m, and the optimal temperature values of PGA, but the activation energy of the enzyme was slightly decreased and k cat value slightly increased after cross-linking.