Preparation of protein conjugates via intermolecular hydrazone linkage

Proteins can be modified at their amino groups under gentle conditions to contain an average of three to six aryl aldehyde or acyl hydrazide groups. These two types of modified proteins at about 10 microM concentration condense with each other at pH approximately 5 to form conjugates linked by hydrazone bonds. Under proper conditions conjugates mainly of dimers and trimers in size or, if desired, higher oligomers can be obtained. The conjugates can be dissociated to their individual protein components by an exchange reaction with an excess of acetyl hydrazide. The reversible hydrazone bonds of conjugates can be reduced with NaCNBH3 to give stable hydrazide bonds. The stability of protein-hydrazone conjugates was found to be significantly greater than that of the model compound, the N-acetylhydrazone of p-carboxybenzaldehyde. This difference is believed to result from the presence of multiple hydrazone linkages in protein conjugates.     

Microinjection of ubiquitin: intracellular distribution and metabolism in HeLa cells maintained under normal physiological conditions

Radioiodinated ubiquitin was introduced into HeLa cells by erythrocyte-mediated microinjection. Subsequent electrophoretic analyses revealed that the injected ubiquitin molecules were rapidly conjugated to HeLa proteins. At equilibrium, 10% of the injected ubiquitin was conjugated to histones and 40% was distributed among conjugates of higher molecular weight. Although the remaining ubiquitin molecules appeared to be unconjugated, the free pool of ubiquitin decreased by one-third and additional conjugates were present when electrophoresis was performed at low temperature under nonreducing conditions. Molecular weights of these labile conjugates suggest that they are ubiquitin adducts in thiolester linkage to activating enzymes. Despite the fairly rapid degradation of injected ubiquitin (t1/2 approximately 10-20 h), the size distribution of ubiquitin conjugates within interphase HeLa cells remained constant for at least 24 h after injection. The intracellular locations of ubiquitin and ubiquitin conjugates were determined by autoradiography, by differential sedimentation of subcellular fractions in sucrose, and by extraction of injected cells with buffer containing Triton X-100. Free ubiquitin was found mostly in the cytosolic or Triton X-100-soluble fractions. As expected, histone conjugates were located predominately in the nuclear fraction and exclusively in the Triton X-100-insoluble fraction. Although high molecular weight conjugates were enriched in the Triton X-100-insoluble fraction, their size distribution was similar to that of soluble conjugates. When injected HeLa cells were exposed to cycloheximide to inhibit protein synthesis, the size distribution of ubiquitin conjugates was similar to that found in untreated cells. Moreover, high molecular weight conjugates decreased less than 20% after inhibition of protein synthesis. These results indicate that most ubiquitin conjugates are not newly synthesized proteins which have been marked for destruction.     

Transformation from block-type to graft-type oligonucleotide-glycopolymer conjugates by self-organization with half-sliding complementary oligonucleotides and their lectin recognition

Block-type oligonucleotide-glycopolymer conjugates bearing alpha-mannosides and beta-galactosides were prepared by coupling 5'-thiol-modified oligonucleotides with iodoacetamidated glycopolymers that were synthesized by telomerization. The conjugates minimally affected the DNA conformation and melting behavior of the duplex. Their self-organization via hybridization with the half-sliding complementary oligonucleotides produced graft-type conjugates or macromolecular gapped DNA duplexes grafted with glycopolymers at regular intervals, which was confirmed using size exclusion chromatography and electrophoresis. The binding affinity of block-type and self-organized graft-type conjugates to lectins was investigated using fluorometry. The affinity of the graft-type duplex assembly bearing mannosides to Con A was approximately 2 times stronger than that of block-type single-stranded or double-stranded conjugates with full complementary oligonucleotides. The organization strategy of DNA-glycopolymer conjugates might be useful for constructing novel glyco-clusters and also for developing a new methodology for gene therapy.     

Impact of aldehyde content on amphotericin B-dextran imine conjugate toxicity

The biocompatibility of oxidized dextran (40 kDa) was investigated in vitro. The contribution of aldehyde groups to the toxicity of polymer-drug conjugates, such as dextran-amphotericin B (AmB) was evaluated. Oxidized dextran was proved to be toxic against the RAW 264.7 cell line with an IC50 of 3 micromol/mL aldehydes. Modification of aldehyde groups and their reaction with ethanolamine reduced the toxicity at least 15-fold. Accordingly, the antifungal and antileishmanial dextran-AmB imine conjugate, which contains unreacted aldehyde groups, was modified with ethanolamine and compared to dextran-AmB amine and imine conjugates. Modification of the imine conjugate with ethanolamine reduced its toxicity toward the RAW cell line by 100%. The effect on Leishmania major parasites was 5 times higher than that of the dextran-AmB amine conjugate. The dextran-AmB-ethanolamine conjugate was at least 15 times less hemolytic than free AmB. Stability and drug release profiles in buffer solution were investigated. The imine conjugates released free AmB while the amine conjugate did not. It is concluded that aldehyde groups may contribute to cell toxicity. This toxicity is reduced by converting the aldehyde groups into imine conjugates with ethanolamine. The results have direct implications toward the safety of AmB-polysaccharide conjugates used against fungal and leishmanial infections.     

Microinjection of ubiquitin: changes in protein degradation in HeLa cells subjected to heat-shock

Ubiquitin was radiolabeled by reaction with 125I-Bolton-Hunter reagent and introduced into HeLa cells using erythrocyte-mediated microinjection. The injected cells were then incubated at 45 degrees C for 5 min (reversible heat-shock) or for 30 min (lethal heat-shock). After either treatment, there were dramatic changes in the levels of ubiquitin conjugates. Under normal culture conditions, approximately 10% of the injected ubiquitin is linked to histones, 40% is found in conjugates with molecular weights greater than 25,000, and the rest is unconjugated. After heat-shock, the free ubiquitin pool and the level of histone-ubiquitin conjugates decreased rapidly, and high molecular weight conjugates predominated. Formation of large conjugates did not require protein synthesis; when analyzed by two-dimensional electrophoresis, the major conjugates did not co-migrate with heat-shock proteins before or after thermal stress. Concomitant with the loss of free ubiquitin, the degradation of endogenous proteins, injected hemoglobin, BSA, and ubiquitin was reduced in heat-shocked HeLa cells. After reversible heat-shock, the decrease in proteolysis was small, and both the rate of proteolysis and the size of the free ubiquitin pool returned to control levels upon incubation at 37 degrees C. In contrast, neither proteolysis nor free ubiquitin pools returned to control levels after lethal heat-shock. However, lethally heat-shocked cells degraded denatured hemoglobin more rapidly than native hemoglobin and ubiquitin-globin conjugates formed within them. Therefore, stabilization of proteins after heat-shock cannot be due to the loss of ubiquitin conjugation or inability to degrade proteins that form conjugates with ubiquitin.     

Characterization of effector-target conjugates for cloned human natural killer and human lymphokine activated killer cells by flow cytometry.

The present studies demonstrate that the intracellular fluorochromes calcein and hydroethidine can be used for quantification of effector-target conjugates involving cloned human natural killer (NK) or interleukin-2 (IL-2) activated human lymphokine activated killer (LAK) cells by dual color flow cytometry without potential artifacts that might result from extensive modification of effector and/or target cell membranes. Cloned NK cells and LAK cells form conjugates with cultured cell lines regardless of susceptibility to lysis. The strength of the interactions in these conjugates was investigated using a variable speed vortexer. Even relatively gentle vortexing disrupted most conjugates involving fresh human peripheral blood lymphocytes (PBL) but only about one-fourth of conjugates between K-562 cells and human PBL that had been cultured with or without IL-2 by this treatment. The rate of conjugate formation for LAK cells was determined to be about 3 times faster than for cloned NK cells, and both rates are considerably faster than the reported rate of formation of cytotoxic T lymphocyte (CTL) target conjugates. The differences in the rate of conjugate formation are apparently not related to target cell specificity, since LAK cells form conjugates with susceptible and resistant cell lines at comparable rates. When effector-target conjugates are incubated at 37 degrees C in the absence of calcium--thereby precluding lysis--the percentage of conjugated LAK or cloned NK cells decreases logarithmically with time. These results suggest that an initial equilibrium between free and conjugated lymphocytes gradually shifts in favor of unconjugated cells.     

Functional Casein-Poly saccharide Conjugates Prepared by Controlled Dry Heating

Casein was conjugated with dextran and galactomannan in a controlled dry state at a relative humidity of 79% and at 60°C for 24 hr. The covalent attachment of polysaccharides to casein was confirmed by SDS-PAGE and HPLC. The emulsifying activity of the casein-dextran and casein-galactomannan conjugates was 1.5 times higher than that of casein. The emulsion stability of the casein-dextran and casein-galactomannan conjugates was 10 times higher than that of casein. The improvement in these emulsifying properties reached a steady state when the conjugation of casein with polysaccharide was done for 24 hr in a controlled dry state, suggesting the rapid formation of conjugates through a Maillard reaction in the case of casein. Compared to commercial emulsifiers, the casein-polysaccharide conjugates showed better emulsifying properties in acidic and high-salt concentration systems.     

Cellular uptake of covalent conjugates of oligonucleotide with membrane-modifying peptide, peptaibol

Using the membrane-modifying peptide, trichorovin-XIIa (TV-XIIa), which is an 11-residual peptaibol isolated from the fungus Trichoderma viride, we synthesized covalent conjugates of 20-mer oligonucleotide with TV-XIIa to examine the potential use of TV-XIIa in cellular delivery. The results indicated that the conjugates were progressively taken up by human lung carcinoma A549 cells. Next, the antisense effects of the conjugates on p53 protein expression were examined. The p53 expression was significantly decreased by ca. 20-50% in the presence of the conjugates upon treatment with the transfection solution at the concentration of 5 μM.     

Antibody conjugates mimic specific B cell presentation of antigen: relationship between T and B cell specificity

We developed antibody conjugates by covalently coupling antibodies against mouse mu-chain and monoclonal antibodies against nominal antigen, myoglobin, as a tool for antigen presentation and as a model of specific presentation of antigen by antigen-specific B cells and T-B interaction. In the presence of the antibody conjugates, myoglobin-specific Iad-restricted cloned T cells proliferated at 1000-fold lower concentration of myoglobin than the stimulatory concentration without the conjugates. This enhanced presentation was observed only when Iad spleen cells were 1000 R-irradiated but not 3300 R-irradiated, consistent with B cell presentation. The simple mixture of each component of the conjugates had no enhancement effects. The conjugates per se had no mitogenic effects on either splenic B cells or the cloned T cells at concentrations employed for antigen presentation. The conjugates reduced the number of antigen-presenting cells required for the maximal response but did not change the kinetics of response. The enhanced presentation by the conjugates required a genetically restricted interaction with B cells. Antigen specificity of the enhanced presentation was confirmed by using various T cell clones or lines with different antigen specificities and different conjugates constructed with monoclonal antibodies of known epitope specificity. The enhanced presentation was significantly inhibited by competition with exogenous mouse IgM or anti-mouse mu-chain but was not significantly inhibited by monoclonal antibodies against Fc receptor. Thus, conjugate-coated B cells serve as models for myoglobin-specific B cells in that they can take up specific antigens at extremely low concentration and can present the antigen to specific T cells. This model system can be applied to any antigen and any species without the need to develop antigen-specific B cell clones, which is not yet possible for most antigens and species of experimental animals. This system allowed us to investigate the relationship between T cell epitope and B cell epitope when these cells interact with each other in an antigen-specific and Ia-restricted manner. Experiments using antibody conjugates of different monoclonal antibodies against myoglobin and various myoglobin-specific cloned T cells of known antigen specificity revealed that there are some particular combinations in which much more limited enhancement of antigen presentation is observed.(ABSTRACT TRUNCATED AT 400 WORDS)     

Liposomes with differential lipid components exert differential adjuvanticity in antigen-liposome conjugates via differential recognition by macrophages

We previously reported that liposomes having differential lipid components displayed differential adjuvant effects when antigen was coupled with liposomes via glutaraldehyde. In the present study, antigen-liposome conjugates prepared using liposomes having differential lipid components were added to the macrophage culture, and phagocytosis and the antigen digest of liposome-coupled antigen by macrophages were then investigated. Antigen presentation by macrophages to an antigen-specific T-cell clone was further investigated using the same conjugates. Antigen-liposome conjugates which induced higher levels of antibody production in vivo were recognized more often, and the liposome-coupled antigen was digested to a greater degree by macrophages than antigen-liposome conjugates which induced lower levels of antibody production. These results correlated closely with those regarding antigen presentation by macrophages; when antigen was coupled to liposomes showing higher adjuvant effect, macrophages cocultured with antigen-liposome conjugates activated antigen-specific T-cells at a higher degree. The concentration of OVA in the macrophage culture added as antigen-liposome conjugates was approximately 32 microg/mL. However, the extent of T-cell activation was almost equal to that when 800 microg/mL of soluble OVA was added to the culture. The results of the present study demonstrated that the adjuvant activity of liposomes observed primary in vivo correlated closely with the recognition of antigen-liposome conjugates and antigen presentation of liposome-coupled antigen by macrophages, suggesting that the adjuvant effects of liposomes are exerted at the beginning of the immune response, i.e., recognition of antigen by antigen-presenting cells.     

Quantitative comparison of long-wavelength Alexa Fluor dyes to Cy dyes: fluorescence of the dyes and their bioconjugates.

Amine-reactive N-hydroxysuccinimidyl esters of Alexa Fluor fluorescent dyes with principal absorption maxima at about 555 nm, 633 nm, 647 nm, 660 nm, 680 nm, 700 nm, and 750 nm were conjugated to antibodies and other selected proteins. These conjugates were compared with spectrally similar protein conjugates of the Cy3, Cy5, Cy5.5, Cy7, DY-630, DY-635, DY-680, and Atto 565 dyes. As N-hydroxysuccinimidyl ester dyes, the Alexa Fluor 555 dye was similar to the Cy3 dye, and the Alexa Fluor 647 dye was similar to the Cy5 dye with respect to absorption maxima, emission maxima, Stokes shifts, and extinction coefficients. However, both Alexa Fluor dyes were significantly more resistant to photobleaching than were their Cy dye counterparts. Absorption spectra of protein conjugates prepared from these dyes showed prominent blue-shifted shoulder peaks for conjugates of the Cy dyes but only minor shoulder peaks for conjugates of the Alexa Fluor dyes. The anomalous peaks, previously observed for protein conjugates of the Cy5 dye, are presumably due to the formation of dye aggregates. Absorption of light by the dye aggregates does not result in fluorescence, thereby diminishing the fluorescence of the conjugates. The Alexa Fluor 555 and the Alexa Fluor 647 dyes in protein conjugates exhibited significantly less of this self-quenching, and therefore the protein conjugates of Alexa Fluor dyes were significantly more fluorescent than those of the Cy dyes, especially at high degrees of labeling. The results from our flow cytometry, immunocytochemistry, and immunohistochemistry experiments demonstrate that protein-conjugated, long-wavelength Alexa Fluor dyes have advantages compared to the Cy dyes and other long-wavelength dyes in typical fluorescence-based cell labeling applications.     

Direct determination of anabolic steroid conjugates in human urine by combined high-performance liquid chromatography and tandem mass spectrometry

A novel screening procedure for the sulfate and glucuronide conjugates of testosterone (T) and epitestosterone (E) in human urine was developed based on liquid-solid extraction and microbore high-performance liquid chromatography combined on-line with ion-spray tandem mass spectrometry. Confirmation of the sulfate and glucuronide conjugates of testosterone and epitestosterone isolated frrm normal human urine was acheived by selected reaction monitoring of characteristic product ions of the parent compounds. Endogenous levels of the steroid conjugates are detected in normal male urine and an increase is observed when the sample is fortified with authentic analytical standards of the conjugates. Calibration curves of all steroid conjugates in urine are linear over a range of twenty. Deuterated internal standards of testosterone glucuronide and epitestosterone sulfate were used for quantitation of the endogenous conjugates. T/E ratios were determined based on the glucuronide fractions of six replicates from a normal male and were shown to be statistically reproducible and below the accepted T/E threshold of 6:1. Sulfate conjugates were shown to be present at significantly lower levels in the urine. The method has potential as an alternative for monitoring anabolic steroid conjugates in human urin.     

Nanoparticulate platinum(II) anticancer drug: synthesis and characterization of amphiphilic cyclotriphosphazene-platinum(II) conjugates

Novel cyclotriphosphazene-platinum(II) conjugates were prepared by hydrolysis and platination of the amphiphilic cyclotriphosphazenes grafted with equimolar hydrophilic methoxy-poly(ethylene glycol) (MPEG) and hydrophobic oligopeptide. These macromolecular conjugates were found to form stable nanoparticles with a mean diameter of approximately 90-200 nm depending on the hydrophobicity of the conjugated (diamine)platinum moieties. The nanoparticulate platinum(II) conjugates have shown temperature and concentration dependent particle sizes. However, the particle sizes of the conjugates were found to decrease to a certain size as the solution concentration was decreased but remained stable even at 10 microM, which is enough for systemic delivery by injection. The conjugates exhibited lower in vitro cytotoxicity than cisplatin but reasonably good activity against selected human tumor cell lines.     

Chiral nature of covalent methylphosphonyl conjugates of acetylcholinesterase

This paper examines the chiral nature of the covalent conjugates formed upon reaction of acetylcholinesterase (AchE) with enantiomeric cycloheptyl, isopropyl, and 3,3-dimethylbutyl methylphosphonyl thiocholines. With the exception of the conjugate formed from reaction of AchE with RP-cycloheptyl methylphosphonyl thiocholine, all enantiomeric conjugates underwent oxime reactivation at rates that were within 2-3-fold of each other. Oxime reactivation was, therefore, independent of both initial configuration about phosphorus and the alkyl phosphonyl ester (-OR) moiety. Aging of the enantiomeric cyclopheptyl and isopropyl methylphosphonyl conjugates occurred exclusively for the conjugate formed from the SP-enantiomer and therefore displayed an absolute dependence on the initial configuration of the methylphosphonyl group. Equilibrium titrations with decidium, a fluorescent bisquaternary competitive inhibitor of AchE, provided an index of aging and enantiomeric configuration of the conjugates independent of enzyme activity. Decidium association with the enantiomeric conjugates (prior to aging) showed no marked dependence on the initial configuration about phosphorus but was measurably dependent on nature of the -OR moiety. These results are interpreted with respect to symmetry and nonrigidity of the organophosphonyl conjugates and are consistent with formation of final methylphosphonyl conjugates that are enantiomerically pure and of opposite configuration. These studies indicate that the active center of AchE comprises at least two kinetically distinct environments separate from the esteratic region but located within 5 A of the nucleophilic serine and differing in dipolar characteristics that promote charge separation and general acid catalysis.     

Design, synthesis, and characterization of pH-sensitive PEG-PE conjugates for stimuli-sensitive pharmaceutical nanocarriers: the effect of substitutes at the hydrazone linkage on the ph stability of PEG-PE conjugates

A set of aliphatic and aromatic aldehyde-derived hydrazone (HZ)-based acid-sensitive polyethylene glycol-phosphatidylethanolamine (PEG-PE) conjugates was synthesized and evaluated for their hydrolytic stability at neutral and slightly acidic pH values. The micelles formed by aliphatic aldehyde-based PEG-HZ-PE conjugates were found to be highly sensitive to mildly acidic pH and reasonably stable at physiologic pH, while those derived from aromatic aldehydes were highly stable at both pH values. The pH-sensitive PEG-PE conjugates with controlled pH sensitivity may find applications in biological stimuli-mediated drug targeting for building pharmaceutical nanocarriers capable of specific release of their cargo at certain pathological sites in the body (tumors, infarcts) or intracellular compartments (endosomes, cytoplasm) demonstrating decreased pH.     

Quantitative measurements of the specificity and kinetics of conjugate formation between cloned cytotoxic T lymphocytes and splenic target cells by dual parameter flow cytometry

The formation of conjugates between cloned anti-H-2Kb and Dd cytotoxic T lymphocytes (CTL) and splenic target cells has been studied by dual parameter flow cytometry. By varying effector-target combinations and by blocking with anti-MHC class I monoclonal antibodies, we found that the specificity of conjugate formation, in general, paralleled that expected from cytotoxicity studies; however, a significant number of "nonspecific" conjugates was always observed. As expected from previous studies, conjugate formation did not occur below 10 degrees C and was inhibited by cytochalasin B, EDTA, and anti-Lyt-2 antibodies. Conjugate formation followed first-order kinetics. The rate of formation of conjugates increased with temperature from 24 degrees to 37 degrees C; at 37 degrees C, the half-time was 1.4 min. After a 6-min lag period, lysis of target cells could be detected at 37 degrees C but not at 30 degrees C or below. Because target cell lysis proceeded during a period of time when the number of conjugates remained constant, considerable effector cell recycling must have occurred. Comparisons of the fluorescence emissions from conjugated effector or target cells with those from unconjugated cells demonstrated that nearly all conjugates contained one effector cell and one target cell, independent of the ratio of the two cell types in the original mix. Once formed, anti-H-2Kb conjugates were stable when diluted into medium alone, but rapidly disaggregated in medium containing either anti-Lyt-2 or anti-Kb monoclonal antibodies, both of which blocked conjugate formation. This finding suggests that conjugates are normally stabilized by intercellular bonds that are constantly breaking and reforming at the cell:cell interface, and that the antibodies disrupt the conjugates by preventing the reformation of broken bonds.     

Quantitative analysis of protein synthesis inhibition and recovery in CRM107 immunotoxin-treated HeLa cells

Previously a mathematical model was proposed that quantitatively related protein synthesis inhibition kinetics of antitransferrin receptor-gelonin immunotoxins to the cellular trafficking of the targeting agent. That work is here extended to describe protein synthesis inhibition kinetics of immunotoxins containing the diphtheria toxin mutant CRM107. CRM107 differs from gelonin in both translocation and ribosomal inactivation mechanisms. Targeting agents used were antitransferrin monoclonal antibodies 5E9 and OKT9, OKT9Fab, and transferrin. CRM107 conjugates inhibited protein synthesis at substantially lower concentrations than gelonin conjugates; this effect was attributed to substantially higher translocation rates for CRM107. However, under certain conditions, CRM107 immunotoxin-treated cells were able to recover completely; this behavior was never observed with gelonin immunotoxins. To quantitatively capture this phenomenon, extracellular and cytosolic degradation of the toxin as well as growth-related recovery from toxin-induced damage were incorporated into the mathematical model. Translocation and cytosolic degradation rate constants were determined for each immunotoxin. Unlike the gelonin conjugates, the translocation rate of CRM107 conjugates depended on the targeting molecule. This provided indirect evidence that CRM107 remains disulfide linked to the targeting agent for at least part of the translocation process. Although the CRM107 conjugates all had higher translocation rates and inhibited protein synthesis at lower concentrations than the gelonin conjugates, the cells' ability to recover from protein synthesis inhibition at low immunotoxin concentrations limits the utility of CRM107 conjugates for targeted cell killing.     

Synthesis and testing of a binary catalytic system for imaging of signal amplification in vivo

We report a binary targeted enzymatic system that is composed of two covalent monoclonal antibody conjugates for specific labeling of cellular targets in vivo. The system utilizes low-molecular weight peroxidase-reducing substrates synthesized by linking 5-hydroxytryptamine (serotonin) with DTPA (5HT-DTPA) for magnetic resonance and radionuclide imaging or with Cy5.5 for near-infrared optical imaging. Initially, the conjugation reaction conditions were optimized to achieve a low level of antiepidermal growth factor receptor (EGFR) antibody (EMD 72000) modification with the N-hydroxysuccinimide ester of 4-hydrazinonicotinate acetone hydrazone (SANH), yielding mAb-HNH conjugate. The resultant modified antibodies were incubated with the periodate-oxidized peroxidase (HRP) or 4-formylbenzoyl-conjugated glucose oxidase (GO), followed by the purification of the resultant mAb-enzyme conjugates by size-exclusion HPLC. The conjugates were further characterized by electrophoresis and were tested by cross-titration on A431 EGFR+ squamous carcinoma or SW620 adenocarcinoma cells (negative control). The conjugates at the optimized concentration ratios were further tested using near-infrared fluorescence microscopy in the presence of Cy5.5 monocarboxy-5-hydroxytryptamide. Further in vitro experiments demonstrated that (1) antibody binding was specific and could be inhibited by free antibody; (2) both antibody conjugates exhibited high enzymatic activity after the binding to the cells; (3) 111In-labeled 5-HT-DTPA was avidly binding to EGFR-positive cells only if both HRP- and GO-conjugates were bound to the cells. The conjugates were tested in vivo using a SPECT imaging experiment, which demonstrated the accumulation of 111In-labeled 5-HT-DTPA substrate at the site containing both conjugates.     

Preparation and characterization of soluble conjugates of defined molecular sizes prepared by linkage of pepsin and carboxypeptidase A to dextrans

Soluble conjugates of pepsin and carboxypeptidase A were prepared by covalent linkage of the enzymes to an amino derivative of dextran. By fractionating the dextran derivatives before and after enzyme coupling, three conjugates, with median Stokes radii between 4.0 and 11.7 nm and with a range of 25% of the median, were prepared from each enzyme. The pepsin and carboxypeptidase A conjugates contained about 35% and 3% protein, respectively. Both types had specific activities close to those of the native enzymes and were stable at -20 degrees C. The pH-activity curve was unaffected by linkage of either enzyme to dextran. The stabilities at 30 degrees C of pepsin at pH 6-7 and carboxypeptidase A at pH 3.5-9.0 were increased by linkage to dextran. No significant amount of unbound enzyme was released from either type of conjugate in skim milk. The molecular sizes, deduced from the intrinsic viscosities and the diffusion coefficients of all conjugates, were close enough to the Stokes radii to indicate that the molecules were approximately spherical. Physical measurements also indicated that the molecules were dextranlike and highly hydrated.     

Cleavage of disulfide-linked fetuin-bisphosphonate conjugates with three physiological thiols

An effective therapeutic agent for treatment of bone diseases is expected to exhibit a high affinity to bone. Conjugating proteins to bisphosphonates (BPs), a class of molecules with an exceptional affinity to bone mineral hydroxyapatite (HA), is a feasible means to impart such a bone affinity. Protein-BP conjugates with cleavable linkages, which allow protein release from the mineral, are preferable over conjugates with stable linkages. To this end, 2-(3-mercaptopropylsulfanyl)-ethyl-1,1-bisphosphonic acid (thiolBP) was conjugated onto fetuin, a model protein, using N-succinimidyl-3-(2-pyridyldithio)propionate to create disulfide-linked conjugates. Although the fetuin-thiolBP conjugates were stable under aqueous conditions, the disulfide linkage was readily cleaved in the presence of the physiological thiols l-cysteine, dl-homocysteine, and l-glutathione. dl-Homocysteine exhibited the highest cleavage of the disulfide linkage among these thiols. The imparted bone affinity as a result of thiolBP conjugation, as assessed by HA binding in vitro, was eliminated upon cleavage of the disulfide linkage. The cleavage of the conjugates bound to HA was as effective as the conjugate cleavage in solution, and even more so at high concentrations of l-glutathione. In conclusion, disulfide-linked fetuin-thiolBP conjugates exhibited a high affinity to HA, which was readily lost upon cleavage with thiols found in physiological milieu.