Effect of dextran and dextran modifications on the thermal and proteolytic stability of conjugated bovine testis beta-galactosidase and human serum albumin

In order to study carbohydrate-induced protein stabilization bovine testis beta-galactosidase and human serum albumin were conjugated with dextran, partially acetylated dextran and partially methylated dextran. The conjugates and the free proteins were compared with respect to thermal stability at 50 degrees C and resistance to proteolytic digestion by subtilopeptidase A. Both beta-galactosidase and serum albumin were stabilized by conjugation with polysaccharide. However, higher stability was achieved by conjugating the proteins with the hydrophilic polysaccharides, dextran and acetylated dextran, than by conjugation with the hydrophobic polysaccharide, methylated dextran. The results are discussed in relation to possible explanations of carbohydrate-induced protein stabilization.     

Comparative study of the sensitizing activity of heterologous serum preparations

Experiments on guinea pigs and adrenalectomized mice showed that the anaphylactogenic properties xenogenic immunoglobulin preparations were 2- to 2.5-fold higher than those of antitoxic sera "Diaferm-3". The partial acid-enzymatic hydrolysis of immunoglobulin preparations obtained by ethanol fractionation permitted a considerable decrease in their sensitizing activity and anticomplementary properties.     

Covalent Fixation of Soluble Derivatized Dextrans to Model Proteins in Low-Concentration Medium: Application to Factor IX and Protein C

Factor IX and protein C are zymogens implicated in blood clotting, and an increase in their plasmatic residence time would be of interest for the treatment of the disorders caused by their deficiency. In this context, the conjugation of these proteins to polymers such as modified dextrans could be used to approach the problem. Conjugate formation in concentrated medium ([protein]>50 g/L) is well documented, whereas drastic dilution ([protein] <1 g/L) is quite unfavorable. Before studying the binding of factor IX and protein C to polymers, the coupling of model proteins (human hemoglobin, Hb; human serum albumin, HSA) in low-concentration medium to benzenetetracarboxylate dextran (BTC-dextran) and dialdehyde dextran was investigated. To obtain soluble benzenetetracarboxylate dextran-based conjugates, the conditions of coupling were optimized; the use of sulfo-NHS was necessary to form a conjugate with benzenetetracarboxylate dextran. In fact, the O-acylurea intermediate formed between coupling agent [l-ethyl-3(3-dimethylaminopropyl) carbodiimide, EDC] and BTC-dextran must be stabilized. Concerning dialdehyde dextran, a more oxidized polymer and a higher pH of the buffer of coupling than for highly concentrated solution must be used to obtain a conjugate. Whatever polymer is used, HSA appeared clearly less reactive than Hb, which can be attributed to the better reactivity of N-terminal amino groups in this latter protein and to the marked affinity of benzenetetracarboxylate dextran for it. No soluble conjugate was formed between the same dextran derivatives and factor IX or protein C. Moreover, the activity of both coagulation factors was dramatically decreased by contact with EDC and glutaraldehyde, a small molecule. Thus, bad accessibility of protein amino groups is probably responsible for this lack of reactivity. Nevertheless, it could be shown that carboxylate and amino groups were essential to the activity of factor IX and protein C.     

Acellular pertussis vaccine

Protective, immunogenic, toxic, and sensitizing properties of acellular pertussis vaccine (aPV) developed according to original technology were studied, aPV had marked protective activity which lasted more than 2 years. Sera of mice immunized by aPV also possess protective properties, and they were more prominent than in sera of mice immunized by pertussis bacteria suspension (PS). Immune sera to aPV neutralized cytopathogenic effect of pertussis toxin (PT) on ovarian Chinese hamster cells in 1:250 dilution, whereas neutralizing activity of sera to PS was very low. Level of antibodies to PT was higher in rabbits immunized, according to schedules and dosage recommended for children, by aPV than by PS. High immunogenicity of aPV was proved also by levels of IgG to PT in sera of mice immunized three times by aPV in human dosage. During experiments on mice and guinea pigs aPV had mild toxicity, did not induce autoimmune process, did not have anaphylactogenic properties compared with bacterial suspension characterized by high anaphylactogenic activity. Histamine-sensitizing abilityof aPVwas 40 times lower than that of PS. Assessment of pyrogenic properties of aPV and PS performed on rabbits showed that aPV was 1,000 times less pyrogenic than PS. Obtained results demonstrate high protective and immunogenic properties of domestic acellular pertussis vaccine and its low toxic and sensitizing characteristics.     

Soybean Bowman–Birk Inhibitor Conjugates with Clinical Dextran. Synthesis and Antiproteolytic Activity

Conjugates of the classical soybean Bowman-Birk inhibitor (BBI) with clinical dextran were synthesized. Clinical dextran was preliminarily oxidized with periodate to dialdehydedextran (DAD). The effect of the degree of oxidation of DAD on coupling of the inhibitor was evaluated. The binding of the protein was shown to increase with increasing degree of DAD oxidation (5, 10, 20%). Total coupling of the inhibitor occurred when the degree of oxidation of the dextran was 20%. The BBI-DAD (20%) conjugate contained 13% protein with BBI/DAD molar ratio 1 : 1. The conjugates retained the ability to inhibit trypsin (Ki = 0.2-0.3 nM) and alpha-chymotrypsin (Ki = 15-30 nM). Thus, the coupling of BBI with the polymeric carrier caused practically no decrease in the antiproteolytic activity of the inhibitor.     

Antigenicity of dextran-protein conjugates in mice. Effect of molecular weight of the carbohydrate and comparison of two modes of coupling

Protein conjugates of polysaccharides or their breakdown products are being used as improved "T-dependent" vaccines. We tried to define optimal characteristics of future conjugate vaccines by testing the immunogenicity of thirteen conjugates of alpha 1-6 dextran and chicken serum albumin in mice (BALB/c and CBA). All conjugates induced stronger antidextran antibody responses than the polysaccharide, and a fair proportion of these antibodies were IgG. However, there was a range of antigenicities. Consistently strong responses were obtained with conjugates that carried small dextran molecules (m.w. 1000 to 4000) coupled to the protein via the reducing end. Modification of such an "optimal" conjugate either by increasing the size of the saccharide to 40,000 Da, or by permitting multiple attachments of the saccharide molecule to the protein, reduced its antigenicity. Carbohydrate/protein ratios varying from 0.17 to 0.49 were associated with excellent antidextran responses.     

Molecular characteristics of bovine serum albumin-dextran conjugates

Bovine serum albumin (BSA)-dextran conjugates were prepared by using the Maillard reaction; depending on the ratio of dextran to BSA used, about 0.5-1 mol of dextran could be bound to 1 mol of native BSA. SDS-PAGE patterns revealed that BSA and dextran had been covalently bonded. Structural analyses by fluorescence spectroscopy and circular dichroism indicated that the BSA surface in each conjugate was covered with dextran without any great disruption of the native conformation. The conjugates could be grouped into two fractions on the basis of the weight-average molecular mass measured: the main fraction at 1.95-2.35 x 10(5) g/mol and a less-abundant fraction with aggregates greater than 1.50 x 10(6) g/mol. High-performance size-exclusion chromatography in conjunction with multi-angle laser light scattering detection revealed that the BSA-dextran conjugates prepared by using the Maillard reaction had various molar masses and radii.     

Molecular Characteristics of Bovine Serum Albumin-Dextran Conjugates

Bovine serum albumin (BSA)-dextran conjugates were prepared by using the Maillard reaction; depending on the ratio of dextran to BSA used, about 0.5–1 mol of dextran could be bound to 1 mol of native BSA. SDS–PAGE patterns revealed that BSA and dextran had been covalently bonded. Structural analyses by fluorescence spectroscopy and circular dichroism indicated that the BSA surface in each conjugate was covered with dextran without any great disruption of the native conformation. The conjugates could be grouped into two fractions on the basis of the weight-average molecular mass measured: the main fraction at 1.95–2.35×10⁵ g/mol and a less-abundant fraction with aggregates greater than 1.50×10⁶ g/mol. High-performance size-exclusion chromatography in conjunction with multi-angle laser light scattering detection revealed that the BSA-dextran conjugates prepared by using the Maillard reaction had various molar masses and radii.     

Permeability characteristics of human endothelial monolayers seeded on different extracellular matrix proteins

OBJECTIVE: To investigate whether endothelial monolayer permeability changes induced by inflammatory mediators are affected by the extracellular matrix protein used for cell seeding. METHODS: Human umbilical venular endothelial cells (HUVEC) were grown to confluent monolayers on membranes coated with either collagen, fibronectin or gelatin. The permeability to albumin and dextran was then assessed, both under normal conditions and after treatment with tumor necrosis factor-alpha (TNF-alpha) and bacterial lipopolysaccharide (LPS). RESULTS: With any of the three protein coatings, tight junctions were formed all over the monolayers. The permeability of the coated membranes to albumin and dextran was reduced strongly by confluent monolayers; the relative reduction was similar for the three matrix proteins used. Pre-incubation of the monolayers with either TNF-alpha or LPS increased permeability dose dependently. However, the relative increase due to either treatment was independent of the protein used for membrane coating. CONCLUSION: The extracellular matrix protein used for initial seeding of endothelial cultures plays a minor role in determining the permeability changes induced in HUVEC monolayers by inflammatory mediators.     

Synthesis and characterization of dextran-peptide-methotrexate conjugates for tumor targeting via mediation by matrix metalloproteinase II and matrix metalloproteinase IX

We designed and synthesized new dextran-peptide-methotrexate conjugates for tumor-targeted delivery of chemotherapeutics via the mediation of matrix metalloproteinase II (MMP-2) and matrix metalloproteinase IX (MMP-9), both being widely known tumor-associated enzymes. A robust and flexible synthesis procedure and process monitoring chromatography assays were developed. The linker chemistry and the backbone charge were optimized to allow high sensitivity of the conjugates toward the targeted enzymes. The optimal conjugate carries Pro-Val-Gly-Leu-Ile-Gly as the peptide linker, and the charge on the dextran backbone is fully neutralized. In the presence of the targeted enzymes, the peptide was cleaved and peptidyl methotrexate was released, with a kcat/Km value of 1.21 x 10(5) M(-1) s(-1) for MMP-2 and 3.60 x 10(3) M(-1) s(-1) for MMP-9, respectively. Satisfactory stability of the new conjugates was demonstrated in serum containing conditions, suggesting the conjugates can remain intact in systemic circulation. These findings supported the tumor targeting capability of the new conjugates and warranted further investigation with in vivo study.     

Conjugation of proteins and enzymes with hydrophilic polymers and their applications.

The efficacy of a number of therapeutically active proteins and peptides is severely limited due to their instability in circulation. Of the various approaches used to stabilise these proteins, the one more successful is covalent modification of the protein or enzyme with some hydrophilic polymers such as dextran or PEG. These conjugates are more stable than the native protein both in vitro as well as in vivo. They exhibit enhanced resistant to proteolytic degradation, have a long-life in circulation and exhibit reduced immunogenicity. The therapeutic efficacy of these conjugates is also greatly enhanced compared to the native protein or enzyme.     

Synthesis, characterization, and in vitro activity of dendrimer-streptokinase conjugates

Dendrimer conjugation with low molecular weight drugs has been of increasing interest recently for improving pharmacokinetics, targeting drugs to specific sites, and facilitating cellular uptake. Opportunities for increasing the performance of relatively large therapeutic proteins such as streptokinase (SK) using dendrimers are being explored in this study. Using the active ester method, a series of streptokinase-poly(amido amine) (PAMAM) G3.5 conjugates were synthesized with varying amounts of dendrimer-to-protein molar ratios. Characterization of these conjugates by GPC, IEC, and native-PAGE suggested that the conjugation reaction was successful, resulting in relatively pure SK-dendrimer conjugates. The conjugate made with an equimolar ratio of dendrimer to streptokinase (1:1) exhibited the highest enzymatic activity retention ( approximately 80% retained) that has been reported so far for conjugated streptokinase with macromolecules such as PEG or dextran. SK conjugates with higher streptokinase-to-dendrimer molar ratios (1:10 and 1:20) exhibited lower initial enzymatic activities. However, these conjugates showed sustained thrombolytic activity in plasma, perhaps due to the release of SK from the conjugate. All of the SK conjugates displayed significantly improved stability in phosphate buffer solution, compared to free SK. The high coupling reaction efficiencies and the resulting high enzymatic activity retention achieved in this study could enable a desirable way for modifying many bioactive macromolecules with dendrimers.     

Evaluation of Fluorescein Isothiocyanate-labeled Whole Antiserum in the Immunofluorescent Identification of Microorganisms

Portions of a whole antiserum to Histoplasma capsulatum were reacted with amounts of fluorescein isothiocyanate (FITC) that ranged from 50 to 400 mug/mg of protein. Portions of the globulin from the same antiserum were reacted with amounts of FITC that ranged from 12.5 to 50 mug of FITC per mg of protein. The globulin conjugates (postlabeled globulins), the whole serum conjugates, and the globulins from the whole serum conjugates (prelabeled globulins) were compared with respect to their fluorescein-protein (F:P) ratios and fluorescent-antibody (FA) activities. The whole serum sample treated with 50 mug of FITC per mg of protein was least reactive in FA tests, and its globulin had the lowest F:P. All other conjugates had globulins with F:P ratios that were considered to be adequate for high FA activity. It was found, however, that the prelabeled globulins were considerably less reactive than the postlabeled globulins or the whole serum conjugates. A larger amount of brightly staining reagent per milliliter of original serum could be obtained from labeled whole serum than from postlabeled globulin. Lissamine-rhodamine conjugated to bovine serum albumin (LRBSA) was evaluated as a counterstain to be used in conjunction with FITC-labeled whole antisera. The counterstain was effective in masking nonspecific FITC fluorescence in Formalin-fixed tissues and in culture smears of fungi. Masking was incomplete in culture smears of a bacterium and in blood smears containing a protozoan.     

Select Alterations in Protein Kinases and Phosphatases During Apoptosis of Differentiated PC12 Cells

Abstract: The involvement of cell cycle-regulatory proteins in apoptosis of neuronally differentiated PC12 cells induced by the removal of nerve growth factor and serum was examined. Three major findings are presented. (1) Cdc2 kinase protein levels increased fivefold in apoptotic PC12 cells by day 3 of serum and nerve growth factor deprivation. Histone H1 kinase activity was increased significantly in p13^suc1 precipitates of apoptotic PC12 cells, which was due to increased activation and/or expression of cdc2 kinase. (2) The protein levels of cyclin-dependent kinase 4, cyclin D, and proliferating cell nuclear antigen that are normally expressed in the cell cycle were increased during neuronal PC12 cell apoptosis. (3) The levels of the catalytic subunit, but not the regulatory subunit of the calcium/calmodulin-dependent protein phosphatase 2B, decreased significantly concomitant with a significant decrease in protein phosphatase 2B activity early in the apoptotic process. Protein phosphatase 2A activity decreased slightly but significantly after 3 days of serum and nerve growth factor deprivation, and no alterations in protein phosphatase 1 were observed during the apoptotic process. These data demonstrate that certain cell cycle-regulatory proteins are inappropriately expressed and that alterations in specific phosphorylation events, as indicated by the increase in histone H1 kinase activity and the decrease in protein phosphatase 2B activity, are most likely occurring during apoptosis of PC12 cells. These observations support the hypothesis that apoptosis may be due in part to a nondividing cell's uncoordinated attempt to reenter and progress through the cell cycle.     

Molecular weight determination and compositional analysis of dextran-protein conjugates using low-angle laser light scattering technique combined with high-performance gel chromatography.

The low-angle laser light scattering technique combined with high-performance gel chromatography was applied for characterization of the dextran-ovalbumin and dextran-lysozyme conjugates obtained from the mild heating in dry state, which is attracting interest as a way leading to stabilization of proteins and to production of proteins with excellent emulsifying or antimicrobial ability (Nakamura, S., Kato, A. and Kobayashi, K. (1991) J. Agric. Food Chem. 39, 647-650). According to the above technique, providing the information about the molecular weight distribution and the composition of the conjugates, one or two dextran molecules were found to be linked to one molecule of the proteins. In addition, each of the conjugates was shown to exist as an oligomeric assembly of which formation is promoted by an increase in the salt concentration of buffer. The observations suggest that the increase in the hydrophobicity of the protein moiety as a result of partial denaturation and the introduction of the hydrophilic dextran chain affords the conjugate an amphiphilic property.     

Assessment of the composition and structure of covalent complexes of superoxide dismutase with aldehyde dextran by analytical ultracentrifugation]

Superoxide dismutase was covalently coupled wih aldehyde dextran, a polymeric carrier of molecular mass of 70 kDa. Modification produced an increase in the enzyme thermostability. Modified preparations retained a high specific activity. The composition of the thus obtained conjugates was analyzed by the ultracentrifugation and diffusion methods. The protein induced the destruction of aldehyde dextran, the enzyme being modified by its fragments. The presence of aldehyde dextran excess in the incubation medium promoted superoxide dismutase dissociation into individual subunits. At the enzyme/carrier ratio of 1:02 modification occurred as covalent coupling of the biocatalyst subunits and its one-point modification.     

Iron dextran treatment does not induce serum protein carbonyls in the newborn pig

Oxidation of serum proteins can lead to carbonyl formation that alters their function and is often associated with stress-related diseases. As it is recommended that all pigs reared in modern production facilities be given supplemental iron at birth to prevent anemia, and metals can catalyze the carbonylation of proteins, the primary objective of this study was to determine whether standard iron dextran treatment was associated with enhanced serum protein oxidation in newborn piglets. Piglets were treated with 100 mg of iron dextran intramuscularly either on the day of birth, or on the third day after birth. Blood samples were collected from piglets 48 or 96 h after treatment and serum was harvested. For quantification, serum protein carbonyls were converted to hydrazones with dinitrophenyl hydrazine and analyzed spectrophotometrically. To identify and determine relative distribution of carbonylated proteins, serum protein carbonyls were derivatized with biotin hydrazide, separated by two-dimensional polyacrylamide gel electrophoresis, stained with avidin-fluorescein and identified by mass spectrometry. The standard iron dextran treatment was associated with no increase in total oxidized proteins if given either on the first or third day of life. In addition, with a few noted exceptions, the overall distribution and identification of oxidized proteins were similar between control and iron dextran-treated pigs. These results indicate that while iron dextran treatment is associated with a marked increase in circulating iron, it does not appear to specifically induce the oxidation of serum proteins.     

Fluorescent immunoanalysis. Synthesis, spectro-fluorescent and immunologic properties of conjugates of coproporphyrin I with antibodies

The synthesis of protein conjugates with the new high-efficient fluorescent labile coproporphyrin-I was optimized. A number of conjugates of monoclonal antibodies with different coproporphyrin-I content were synthesized, and their spectral properties were studied in water and micellar solutions, i.e. adsorption, excitation and emission spectra, fluorescence quantum yields, fluorescence pH-dependences. The binding constants of coproporphyrin-I and its protein conjugates with serum albumin were determined. The antibodies labelled with coproporphyrin-I retain the functional activity and photochemically stable in water solutions. The sensitivity of fluorometric detection of coproporphyrin-I and its conjugates with proteins is more than 10 times greater than in case of FITC.     

Effect of combination of high-intensity ultrasound treatment and dextran glycosylation on structural and interfacial properties of buckwheat protein isolates

This study investigated the effects of high-intensity ultrasound and glycosylation on the structural and interfacial properties of the Maillard reaction conjugates of buckwheat protein isolate (BPI). The covalent attachment of dextran to BPI was confirmed by examination of the Fourier-transform infrared spectra. Emulsifying properties of the conjugates obtained by ultrasound treatment were improved as compared to those obtained by classical heating. Structural feature analyses suggested that conjugates obtained by ultrasound treatment had less α-helix and more random coil, higher surface hydrophobicity and less compact tertiary structure as compared to those obtained by classical heating. The surface activity measurement revealed that the BPI–dextran conjugates obtained by ultrasound treatment were closely packed and that each molecule occupied a small area of the interface. Combination of ultrasonic treatment and glycosylation was proved to be an efficient way to develop new stabilizers and thickening agents for food in this study.     

Induction of high levels of epitope-specific antibodies by epitope/peptide candidate vaccines against human immunodeficiency virus type-1 (HIV-1)

To test the immunogenicity of GPGRAFY-epitope-based candidate vaccines, a peptide with four repetitive GPGRAFY epitopes, V3-P1 [C-(GPGRAFY)4], and a peptide (PND) of the principal neutralizing domain (V3 loop: amino acid 301-328: C-TRPNNNTRKSIRIQRGPGRAFYTIGKI) on gp120 were synthesized and covalently coupled to a carrier protein BSA. Immunization of BALB/c mice and New Zealand White Rabbits with these conjugate vaccines engendered strong antibody responses against the PND (mouse serum titer by 1:12,800-25,600; rabbit serum titer by 1:6,400-12,800). Interestingly, the V3-P1-BSA conjugates and the PND-BSA conjugates could induce high levels of GPGRAFY-epitope-specific antibodies in the mice and rabbits (mouse serum titer by 1:25,600; rabbit serum titer by 1:12,800-25,600), while a recombinant gp160 subunit vaccine induced a low level of GPGRAFY-epitope-specific antibodies (serum titer by 1:400-1,600 in mice and rabbits). To confirm the above results, GPGRAFY-epitope-specific antibodies were isolated from rabbit sera induced by V3-P1-BSA, PND-BSA conjugates and rgp160 vaccine. In fact, 23-38 and 13-22 microg epitope-specific antibodies per milliliter serum were isolated from rabbit sera induced by V3-P1-BSA and PND-BSA conjugate, respectively, while 1.34 microg epitope-specific antibodies per milliliter serum were identified in rabbit serum induced by rgp160 vaccine. In the control group, only 0.069 microg proteins per milliliter serum were found in pooled pre-immune serum (normal serum). These results from mouse and rabbit experiments indicate that epitope and peptide vaccines both induce high levels of GPGRAFY-epitope-specific antibodies in comparison with rgp160 subunit vaccine, suggesting that epitope/peptide vaccines may be a new strategy to induce protective activity.