Characterization of morphine-glucose-6-phosphate dehydrogenase conjugates by mass spectrometry

A key characteristic of the analyte-reporter enzyme conjugate used in the enzyme-multiplied immunoassay technique (EMIT) is the inhibition of the conjugate enzyme upon anti-analyte antibody binding. To improve our understanding of the antibody-induced inhibition mechanism, we characterized morphine-glucose-6-phosphate dehydrogenase (G6PDH) conjugates as model EMIT analyte-reporter enzyme conjugates. Morphine-G6PDH conjugates were prepared by acylating predominantly the primary amines on G6PDH with morphine 3-glucuronide NHS ester molecules. In this study, morphine-G6PDH conjugates were characterized using a combination of methods, including tryptic digestion, immunoprecipitation, matrix-assisted laser desorption ionization mass spectrometry, and electrospray ionization tandem mass spectrometry. Twenty-six conjugation sites were identified. The identified sites all were found to be primary amines. The degree of conjugation was determined to be less than the number of conjugation sites, suggesting heterogeneity within the morphine-G6PDH conjugate population. Two catalytically important residues in the active site (K22 and K183) were among the identified conjugation sites, explaining at least partially the cause of loss of activity due to the coupling reaction.     

DURATIONS OF GAMETE MOTILITY AND CONJUGATION ABILITY OF ULVA COMPRESSA (ULVOPHYCEAE)1

The present study was designed to develop a technique for crossing and to gain insight into how sexual reproduction contributes to the maintenance of local populations of Ulva compressa L. To examine the durations of gamete motility and conjugation ability, freshly released gametes were incubated for various periods of time prior to mixing both mating types. The conjugation ability of the gametes gradually declined after being released from the thalli when the gametes were incubated without mixing with the opposite mating type. The ability to conjugate decreased by half after 6 h, although most of the gametes remained motile. The gametes released 4 h later had the same level of conjugation ability when mixed immediately after releasing. When the mature thalli were wrapped in a moist paper towel to prevent gametes from being released, the gametes were preservable for 7 h without a significant decrease in their conjugation ability. Conjugation occurred soon after mixing gametes of both mating types and reached a plateau after 30 s. However, conjugation rates did not exceed a rate of ～70%, even though freshly released gametes were used. Interestingly, a portion of the gametes newly conjugated 30 min after mixing both mating types, and conjugation rates reached a second plateau at ～90%. Gametes with delayed conjugation are provided some period of time that allows them to be transported away and increases their chances of mating with more distant populations, thus contributing to the maintenance of genetic variation.     

Conjugating oligosaccharides to proteins by squaric acid diester chemistry: rapid monitoring of the progress of conjugation, and recovery of the unused ligand

Samples that are periodically withdrawn from the mixture of a conjugation reaction can be analyzed on a picomolar scale without any work-up or pre-purification using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) in combination with the ProteinChip^® System. The technique provides rapid information about the increasing molecular mass of the glycoconjugate formed, thereby allowing termination of the process when the desired incorporation of the ligand onto the carrier protein is achieved. The excess oligosaccharide used at the onset of conjugation can be recovered and used in preparation of a similar neoglycoconjugate. The overall economy of conjugations, which often involve labor-intensive linker-equipped oligosaccharides, can be markedly increased in this way.     

Biochemical and cellular changes occuring during conjugation in Hansenula wingei

Brock, Thomas D. (Indiana University, Bloomington). Biochemical and cellular changes occurring during conjugation in Hansenula wingei. J. Bacteriol. 90:1019-1025. 1954.-A technique has been devised for deagglutinating mixed populations of conjugating cells so as to be able to visualize microscopically early stages of the conjugation process. A cell can form a conjugation tube only when in contact with a cell of opposite mating type, but may do so even if the mate is unresponsive or ultraviolet-inactivated. Cell fusion occurs, however, only when both cells are able to form conjugation tubes in a region of contact. Fusion begins almost as soon as the two cells begin to form protuberances, and long before any dissolution of cell-wall material between the cells occurs. A cell which has conjugated in one region of its cell wall is still able to conjugate with another cell in another region, so that triply and quadruply conjugated cells are occasionally formed. There is no significant net increase in deoxyribonucleic acid, ribonucleic acid, protein, or carbohydrate which might be related to the conjugation process, because any minor changes that occur in these components are also detected when cells of only one mating type are incubated or when the conjugation process is inhibited with the antibiotic cycloheximide. Changes in activity of beta-1,3-glucanase (with laminarin as substrate) and beta-1,6-glucanase (with pustulan as substrate) have been measured during the conjugation process, in addition to changes in the activity of several control enzymes which would not be expected to be related to the conjugation process. Significant increases in invertase (sucrase), laminarinase, and pustulanase were detected, and minimal increases occurred in beta-glucosidase and acid phosphatase. However, these same increases were also observed in controls involving only one mating type; thus, these increases are probably not related to the conjugation process, but may be a result of other processes which probably occur during incubation in the conjugation medium.     

Photoreduction of monoclonal antibodies for conjugation and fragmentation

The conjugation of enzymes, fluorescent or radioactive labels, cross-linkers and other moieties to antibodies is a commonly performed procedure in biochemical research. Using reduced disulphides, conjugation can be an inconvenient, multistep, time- and material-consuming process. We have developed a reduction technique based on UV irradiation, which lacks these drawbacks. Antibodies are irradiated in a sealed vial for a few minutes by a common laboratory UV source in the presence of stannous ions, following the depletion of atmospheric oxygen. The preparation may subsequently be conjugated with thiol-reactive probes such as maleimide derivatives, with no need for any prior purification or concentration. This simple, rapid and effective reduction and conjugation process results in a fully functional immunoglobulin conjugate that can be used for a variety of biochemical applications.     

One-pot synthesis, purification, and formulation of bionanoparticle-CpG oligodeoxynucleotide hepatitis B surface antigen conjugate vaccine via tangential flow filtration

The synthesis and characterization of a Hepatitis B virus vaccine (HBsIC-ISS) candidate composed of Hepatitis B surface antigen (HBsAg) bionanoparticles conjugated to multiple copies of immunostimulatory sequence oligodeoxynucleotides is presented. An efficient tangential flow filtration (TFF) method has been developed to purify the conjugated bionanoparticles from the excess conjugation reagents. The TFF technique presented can serve as a rapid and convenient alternative to current methods like ultracentrifugation for the separation of excess small molecule/polymeric conjugation reagents from chemically modified viruses and other viruslike particles.     

Genetic interactions in Streptomyces rimosus mediated by conjugation and by protoplast fusion

The development of a protoplast fusion technique for oxytetracycline-producing Streptomyces rimosus strains, and its evaluation for the application for a breeding programme, has been described. Treatment of S. rimosus protoplasts with 40% (w/v) PEG 1550 for 30 min gave optimal numbers of recombinants ranging from 1 to 10% of the total progeny. Therefore, by comparison with conjugation, protoplast fusion increased the frequency of recombination by two to three orders of magnitude. The proportion of multiple crossover classes amongst recombinants was higher, by a factor of ten, after protoplast fusion (13.3%) than after conjugation (1.5%). Participation of less frequent complementary genotype doubled from 9.0% in conjugation to 17.9% in protoplast fusion. Overall, this suggested that the opportunities for crossing over in a fusion of S. rimosus protoplasts were spatially and/or temporally extended leading to a loosening of linkage with a near-random assortment of genotypes in a cross. However, by minimizing the multiple crossover classes and calculating allele frequency gradients, it was shown that the protoplast fusion technique allows arrangement of genetic markers on the S. rimosus chromosome. These are ideal characteristics for the recombination of divergent lines in a strain improvement programme.     

Combined physical and chemical immobilization of glucose oxidase in alginate microspheres improves stability of encapsulation and activity

Chemical sensors utilizing immobilized enzymes and proteins are important for monitoring chemical processes and biological systems. In this study, calcium-cross-linked alginate hydrogel microspheres were fabricated as enzyme carriers by an emulsification technique. Glucose oxidase (GOx) was encapsulated in alginate microspheres using three different methods: physical entrapment (emulsion), chemical conjugation (conjugation), and a combination of physical entrapment and chemical conjugation (emulsion-conjugation). Nano-organized coatings were applied on alginate/GOx microspheres using the layer-by-layer self-assembly technique in order to stabilize the hydrogel/enzyme system under biological environment. The encapsulation of GOx and formation of nanofilm coating on alginate microspheres were verified with FTIR spectral analysis, zeta-potential analysis, and confocal laser scanning microscopy. To compare both the immobilization properties of enzyme encapsulation techniques and the influence of nanofilms with uncoated microspheres, the relationship between enzyme loading, release, and effective GOx activity (enzyme activity per unit protein loading) were studied over a period of four weeks. The results produced four key findings: (1) the emulsion-conjugation technique improved the stability of GOx in alginate microspheres compared to the emulsion technique, reducing the GOx leaching from microsphere from 50% to 17%; (2) the polyelectrolyte nanofilm coatings increased the GOx stability over time, but also reduced the effective GOx activity; (3) the effective GOx activity for the emulsion-conjugation technique (about 3.5 x 10(-)(5) AU microg(-)(1) s(-)(1)) was higher than that for other methods, and did not change significantly over four weeks; and (4) the GOx concentration, when compared after one week for microspheres with three bilayers of poly(allylamine hydrochloride)/sodium poly(styrene sulfonate) ({PAH/PSS}) coating, was highest for the emulsion-conjugation technique. As a result, the comparison of these three techniques showed the emulsion-conjugation technique to be a potentially effective and practical way to fabricate alginate/GOx microspheres for implantable glucose biosensor application.     

Low modulus biomimetic microgel particles with high loading of hemoglobin

We synthesized extremely deformable red blood cell-like microgel particles and loaded them with bovine hemoglobin (Hb) to potentiate oxygen transport. With similar shape and size as red blood cells (RBCs), the particles were fabricated using the PRINT (particle replication in nonwetting templates) technique. Low cross-linking of the hydrogel resulted in very low mesh density for these particles, allowing passive diffusion of hemoglobin throughout the particles. Hb was secured in the particles through covalent conjugation of the lysine groups of Hb to carboxyl groups in the particles via EDC/NHS coupling. Confocal microscopy of particles bound to fluorescent dye-labeled Hb confirmed the uniform distribution of Hb throughout the particle interior, as opposed to the surface conjugation only. High loading ratios, up to 5 times the amount of Hb to polymer by weight, were obtained without a significant effect on particle stability and shape, though particle diameter decreased slightly with Hb conjugation. Analysis of the protein by circular dichroism (CD) spectroscopy showed that the secondary structure of Hb was unperturbed by conjugation to the particles. Methemoglobin in the particles could be maintained at a low level and the loaded Hb could still bind oxygen, as studied by UV-vis spectroscopy. Hb-loaded particles with moderate loading ratios demonstrated excellent deformability in microfluidic devices, easily deforming to pass through restricted pores half as wide as the diameter of the particles. The suspension of concentrated particles with a Hb concentration of 5.2 g/dL showed comparable viscosity to that of mouse blood, and the particles remained intact even after being sheared at a constant high rate (1000 1/s) for 10 min. Armed with the ability to control size, shape, deformability, and loading of Hb into RBC mimics, we will discuss the implications for artificial blood.     

The use of the plasmid pTH10 for isolating the donor strains of Pseudomonas mallei

The plasmid pTH10 was transfered by conjugation into the Pseudomonas mallei strains. An attempt to construct the donor strains using the widely known technique employing the homology between the plasmid and chromosome due to the transposon Tn1 carried by the plasmid was unsuccessful. Among the clones resistant to bacteriophage PRD1 the variants were selected with the supposed integration of the plasmid into the chromosome. The latter clones required the ability to transfer the auxotrophic chromosomal markers in conjugation after the repeated conjugational transfer of the plasmid pTH10 into them.     

Arginine-rich peptide conjugation to morpholino oligomers: effects on antisense activity and specificity

Noncharged antisense compounds, such as phosphorodiamidate morpholino oligomers (PMOs), do not readily enter mammalian cells in culture. A simple and effective means for cellular delivery of PMOs is through their conjugation to arginine-rich peptides. Understanding the effect of peptide conjugation on the efficacy, toxicity, and specificity of PMOs is important to the successful application of this antisense delivery method. We investigated the effects of conjugation of arginine-rich peptides to PMO on the thermal stability, efficacy and specificity for targeted RNA of the resulting compound. In vitro translation assays showed that (1) R9F2-PMO generated antisense activity 3-25-fold higher than corresponding nonconjugated PMO, (2) the level of antisense activity enhancement by R9F2-PMO over a corresponding nonconjugated PMO is related to the GC content of the PMO sequence, (3) R9F2 conjugation reduced the minimum length of a PMO required to inactivate a target RNA from 20 bases to 14 bases, and (4) nonspecific effects of R9F2-PMO occur at lower concentrations than corresponding PMO alone. Thermal stability of heteroduplexes of PMO and complementary RNA were increased by conjugation of PMO to R9F2 peptide, likely accounting for the increased specific antisense activity of conjugated over nonconjugated PMO. A cell-culture based assay demonstrated that while conjugation to unnatural peptides increased PMO efficacy without causing nonspecificity at concentrations < or = 10 microM, only L-peptide conjugation retained high specificity at higher concentrations. This study demonstrates that conjugation of PMO to an arginine-rich peptide generally increases the binding affinity of the PMO to complementary RNA and increases its antisense potency. Additionally, it is shown that the enzymatic stability of an L- or unnatural peptide used for PMO conjugation affects the antisense properties of the resulting compound.     

The Zinc Finger Protein Zfr1p Is Localized Specifically to Conjugation Junction and Required for Sexual Development in Tetrahymena thermophila

Conjugation in Tetrahymena thermophila involves a developmental program consisting of three prezygotic nuclear divisions, pronuclear exchange and fusion, and postzygotic and exconjugant stages. The conjugation junction structure appears during the initiation of conjugation development, and disappears during the exconjugant stage. Many structural and functional proteins are involved in the establishment and maintenance of the junction structure in T. thermophila. In the present study, a zinc finger protein-encoding gene ZFR1 was found to be expressed specifically during conjugation and to localize specifically to the conjugation junction region. Truncated Zfr1p localized at the plasma membrane in ordered arrays and decorated Golgi apparatus located adjacent to basal body. The N-terminal zinc finger and C-terminal hydrophobic domains of Zfr1p were found to be required for its specific conjugation junction localization. Conjugation development of ZFR1 somatic knockout cells was aborted at the pronuclear exchange and fusion conjugation stages. Furthermore, Zfr1p was found to be important for conjugation junction stability during the prezygotic nuclear division stage. Taken together, our data reveal that Zfr1p is required for the stability and integrity of the conjugation junction structure and essential for the sexual life cycle of the Tetrahymena cell.     

Isolation of the R'his plasmids of Vibrio cholerae

V. cholerae strain VT5104 capable of donor activity in conjugation has been constructed by the genetic technique based on plasmid RP4::Mucts62 integration into V. cholerae chromosome due to plasmid homology with Mucts62 inserted into the chromosome. The gene for histidine synthesis has been mobilized and transferred into the recipient cells from VT5104 donor. The conjugants obtained are able to efficiently transfer his+ gene included into the plasmid structure in conjugation with eltor recipient. Thus, the constructed strain VT5104 generates R' plasmids carrying V. cholerae chromosomal genes.     

Preparation of poly(ethylene glycol) protected nanoparticles with variable bioconjugate ligand density

Maleimide-functional poly(ethylene glycol)-b-poly(epsilon-caprolactone) nanoparticles (NPs) were prepared via the Flash NanoPrecipitation technique. Subsequent reaction with a model ligand, bovine serum albumin (BSA), was conducted using thiol-maleimide conjugation. Reaction of up to 22% of NP surface maleimide-PEG tethers was obtained, with the percent conversion being essentially independent of the ratio of maleimide-PEG to methyl-PEG over the range 30-100%, respectively. At the highest surface coverage, BSA is calculated to essentially cover the NP surface area. Reaction parameters (reaction order and docking constant) describing the extent of ligand conjugation were determined. The reaction order is applicable to the conjugation of ligands presenting free thiol functionalities, while the value of the docking constant is ligand-dependent and accounts for physical and dynamic properties of the ligand-PEG interaction. Jointly, the particle formation process, using block copolymer-directed kinetically controlled assembly and surface functionalization represent a versatile new platform for the preparation of bioconjugated NPs with accurate control of ligand density and minimal processing steps.     

Mouse Apg10 as an Apg12-conjugating enzyme: analysis by the conjugation-mediated yeast two-hybrid method

Autophagosome formation is a central event in macroautophagy. The Apg12-Apg5 conjugate, which is essential in this process, is generated by a ubiquitin-like protein conjugation system. In yeast, Apg12, following activation by the E1-like Apg7, forms a thioester with Apg10 (E2-like). Apg12 is finally conjugated to Apg5 via an isopeptide bond. The possible requirement of an E3-like protein for the conjugation, however, has not yet been confirmed. The Apg12 system is conserved among eukaryotes, although a mammalian counterpart of Apg10 has not yet been identified. Here, we report the identification and characterization of the mouse Apg10 ortholog. A yeast two-hybrid screen using the mouse Apg5 (mApg5) as bait identified a novel protein with 19% identity to yeast Apg10. We designated this protein mouse Apg10 (mApg10). We demonstrated by a modified yeast two-hybrid assay that mApg10 mediates the conjugation of mApg12 and mApg5. The in vivo interaction of mApg12 with mApg10 in HeLa cells suggests that mApg10 is an Apg12-conjugating enzyme, likely serving as an Apg5-recognition molecule in the Apg12 system. This novel two-hybrid method, which we have named 'conjugation-mediated yeast two-hybrid', proves to be a simple and useful technique with which to analyze protein-protein conjugation.     

Ratiometric Raman spectroscopy for quantification of protein oxidative damage

A novel ratiometric Raman spectroscopic (RMRS) method has been developed for quantitative determination of protein carbonyl levels. Oxidized bovine serum albumin (BSA) and oxidized lysozyme were used as model proteins to demonstrate this method. The technique involves conjugation of protein carbonyls with dinitrophenyl hydrazine (DNPH), followed by drop coating deposition Raman spectral acquisition (DCDR). The RMRS method is easy to implement because it requires only one conjugation reaction, uses a single spectral acquisition, and does not require sample calibration. Characteristic peaks from both protein and DNPH moieties are obtained in a single spectral acquisition, allowing the protein carbonyl level to be calculated from the peak intensity ratio. Detection sensitivity for the RMRS method is approximately 0.33 pmol carbonyl per measurement. Fluorescence and/or immunoassay-based techniques only detect a signal from the labeling molecule and, thus, yield no structural or quantitative information for the modified protein, whereas the RMRS technique allows protein identification and protein carbonyl quantification in a single experiment.     

Preparation of glycoconjugates by dialkyl squarate chemistry revisited

The methyl 6-hydroxyhexanoyl glycoside of lactose was treated with each of 1,2-diaminoethane or hydrazine hydrate, and the corresponding amino amide 4 and acyl hydrazide 13, were treated with each of squaric acid dimethyl, diethyl, dibutyl, and didecyl esters. The monoesters were conjugated to bovine serum albumin (BSA) at different concentrations of hapten using 0.05 and 0.5M pH 9 borate buffer. Maximum loading was achieved faster, and the conjugation efficiency was higher, when the conjugation was conducted at higher concentrations of both hapten and buffer. Conjugations involving haptens 14-17 prepared from hydrazide 13 were generally slower and less efficient than those with compounds 5-8, which were made from amino amide 4. Maintaining pH 9 during conjugation was found to be the most important factor in ensuring that the conjugation was a fast, highly efficient, and reproducible process. When the pH of the conjugation mixture fell during the reaction, resulting in decreased reaction rate or even termination of the conjugation process, the normal course of the conjugation process could be restored by addition of buffer salts. Hydrolysis studies with monoesters formed from amino amide 4 under conjugation conditions showed that decyl ester 8 was the most stable and that the methyl compound 5 was the one most readily hydrolyzed. The stability of monoesters prepared from hydrazide 13 was similar and comparable to the decyl ester prepared from 4. No definite advantage was found for the use of any of the four dialkyl squarate reagents (methyl-, ethyl-, butyl-, and decyl-) for conversion of carbohydrate derivatives to species amenable for conjugation. Nevertheless, dimethyl squarate seemed to be the most convenient reagent because it is a crystalline, easy to handle, and commercially available material with very good reactivity.