Antigenicity of recombinant proteins after regioselective immobilization onto polyanhydride-based copolymers

We previously demonstrated that the introduction of a tag consisting of several contiguous lysines at the N- or C-terminus of a recombinant protein greatly improved the covalent grafting of the protein onto negatively charged maleic anhydride-alt-methyl vinyl ether (MAMVE) copolymer, under many different experimental conditions (Ladavière, C., et al. (1998) Bioconjugate Chem. 9, 655; Allard, L., et al. (2002) Biotechnol. Bioeng. 80, 341). The grafting efficiency was dependent on the charge and amine density of the tag, characteristics which were determined by the tag composition. The six lysine tag (Lys6) was found to be the most efficient (Allard, L., et al. (2001) Bioconjugate Chem. 12, 972). In the present work, the biological activity of Lys6-proteins covalently bound to polymer was investigated. N- or C-terminal Lys6-tagged HIV-1 p24 recombinant proteins (RK24H and RH24K) were grafted onto MAMVE, and the antigenicity each of the bioconjugates was evaluated using six monoclonal antibodies that recognized different epitopes distributed along the protein. We demonstrate that the position of the tag and the hydrolysis rate of the anhydride moieties of the polymer are the two main parameters involved in the conservation of the biological activity of the immobilized protein. We thus present a process which allows an efficient oriented immobilization of proteins onto copolymers with optimal biological activity that is suitable for the controlled production of active bioconjugates.     

Biotin reagents for antibody pretargeting. 5. Additional studies of biotin conjugate design to provide biotinidase stability.

An investigation was conducted in which the stabilities of four structurally different biotin derivatives were assessed with regard to biotinamide bond hydrolysis by the enzyme biotinidase. The biotin derivatives studied contained an extra methylene in the valeric acid chain of biotin (i.e., homobiotin), or contained conjugated amino acids having hydroxymethylene, carboxylate, or acetate functionalities on a methylene alpha to the biotinamide bond. The biotinidase hydrolysis assay was conducted on biotin derivatives that were radioiodinated at high specific activity, and then subjected to diluted human serum at 37 degrees C for 2 h. After incubation, assessment of biotinamide bond hydrolysis by biotinidase was readily achieved by measuring the percentage of radioactivity that did not bind with avidin. As controls, an unsubstituted biotin derivative which is rapidly cleaved by biotinidase and an N-methyl-substituted biotin derivative which is stable to biotinidase cleavage were included in the study. The results indicate that increasing the distance from the biotin ring structure to the biotinamide bond by one methylene only decreases the rate of biotinidase cleavage, but does not block it. The data obtained also indicate that placing a hydroxymethylene, carboxylate, or acetate alpha to the biotinamide bond is effective in blocking the biotinamide hydrolysis reaction. These data, in combination with data previously obtained, which indicate that biotin derivatives containing hydroxymethylene or carboxylate moieties retain the slow dissociation rate of biotin from avidin and streptavidin [Wilbur, D. S., et al. (2000) Bioconjugate Chem. 11, 569-583], strongly support incorporation of these structural features into biotin derivatives being used for in vivo targeting applications.     

Thiolytically cleavable dithiobenzyl urethane-linked polymer-protein conjugates as macromolecular prodrugs: reversible PEGylation of proteins

New thiolytically cleavable dithiobenzyl (DTB) urethane-linked conjugates of methoxypoly(ethylene glycol) (mPEG) and a model protein, lysozyme, were prepared and thoroughly characterized. In contrast to our earlier communication [Zalipsky, et al. (1999) Bioconjugate Chem. 10, 703], in the current study we used a more sterically hindered form of para-DTB urethane linkage containing a methyl group on the alpha-carbon to the disulfide moiety. The new reagent for covalent attachment of mPEG-DTB to amino groups of proteins was synthesized via a seven-step process. As a result of PEG conjugation, the lysozyme was shown to completely lose its bacterial cell wall-lysing activity. However, activity was almost fully restored upon cysteine-mediated cleavage of the PEG component. The conjugate decomposition process was monitored by RP-HPLC and by ion spray LC-MS, which showed the formation of the p-mercaptobenzyl urethane-lysozyme intermediate, and ultimately its conversion to the unmodified lysozyme as the sole protein component. Pharmacokinetic evaluation of (125)I-labeled cleavable and noncleavable PEG-lysozyme given intravenously in rats revealed similar clearance patterns; both cleared in a significantly slower manner compared to that of the native protein. However, subcutaneous administration of the same conjugates showed a significantly larger AUC of the cleavable conjugate, indicating that some cleavage of the DTB urethane may have occurred. Although the DTB-linked PEG-lysozyme exhibited almost the same plasma clearance as the noncleavable counterpart, hinting that methyl-DTB linkage might be stable in the bloodstream, SDS-PAGE examination of the conjugate incubated in plasma showed decomposition at least partially mediated by albumin. These results suggest the potential of PEG-DTB-proteins as macromolecular prodrugs capable of generating fully active native proteins under in vivo conditions.     

Targeted delivery of siRNA to hepatocytes and hepatic stellate cells by bioconjugation

Previously, we successfully conjugated galactosylated poly(ethylene glycol) (Gal-PEG) to oligonucleotides (ODNs) via an acid labile ester linker (Zhu et al., Bioconjugate Chem. 2008, 19, 290-8). In this study, sense strands of siRNA were conjugated to Gal-PEG and mannose 6-phosphate poly(ethylene glycol) (M6P-PEG) for targeted delivery of siRNAs to hepatocytes and hepatic stellate cells (HSCs), respectively. These siRNA conjugates were purified by ion exchange chromatography and verified by gel retardation assay. To evaluate their RNAi functions, the validated siRNA duplexes targeting firefly luciferase and transforming growth factor beta 1 (TGF-β1) mRNA were conjugated to Gal-PEG and M6P-PEG, and their gene silencing efficiencies were determined after transfection into HepG2 and HSC-T6 cells. The disulfide bond between PEG and siRNA was cleaved by dithiothreitol, leading to the release of intact siRNA. Both Gal-PEG-siRNA and M6P-PEG-siRNA conjugates could silence luciferase gene expression by about 40% without any transfection reagents, while the gene silencing effects reached more than 98% with the help of cationic liposomes at the same dose. Conjugation of TGF-β1 siRNA with Gal-PEG and M6P-PEG could silence endogenous TGF-β1 gene expression as well. In conclusion, these siRNA conjugates have the potential for targeted delivery of siRNAs to hepatocytes and hepatic stellate cells for efficient gene silencing in vivo.     

Application of the functionalized congener approach to dendrimer-based signaling agents acting through A2A adenosine receptors

As a continued effort to develop multivalent ligands to enhance the pharmacological effects of monomeric drugs, DITC-APEC, a chemically reactive nucleoside A(2A) adenosine receptor (AR) agonist, was employed to derivatize the surface of third-generation (G3) polyamidoamine (PAMAM) dendrimers. The resulting conjugates carried multiple copies of the agonist attached through a thiourea linkage and differed in the number of attachments and in the presence of a fluorophore or additional surface modification. Computer modeling studies suggested that these DITC-APEC-loaded dendrimers extended the overall diameter of the previously reported PAMAM-CGS21680 dendrimer derivatives (Kim et al., Bioconjugate Chem 2008 19:406-411) by ca. 20 A, potentially increasing the conformational flexibility of the appended ligands to achieve optimal geometry for efficient binding at A(2A) ARs. Increased affinity and selectivity in binding in comparison to the CGS21680 conjugate were envisioned, due to the presence of an extended linker, i.e., a dithioureylenephenyl functionality. In vitro radioligand competition experiments showed effective binding of these PAMAM-DITC-APEC dendrimer conjugates at the human A(2A) and A(3) ARs with submicromolar K (i) values and selectivity in comparison to the human A(1) AR. Furthermore, these nucleoside-loaded dendrimers exhibited an A(2A) AR-mediated inhibitory effect on ADP-induced aggregation of human platelets. The present study demonstrates the potential of applying the functionalized congener concept to engineer dendrimer-based multivalent ligands for G protein-coupled receptors.     

Synthesis of acid-labile diplasmenyl lipids for drug and gene delivery applications.

The low pH environments characteristic of endosomal compartments and ischemic tissues provide an intrinsic pathway for triggering site-specific contents release from appropriately designed delivery vehicles. Accordingly, research in this group has focused on the design, synthesis and application of novel acid-sensitive lipids that will undergo facile lamellar (L alpha) to hexagonal (HII) phase transitions within these acidic sites. Previously, it has been demonstrated that plasmenylcholine-type lipids have excellent acid hydrolysis and contents release kinetics (Gerasimov et al., Biochim. Biophys. Acta. 1324 (1997) 200-214; Rui et al., J. Am. Chem. Soc. 120 (1998) 11213-11218). This paper describes the synthesis of three new acid sensitive lipids, based on a chiral 1,2-di-O-(1Z',9Z'-octadecadienyl)-sn-glycerol (6) platform, displaying phosphocholine (7), poly(ethyleneoxide) (8), and O-carbamoyl-N-diethylen-etriamine (10) headgroups. Intermediate 6 was obtained in 28% overall yield via a six step synthesis from (S)-(+)-2,2-dimethyl-1,2-dioxolane-4-methanol. Subsequent conversion to the final products was acheived in moderate (7 and 10) to excellent yields (8).     

Syntheses and hydrolysis of basic and dibasic ampicillin esters tailored for intracellular accumulation

Readily hydrolysable basic and dibasic esters of ampicillin were synthesised by alkylation of the carboxylate function of ampicillin to obtain prodrugs that may accumulate in cells and allow for an intracellular delivery of ampicillian (Fan et al., Bioorg. Med. Chem. Lett. 1997, 7, 3107). We found that the beta-lactam ring cleavage and the hydrolysis of the ester function were competitive reactions. The prerequisite for biological activity of compounds of this type is therefore that ester hydrolysis proceeds faster than ring opening. Some synthesised compounds show promise as prodrugs since they displayed a reasonable stability and regenerate large quantities of bioactive ampicillin in broth.     

Acid-sensitive polyethylene glycol conjugates of doxorubicin: preparation, in vitro efficacy and intracellular distribution

Coupling anticancer drugs to synthetic polymers is a promising approach of enhancing the antitumor efficacy and reducing the side-effects of these agents. Doxorubicin maleimide derivatives containing an amide or acid-sensitive hydrazone linker were therefore coupled to alpha-methoxy-poly(ethylene glycol)-thiopropionic acid amide (MW 20000 Da), alpha,omega-bis-thiopropionic acid amide poly(ethylene glycol) (MW 20000 Da) or alpha-tert-butoxy-poly(ethylene glycol)-thiopropionic acid amide (MW 70000 Da) and the resulting polyethylene glycol (PEG) conjugates isolated through size-exclusion chromatography. The polymer drug derivatives were designed as to release doxorubicin inside the tumor cell by acid-cleavage of the hydrazone bond after uptake of the conjugate by endocytosis. The acid-sensitive PEG conjugates containing the carboxylic hydrazone bonds exhibited in vitro activity against human BXF T24 bladder carcinoma and LXFL 529L lung cancer cells with IC70 values in the range 0.02-1.5 microm (cell culture assay: propidium iodide fluorescence or colony forming assay). In contrast, PEG doxorubicin conjugates containing an amide bond between the drug and the polymer showed no in vitro activity. Fluorescence microscopy studies in LXFL 529 lung cancer cells revealed that free doxorubicin accumulates in the cell nucleus whereas doxorubicin of the acid-sensitive PEG doxorubicin conjugates is primarily localized in the cytoplasm. Nevertheless, the acid-sensitive PEG doxorubicin conjugates retain their ability to bind to calf thymus DNA as shown by fluorescence and visible spectroscopy studies. Results regarding the effect of an acid-sensitive PEG conjugate of molecular weight 20000 in the chorioallantoic membrane (CAM) assay indicate that this conjugate is significantly less embryotoxic than free doxorubicin although antiangiogenic effects were not observed.     

Synthesis of pH-sensitive amphotericin B-poly(ethylene glycol) conjugates and study of their controlled release in vitro

New intravenous conjugates of amphotericin B (AMB) with poly(ethylene glycols) (PEG) (M=5000, 10,000, 20,000) have been synthesized and characterised. The intermediate PEGs possess a 1,4-disubstituted benzene ring with aldehyde group at the end of the chain. The benzene ring is connected with PEG at its 4-position (with respect to the aldehyde group) by various functional groups (ether, amide, ester). Reaction of terminal aldehyde group of the substituted PEGs with AMB gave conjugates containing a pH-sensitive imine linkage, which can be presumed to exhibit antimycotic effect at sites with lowered pH value. All types of the conjugates are relatively stable in phosphate buffer at physiological conditions of pH 7.4 (37 degrees C), less than 5 mol% AMB being split off from them within 24 h. For a model medium of afflicted tissue was used a phosphate buffer (pH 5.5, 37 degrees C), in which controlled release of AMB from the conjugates takes place. The imine linkage is split to give free AMB with half-lives of 2-45 min. The rate of acid catalysed hydrolysis depends upon substitution of the benzene ring; however, it does not depend on molecular weights of the PEGs used. The conjugates with ester linkage undergo enzymatic splitting in human blood plasma and/or blood serum at pH 7.4 (37 degrees C) with half-lives of 2-5 h depending on molecular weights of the PEGs used (M = 5000, 10,000, 20,000). At first, the splitting of ester linkage produces the relatively stable pro-drug, that is, 4-carboxybenzylideniminoamphotericin B, which is decomposed to AMB and 4-formylbenzoic acid in a goal-directed manner only at pH 7 (t1/2 = 2 min, pH 5.5, 37 degrees C). A goal-directed release of AMB is only achieved by acid catalysed hydrolysis of imine linkage, either from the polymeric conjugate or from the pro-drug released thereof. The LD50 values determined in vivo (mouse) are 20.7 mg/kg and 40.5 mg/kg for the conjugates with ester linkage (M = 10,000 and 5000, respectively), which means that they are ca. 6-11 times less toxic than free AMB.     

In Vitro and In Vivo Antitumor Activity of a Novel pH-Activated Polymeric Drug Delivery System for Doxorubicin

Background

Conventional chemotherapy agent such as doxorubicin (DOX) is of limited clinical use because of its inherently low selectivity, which can lead to systemic toxicity in normal healthy tissue.

Methods

A pH stimuli-sensitive conjugate based on polyethylene glycol (PEG) with covalently attachment doxorubicin via hydrazone bond (PEG-hyd-DOX) was prepared for tumor targeting delivery system. While PEG-DOX conjugates via amid bond (PEG-ami-DOX) was synthesized as control.

Results

The synthetic conjugates were confirmed by proton nuclear magnetic resonance (NMR) spectroscopy, the release profile of DOX from PEG-hyd-DOX was acid-liable for the hydrazone linkage between DOX and PEG, led to different intracellular uptake route; intracellular accumulation of PEG-hyd-DOX was higher than PEG-ami-DOX due to its pH-triggered profile, and thereby more cytotoxicity against MCF-7, MDA-MB-231 (breast cancer models) and HepG2 (hepatocellular carcinoma model) cell lines. Following the in vitro results, we xenografted MDA-MB-231 cell onto SCID mice, PEG-hyd-DOX showed stronger antitumor efficacy than free DOX and was tumor-targeting.

Conclusions

Results from these in vivo experiments were consistent with our in vitro results; suggested this pH-triggered PEG-hyd-DOX conjugate could target DOX to tumor tissues and release free drugs by acidic tumor environment, which would be potent in antitumor drug delivery.     

Synthesis and stability studies of phosphonoformate-amino acid conjugates: a new class of slowly releasing foscarnet prodrugs

Prodrugs of phosphonoformic acid (PFA), an anti-viral agent used clinically as the trisodium salt (foscarnet), are of interest due to the low bioavailability of the parent drug, which severely limits its utility. Neutral PFA triesters are known to be susceptible to P-C bond cleavage under hydrolytic de-esterification conditions, and it was previously found that P,C-dimethyl PFA P-N conjugates with amino acid ethyl esters did not release PFA at pH 7, and could not be fully deprotected under either acid or basic conditions, which led, respectively, to premature cleavage of the P-N linkage (with incomplete deprotection of the PFA ester moiety), or to P-C cleavage. Here we report that novel, fully deprotected PFA-amino acid P-N conjugates 4 can be prepared via coupling of C-methyl PFA dianion 2 with C-ethyl-protected amino acids using aqueous EDC, which gives a stable monoanionic intermediate 3 that resists P-C cleavage during subsequent alkaline deprotection of the two carboxylate ester groups. At 37 degrees C, the resulting new PFA-amino acid (Val, Leu, Phe) conjugates (4a-c) undergo P-N cleavage near neutral pH, cleanly releasing PFA. A kinetic investigation of 4a hydrolysis at pH values 6.7, 7.2, and 8.5 showed that PFA release was first-order in [4a] with respective t(1/2) values of 1.4, 3.8, and 10.6 h.     

Single site catalysis of the F1-ATPase from Saccharomyces cerevisiae and the effect of inorganic phosphate on it

The kinetical characteristics of ATP hydrolysis by mitochondrial F1-ATPase from Saccharomyces cerevisiae (yeast) have been studied under conditions where only a single catalytic site per enzyme molecule bound ATP. Four major features were observed, that is, fast ATP binding to the enzyme, slow product release from the enzyme, an equilibrium close to unity between ATP and products on the enzyme, and promotion of ATP hydrolysis on the second addition of a large excess of ATP (cold chase). These are essentially the same as the kinetical characteristics observed for beef heart mitochondrial F1-ATPase, which were called as unisite catalysis by Grubmeyer et al. (Grubmeyer, C. et al. (1982) J. Biol. Chem. 257, 12092-12100), although the release of a hydrolysis product, Pi, from the yeast enzyme appeared to occur significantly faster than that from the beef enzyme, which resulted in a decreased extent of cold chase promotion of ATP hydrolysis of the yeast enzyme. The yeast F1-ATPase showed unisite catalysis even in the absence of Pi in the reaction mixtures, while it was reported for the beef F1-ATPase that the presence of Pi in the reaction mixture was essential for unisite catalysis (Penefsky, H.S. & Grubmeyer, C. (1984) in H+-ATPase (ATP Synthase) (Papa, S. et al., eds.) pp. 195-204, The ICSU Press). Another difference in the Pi effect on the kinetics was that ATP hydrolysis was initiated without a lag time in the absence of Pi in the case of the yeast enzyme when a 1,000-fold molar excess of ATP per enzyme molecular was mixed with the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

Beryllium binding at neutral pH: the importance of the Be-O-Be motif

Beryllium speciation at physiological conditions is critical to understanding chronic beryllium disease (CBD). The MHC-class II receptor alleles that have been linked to CBD have more than six carboxylates in a short 20 amino acid segment of the binding pocket and it has been suggested that beryllium may bind within the MHC-class II receptor via the carboxylates. Previous reports also show that citric acid binds beryllium significantly stronger than similar carboxylate ligands such as tartaric acid and is one of the few ligands that can compete with hydrolysis to solubilize beryllium across the entire pH range at molar concentrations. We have characterized the binding of Be to citric acid and shown using a combination of NMR, mass spectrometry and ligand competition studies that Be2L and Be4L2 species dominate. A Be-O-Be linkage with the bridging oxygen coming from the aliphatic alcohol is critical to the stability of the complex. We show through competition experiments that the most stable Be-O-Be arrangement has one Be in a five-member ring and the other Be in a six-member ring. The unusual deprotonation of an aliphatic alcohol (pK(a) = 18) at neutral pH has significant ramifications on the potential interactions of Be with biological ligands such as carbohydrates and Ser and Thr residues.     

Norbornene derived doxorubicin copolymers as drug carriers with pH responsive hydrazone linker

The synthesis and complete characterization of both norbornene-derived doxorubicin (mono 1) and polyethylene glycol (mono 2) monomers are clearly described, and their copolymerization by ring-opening metathesis polymerization (ROMP) to get the block copolymer (COPY-DOX) is vividly elaborated. The careful design of these conjugates exhibits properties like well-shielded drug moieties and well-defined nanostructures; additionally, they show solubility in both water and biological medium and also have the important tendency of rendering acid-triggered drug release. The drug release profile suggests the importance of having the hydrazone linker that helps to release the drug exactly at the mild acidic conditions resembling the pH of the cancerous cells. It is also observed that the drug release from micelles of COPY-DOX is significantly accelerated at a mildly acidic pH of 5.5-6, compared to the physiological pH of 7.4, suggesting the pH-responsive feature of the drug delivery system with hydrazone linkages. Confocal laser scanning microscopy (CLSM) measurements indicate that these COPY-DOX micelles are easily internalized by living cells. MTT assays against HeLa and 4T cancer cells showing COPY-DOX micelles have a high anticancer efficacy. All of these results demonstrate that these polymeric micelles that self-assembled from COPY-DOX block copolymers have great scope in the world of medicine, and they also symbolize promising carriers for the pH-triggered intracellular delivery of hydrophobic anticancer drugs.     

Triticum aestivum shows a greater biomass response to a supply of aluminium phosphate than Lupinus albus, despite releasing fewer carboxylates into the rhizosphere

The relationship between carboxylate release and the ability of plants to access phosphorus from AlPO4 and to detoxify aluminium was studied by comparing species with a low and high rate of carboxylate release, Triticum aestivum (wheat) and Lupinus albus (white lupin), respectively. Species were supplied with P at 10, 20, 40 or 100 mg P kg-1 sand in the form of sparingly soluble AlPO4 or soluble KH2PO4; control plants did not receive any P. Triticum aestivum was significantly better than L. albus at accessing P from AlPO4, despite accumulating fewer carboxylates in its rhizosphere. Rhizosphere pH of L. albus did not vary with form or level of P supply, while the rhizosphere pH of T. aestivum increased with the level of P supplied. Based on the evidence in the present study, a model is proposed to explain the poor performance of L. albus, whereby the release of carboxylates and associated protons reduces the chelating ability of exuded carboxylates, thus reducing P acquisition and increasing Al toxicity.     

Enantiomeric discrimination of Ru-substrates by cytochrome P450cam

Molecules with photosensitizers attached to substrates (Wilker et al., Angew. Chem. Int. Ed. 38 (1999) 90-92) or cofactors (Hamachi et al., J. Am. Chem. Soc. 121 (1999) 5500-5506) can rapidly deliver redox equivalents to buried active sites. The structure of cytochrome P450cam (P450) co-crystallized with a prototypal sensitizer-substrate, [Ru-C9-Ad]Cl2, has been determined (Dmochowski et al., Proc. Natl. Acad. Sci. USA 96 (1999) 12987-12990); and, in separate UV-vis absorption and time-resolved luminescence experiments, the binding of the lambda and delta enantiomers of Ru-C9-Ad to P450 has been measured. The results, KD(delta/lambda) approximately 2, indicate that the bipyridyl ligands of the lambda isomer interact more favorably with hydrophobic residues at the entrance to the substrate channel. We conclude that enantiospecific interactions may be exploited in the design of enzyme-metallosubstrate conjugates.     

The stereospecific labilization of the C-4' pro-S hydrogen of pyridoxamine 5'-phosphate is abolished in (Lys258----Ala) aspartate aminotransferase

Reconstitution of wild-type apoaspartate aminotransferase from Escherichia coli with [4'-3H]pyridoxamine 5'-phosphate results in stereospecific release of the pro-S C-4' 3H to the solvent. The reaction follows first-order kinetics (t1/2 = 15 min at pH 7.5 and 25 degrees C), its rate constant being similar to that found previously with mitochondrial aspartate aminotransferase from chicken (Tobler, H.P., Christen, P., and Gehring, H. (1986) J. Biol. Chem. 261, 7105-7108). Substituting the active site residue Lys258 by alanine via site-directed mutagenesis yields a catalytically inactive enzyme (Malcolm, B. A., and Kirsch, J. F. (1985) Biochem. Biophys. Res. Commun. 132, 915-921). This mutant enzyme fails to release any measurable 3H from bound [4'-3H]pyridoxamine 5'-phosphate. The data are consistent with earlier proposals that Lys258 is indispensable for the ketimine/aldimine tautomerization, and corroborate the previous conclusion that 3H exchange from enzyme-bound pyridoxamine 5'-phosphate mechanistically corresponds to the deprotonation at C-4' of the ketimine intermediate during the transamination reaction.     

Function and stability of abscisic acid acyl hydrazone conjugates by LC-MS2 of ex vivo samples

We prepare a biotinylated conjugate of the ubiquitous plant hormone (S)-(+)-abscisic acid via an acyl hydrazone linkage at the C4' position and demonstrate in vivo cleavage of the otherwise stable acyl hydrazone linkage using LC-MS2. As part of a wider chemical genomic study, biological activity of the conjugate was assessed using standard epidermal peel and gravimetric transpiration assays, showing significant activity but at a level lower than the unconjugated hormone. When deuterated samples of the conjugate were fed to the plant, however, it was apparent by LC-MS2 experiments that significant levels of hydrolysis of the acyl hydrazone had taken place, contrary to in vitro stability assays in artificial sap. We conclude that abscisic acid is liberated in sufficient quantities to account for the observed physiological response and that LC-MS2 monitoring of conjugates is a simple and practical method by which such events may be assessed, whether in plants or other organisms.     

Interaction of antibodies and antigens conjugated with synthetic polyanions: on the way of creating an artificial chaperone

Recently we have initiated the use of synthetic polyelectrolytes to mimic the action of chaperones in living cells [Dainiak et al., Biochim. Biophys. Acta 1381 (1998) 279-285]. The next step in this direction is done by the synthesis of conjugates of poly(methacrylic acid) (PMAA) with antigen, denatured glyceraldehyde-3-phosphate dehydrogenase (dGAPDH), and with monoclonal antibodies specific for dGAPDH (but not for the native protein). The pH-dependent properties of the conjugates have been studied using turbidimetry and light scattering. The antibody-PMAA and dGAPDH-PMAA conjugates were shown to interact with free dGAPDH and antibodies respectively as well as with each other. Insoluble aggregates of dGAPDH with antibody-PMAA and of antibodies with dGAPDH-PMAA are formed in acidic media. The same situation occurs in the mixture of antibody-PMAA and dGAPDH-PMAA: precipitation takes place in acidic media, whereas soluble associates are formed in neutral solutions. The size of the soluble associates and the number of conjugates in the associate could be regulated by pH. The competition of free dGAPDH and dGAPDH-PMAA for binding with antibody-PMAA and the dynamic release of refolded GAPDH, with no affinity to antibody-PMAA, into solution could be used for simulating chaperone action.     

Crystal structure of a high-affinity variant of rat alpha-parvalbumin

In model peptide systems, Ca2+ affinity is maximized in EF-hand motifs containing four carboxylates positioned on the +x and -x and +z and -z axes; introduction of a fifth carboxylate ligand reduces the affinity. However, in rat beta-parvalbumin, replacement of Ser-55 with aspartate heightens divalent ion affinity [Henzl, M. T., et al. (1996) Biochemistry 35, 5856-5869]. The corresponding alpha-parvalbumin variant (S55D/E59D) likewise exhibits elevated affinity [Henzl, M. T., et al. (2003) Anal. Biochem. 319, 216-233]. To determine whether these mutations produce a variation on the archetypal EF-hand coordination scheme, we have obtained high-resolution X-ray crystallographic data for alpha S55D/E59D. As anticipated, the aspartyl carboxylate replaces the serine hydroxyl at the +z coordination position. Interestingly, the Asp-59 carboxylate abandons the role it plays as an outer sphere ligand in wild-type rat beta, rotating away from the Ca2+ and, instead, forming a hydrogen bond with the amide of Glu-62. Superficially, the coordination sphere in the CD site of alpha S55D/E59D resembles that in the EF site. However, the orientation of the Asp-59 side chain is predicted to stabilize the D-helix, which may contribute to the heightened divalent ion affinity. DSC data indicate that the alpha S55D/E59D variant retains the capacity to bind 1 equiv of Na+. Consistent with this finding, when binding measurements are conducted in K(+)-containing buffer, divalent ion affinity is markedly higher. In 0.15 M KCl and 0.025 M Hepes-KOH (pH 7.4) at 5 degrees C, the macroscopic Ca2+ binding constants are 1.8 x 10(10) and 2.0 x 10(9) M(-1). The corresponding Mg2+ binding constants are 2.7 x 10(6) and 1.2 x 10(5) M(-1).