Mole Of Bovine Serum Albumin Research Articles

Fluorescence Spectroscopic Studies of in vitro Interactions of Famotidine and Tapentadol Hydrochloride with Bovine Serum Albumin

The in vitro interactions of Famotidine (FT) and Tapentadol hydrochloride (TAP) with bovine serum albumin (BSA) have been studied by fluorescence emission spectroscopy under different conditions. Quenching constants were determined using the Stern-Volmer equation. Two moles FT bound with 1 mole of BSA at 298 K and 3 mole FT bound with 1 mole of BSA at 308 K in presence of TAP. BSA was used for the study as it shows approximately 76% sequence homology to human serum albumin (HSA).Dhaka Univ. J. Pharm. Sci. 15(1): 21-26, 2016 (June)

Synthesis of bifunctional TiO2@SiO2-B(OH)2@Fe3O4@TiO2 sandwich-like nanosheets for sequential selective enrichment of phosphopeptides and glycopeptides for mass spectrometric analysis.

In this work, the bifunctional TiO2@SiO2-B(OH)2@Fe3O4@TiO2 sandwich-like nanosheets were designed and synthesized for the sequential selective enrichment of phosphopeptides and glycopeptides. Due to the bifunctional property of the titanium dioxide and the boronic acid group, the nanosheets were successfully applied to the enrichment of phosphopeptides and glycopeptides sequentially, evaluated by capturing phosphopeptides from tryptic digestion of model phosphoprotein bovine β-casein diluted to 0.02ng/μL (8 × 10(-16) mol/μL) and glycopeptides from tryptic digestion of model glycoprotein horseradish peroxidase (HRP) diluted to 0.1ng/μL (2.5 × 10(-15) mol/μL). The enrichment selectivity of the bifunctional nanosheets was evaluated by capturing phosphopeptides from a peptide mixture of β-casein and bovine serum albumin (BSA) with the molar ratio of 1:1000 (8.3 × 10(-12) mol of β-casein and 8.3 × 10(-9) mol of BSA in 100μL) and glycopeptides from a peptide mixture of HRP and BSA up to the ratio of 1:50 (5.0 × 10(-11) mol of HRP and 2.5 × 10(-9) mol of BSA in 100μL). Graphical Abstract A workflow of the sequential enrichment strategy for phosphopeptides and glycopeptides by the bifunctional TiO2@SiO2-B(OH)2@Fe3O4@TiO2 sandwich-like nanosheets.

Binding of bisphenol A and acrylamide to BSA and DNA: Insights into the comparative interactions of harmful chemicals with functional biomacromolecules

The interactions between bisphenol A (BPA)/acrylamide (AA) and bovine serum albumin (BSA)/deoxyribonucleic acid (DNA) was investigated by the equilibrium dialysis, fluorophotometry, isothermal titration calorimetry (ITC) and circular dichroism (CD). The bindings of BPA and AA to BSA and DNA responded to the partition law and Langmuir isothermal model, respectively. The saturation mole number of AA was calculated to be 24 per mol BSA and 0.26 per mol DNA-P. All the reactions were spontaneous driven by entropy change. BPA stacked into the aromatic hydrocarbon groups of BSA and between adjacent basepairs of DNA via the hydrophobic effect. The interactions of AA with BSA and DNA induced the formation of hydrogen bond and caused changes of their secondary structures. At normal physiological condition, 0.100 mmol/l BPA reduced the binding of vitamin B 2 to BSA by more than 70%, and 2.8 mmol/l AA by almost one half. This work provides an insight into non-covalent intermolecular interaction between organic contaminant and biomolecule, helping to elucidate the toxic mechanism of harmful chemicals.

Use of Biotin Derivatives to Probe Conformational Changes in Proteins

Sulfosuccinimidyl-6-(biotinamido) hexanoate and derivatives thereof covalently bind to the epsilon-amino group of lysine residues. Our observation that access of the biotin derivative to specific lysine residues depends on conformational properties of the entire polypeptide chain prompted us to investigate whether differential biotinylation patterns of a protein can be used as indicators for conformational changes. Bovine serum albumin is a soluble protein with characteristic unfolding kinetics upon exposure to high temperature. First, we show that biotinylation patterns of proteins are highly reproducible. Second, we demonstrate by mass spectrometry and tandem mass spectrometry that unfolding of the protein correlates with the accessibility of the biotin derivative to specific lysine residues. We have applied this experimental strategy to the analysis of a cell-surface protein, viz. the human band 3 anion exchanger of erythrocytes infected with the malaria parasite Plasmodium falciparum. We found that Lys(826) in a highly flexible loop can be biotinylated in non-infected (but not infected) erythrocytes, confirming earlier observations (Winograd, E., and Sherman, I. W. (2004) Mol. Biochem. Parasitol. 138, 83-87) based on epitope-specific monoclonal antibodies suggesting that this region undergoes a conformational change upon infection.

Non-enzymatic phosphorylation of bovine serum albumin by Cr(V) complexes: Role in Cr(VI)-induced phosphorylation and toxicity

Evidence for the non-enzymatic phosphorylation of bovine serum albumin (BSA) by sodium bis(2-ethyl-2-hydroxybutyrato)oxochromate(V), Na[CrVO(ehba)2], 1, sodium bis(2-hydroxy-2-methylbutyrato)oxochromate(V), Na[CrVO(hmba)2], 2 and potassium dichromate, K2Cr2O7, 3 in the presence of labeled adenosine-5'-triphosphate (ATP) under conditions of physiological pH is presented. Aggregation and extent of phosphorylation of BSA mediated by 1, 2 or 3 seems to increase with the concentration and time of incubation of the reaction mixture containing all the reactants. The [gamma-32P] label in ATP is incorporated into aggregates of BSA in the in vitro reaction of the protein with ATP in the presence of 1, 2 or 3. Phosphorylation of BSA by ATP in the absence of 1, 2 or 3 is negligible. Addition of EDTA reverses aggregation of protein and liberates partially the incorporated phosphate label. The stoichiometry of phosphorylation is found to be the highest and is equal to 12.25 mol PO4(3-)/mol BSA in the presence of 500 microM of 1, which decreases to 10.56 mol PO4(3-)/mol BSA after EDTA treatment. Resistance to the removal of phosphate label by EDTA increases with increase in time of incubation. Dialysis of phosphorylated BSA reverses the incorporated [gamma-(32)P] label only partially, indicating the formation of covalent links of phosphate groups to BSA. Evidence for the site of phosphorylation in the reaction mediated by 1, 2 or 3 being hydroxyl side groups of tyrosine and serine/threonine residues has been gained. Based on the results, a possibility that 1, 2 and 3 mimic the function of tyrosine and serine/threonine kinases has been invoked.

Detection of Aspergillus fumigatus mycotoxins: immunogen synthesis and immunoassay development

Immunological detection of secreted low molecular weight toxins represents a potentially novel means of diagnosing infection by the fungus Aspergillus fumigatus. Two such metabolites, gliotoxin and helvolic acid, were selected and conjugated to thyroglobulin for antisera generation in rabbits. Gliotoxin was initially activated using N-[ p-maleimidophenyl] isocyanate (PMPI) and subsequently conjugated to S-acetyl thioglycolic acid N-hydroxysuccinimide-activated thyroglobulin, whereas helvolic acid was activated with N-(3-Dimethylaminopropyl)- N′-ethylcarbodiimide (EDC) in the presence of thyroglobulin prior to immunisation. To facilitate subsequent antisera evaluation, both toxins were similarly conjugated to bovine serum albumin (BSA). Matrix-Assisted Laser Desorption Ionisation-Time Of Flight (MALDI-TOF) mass spectrometry and SDS-PAGE analysis confirmed covalent attachment of toxins to BSA in the ratios of 15 and 2.4 mol per mol BSA for gliotoxin and helvolic acid, respectively. Resultant high titer antisera were capable of detecting both BSA-conjugated toxins (inhibitory concentration (IC) 50: 4–5 μg/ml). Free toxins were also detectable by competitive immunoassay, whereby 10 μg/ml free gliotoxin (30 μM) and helvolic acid (17 μM), respectively, inhibited antibody binding to cognate toxin–BSA previously immobilised on microwells. This work confirms that sensitive and specific antisera can be raised against fungal toxins and may have an application in diagnosing fungal infection.

Emulsifying properties of bovine serum albumin-galactomannan conjugates.

Bovine serum albumin (BSA)-galactomannan conjugates were prepared through the Maillard reaction. To 1 mol of BSA was bound 2.5-7 mol of galactomannan. Conjugates could be grouped into two fractions, on the basis of the weight-average molar mass measured with a multiangle laser light-scattering detector, the main one with 220 000-250 000 Da and the other one with a very small amount of aggregates over 1 000 000 Da. Spectroscopic analysis suggested that the surface of the conjugate was covered with galactomannan and the conformation of the hydrophobic interior and the secondary structure were not significantly changed. The emulsifying activity index of the conjugates increased greatly as compared with that of BSA alone. All conjugates showed better stability than BSA, presumably due to the physical protection introduced by the viscoelastic galactomannan layer. The average particle sizes of the emulsions were similar. The interfacial properties of the BSA-galactomannan conjugates were improved, possibly due to the reduced hydrophobic interaction between the droplets and the viscoelastic interfacial properties of galactomannan.

An improved method for the microscale preparation and characterization of hapten-protein conjugates: the use of cholesterol as a model for nonchromophore hydroxylated haptens.

A minute amount (0.446 micromol) of cholesterol (Chol) was converted into an hemisuccinate derivative (Chol HS) using an excess of succinic anhydride. The optimal conditions for synthesis of Chol HS were explored by checkerboard experiments in which various succinic anhydride/Chol molar ratios ranging from 5:1 to 30:1 were assayed over a wide temperature range (50-85 degrees C) and for various incubation times (3-8 h). Total conversion was obtained at the higher reagent ratios, temperatures, and incubation times. Subsequently, this carboxylic derivative was first covalently linked to bovine serum albumin (BSA) then to various proteins (casein, ovalbumin, and hemocyanins) or to a synthetic homopolymer (poly-DL-Lysine) via a modified version of the mixed anhydride method of Erlanger, performed in a reversed micellar medium. The assessment of the number of haptenic groups per mole of BSA (epitope density) was achieved chromatographically by two methods according to a Chol standard curve established at 207 nm with linearity in the range 0-50 microg. These procedures involving an alkaline hydrolysis of a sample of either the conjugate (direct method) or the unreacted Chol HS (indirect method) yielded an acceptable level of agreement and concordant results in all cases. The influence of the activated hapten/BSA molar ratio on the coupling efficiency was investigated by the direct method within the range 10:1 to 250:1. Using the optimal conditions determined for Chol HS synthesis (a molar reagent ratio of 30:1 with incubation at 65 degrees C for 6 h) and for BSA haptenation (a 100-fold molar excess of activated hapten, with a carrier stock concentration of 5 mg/mL), epitope density of the conjugates lied between 23 and 27. By reacting the same amount of activated hapten ( approximately 216 microg) with identical amounts of various carriers (300 microg), conjugation efficiency was found similar on a microgram of Chol bound per milligram of carrier basis. This simple and reproducible conjugation and analysis procedures should provide a general method applicable to poorly available and weakly immunogenic haptens bearing hydroxyl groups such as polyether-type marine toxins.

Hydroxopentaamminechromium(III) promoted phosphorylation of bovine serum albumin: its potential implications in understanding biotoxicity of chromium

Evidence for chromium(III) induced phosphorylation of a biomarker protein bovine serum albumin (BSA) is presented. Radiolabelled adenosine 5′-triphosphate (ATP) was reacted with BSA in the presence of various Cr(III) salts. While [Cr(NH 3) 5(H 2O)] 3+ brought about phosphorylation of BSA, several Cr(III) complexes, viz. [Cr(bpy) 3] 3+, [Cr(phen) 3] 3+, [Cr(en) 3] 3+, [Cr(salen)(H 2O) 2] + and [Cr(salprn)(H 2O) 2] +, did not phosphorylate BSA. The Cr(III) mediated the transfer of γ- and α-phosphates but not the adenine and the sugar moieties of the ATP molecule to BSA. The observed stoichiometry was 0.75 mol P i to mol BSA for the γ-phosphate and 0.5 mol P i to mol BSA for the α-phosphate of ATP. The presence of serine phosphate and threonine phosphate was detected in the hydrolysate of phosphorylated BSA by means of comparison of R f values with authentic samples of phosphoserine and phosphothreonine after chromatographic separation and autoradiography. [Cr(NH 3) 5(H 2O)] 3+ at pH 7.4 is known to exist as the conjugate base [Cr(NH 3) 5(OH)] 2+ and is capable of ligand substitution involving metal-oxygen bond retention. Such anation reaction of [Cr(NH 3) 5(OH)] 2+ with ATP subsequently leads to the esterification of alcoholic hydroxyl groups of serine and threonine of BSA. Possible consequences of chromium(III) induced in vivo phosphorylation of proteins are discussed.

Generation of anti-peptide antibodies against serotonin 5-HT 2A and 5-HT 2C receptors

Anti-peptide antibodies were generated against several 13–17 amino acid regions of rat serotonin 5-HT 2A and 5-HT 2C receptors. Peptides containing terminal cysteine residues were conjugated to bovine serum albumin (BSA) and ovalbumin (OVA) with the cross-linking reagent sulfo-SMCC (sulfosuccinimidyl 4-( N-maleimidomethyl) cyclohexane-1-carboxylate). Both the carrier protein and the number of peptide molecules per carrier molecule were changed during the immunization schedule. For the early immunizations, immunogens were BSA-peptides at ratios of 8–27 mol peptide per mol BSA. For the later boosts, immunogens were OVA-peptides at ratios of 1–2 mol peptide per mol OVA. The peptide constructs were used to immunize rabbits and chickens. Anti-peptide antibodies were purified from sera (rabbits) or egg yolks (hens) using peptide matrices. Cell lines expressing similar densities of rat 5-HT 2A or 5-HT 2C receptors were used to monitor the specificity of purified antibodies on immunoblots and in immunocytochemistry. A total of five out of the six rabbit antibodies were positive on immunoblots (three anti-5-HT 2A and two anti-5-HT 2C) and four were also positive in immunocytochemistry (three anti-5-HT 2A and one anti-5-HT 2C). None of the anti-peptide chicken antibodies were useful on immunoblots or in immunocytochemistry. Since there is a paucity of high affinity reagents selective for 5-HT 2A or 5-HT 2C receptors, these rabbit antibodies will be useful tools. The methods used to generate site-directed antibodies specific for 5-HT 2A or 5-HT 2C receptors should be applicable to other proteins.

Effects of free fatty acids on the binding of bovine and human serum albumin with steroid hormones

Recent studies have shown that, in addition to free steroid hormones, those bound to albumin in plasma may also be available to tissues. In this report, the effects of free fatty acids (FFA) on the binding of steroids to albumin were compared for the cases of bovine serum albumin (BSA) and human serum albumin (HSA). The apparent association constant, K a, was estimated from the changes in the equilibrium partition coefficient of steroids between the aqueous/hexane phases caused by the addition of albumin to the aqueous phase. In the case of BSA, K a for progesterone and testosterone increased upon binding of FFA (myristic, palmitic and stearic acid) to BSA and the maximum value of K a for these steroids could be attained by 3–4 mol of FFA bound per mol BSA. Furthermore, the elution profiles of gel-filtration chromatography clearly showed that progesterone and testosterone are easily liberated from the steroid/BSA complexes and that FFA potentiates the binding of these steroids to BSA. In the case of HSA, the binding affinities of progesterone and testosterone were not greatly affected by bound FFA. On the other hand, the affinities of ethynylestradiol to both BSA and HSA were unaffected below 4 mol of FFA binding per mol.

Drug–Protein Conjugates: Haptenation of 1‐Methyl‐10α‐methoxydihydrolysergol and 5‐Bromonicotinic Acid to Albumin for the Production of Epitope‐Specific Monoclonal Antibodies Against Nicergoline

Two types of monoclonal antibodies were used for the determination of nicergoline in biological matrices. The antibodies were prepared with the hydrolysis products 5‐bromonicotinic acid and 1‐methyl‐10α‐methoxydihydrolysergol after hemisuccinoylation to haptens. The current amide bond‐generating methods (mixed anhydride‐, carbodiimide‐, carbodiimide/sulfo‐N‐hydroxysuccinimide‐, and dicyclohexylcarbodiimide/N‐hydroxysuccinimide methods) were used in bovine serum albumin (BSA)‐coupling techniques and yielded conjugates that were haptenated to varying extents. The conjugates exhibiting 23mol of 1‐methyl‐10α‐methoxydihydrolysergol (MMD) or 41mol of 5‐bromonicotinic acid (BNA) per mole of BSA were used for both immunization of mice and for coating the wells of the microtiter plates to select hybridomas and investigate specificity of the obtained antibodies. The results of hapten‐inhibition ELISA using antigen‐coated wells indicate that the supernatant of MMD‐specific hybridoma exhibited 50% inhibition of antibody binding at 17 ± 2 μg of MMD and at 24.5 ± 2 μg of nicergoline, and the BNA‐specific hybridoma exhibited similar inhibition at 147 ± 6 μg of BNA and 500 ± 30 μg of nicergoline. A main requirement for analytical purposes is that two different types of monoclonal antibodies recognize two different epitopes on nicergoline and its main metabolite, as shown by hapten‐inhibition ELISA.

Interaction Between Drugs and Water-Soluble Polymers. VII. Binding of Berberine with Bovine Serum Albumin

Abstract The interaction between berberine chloride (BC) and bovine serum albumin (BSA) was studied by equilibrium dialysis. Since the number (n) of binding sites per mole of BSA was large, the binding was nonspecific. Then the binding capacity (nK) was evaluated instead of the binding constant (K). According to the dependence of the NK value on ionic strength, the interaction was due to hydrophilic binding. Intermolecular interaction of BC was observed by the concentration (1–10 mmol/L) dependence of 1H-NMR parameters; that is, the respective signals shifted to a higher magnetic field, the spin-lattice relaxation time (T 1) decreased, and the spin-spin relaxation rate (T 2 −1) increased. The interaction of BC with BSA (1.45 × 10−4 mol/L) resulted in a decrease in T 1, an increase in T 2 −1, and little variation of the chemical shift. On the basis of the ratio of the spin-spin relaxation rate of bound BC to free BC (T 2b −1/T 2f −1). the binding position of BC to BSA was considered to be spread over the e...

α-Lipoate Can Protect Against Glycation of Serum Albumin, But Not Low-Density Lipoprotein

Protein glycation may play a role in the pathogenesis of diabetic complications. α-Lipoate (1,2-dithiolane-3-pentanoate) has been reported to prevent glycation and structural modification of bovine serum albumin (BSA). To elucidate the protective mechanism, we tested the effects of enantiomerism, thiol moiety and hydrophobicity of α-lipoate on glycation of BSA and low density lipoprotein(LDL). When BSA (1 mM) was incubated with 500 mM glucose in the presence of α-lipoate homologues or dihydrolipoate (6,8-dimercaptooctanoate, DHLA) at 37°C for 72 h, both α-lipoate (racemic, R- and S-forms) and DHLA inhibited BSA glycation similarly, but tetranorlipoate (1,2-dithiolane-3-carboxylate) did not. However, under similar conditions, α-lipoate did not inhibit LDL glycation. Scatchard plot analysis demonstrated that 6 mol of α-lipoate bind to 1 mol of BSA with a formation constant of 8.7 × 104 M−1. Therefore, we concluded that α-lipoate protects BSA glycation by hydrophobic binding near the glycation sites of BSA.

Binding of arachidonate and oleate to bovine serum albumin.

Using the "ghost method" previously applied to study the binding of palmitate to bovine serum albumin (BSA) (Bojesen, I. N., and E. Bojesen. 1992. J. Lipid Res. 33: 1327-1334) we have measured the water-phase concentrations of arachidonate (AR) and oleate (OL) in 165 mM KCl and in 165 mM NaCl, 2 mM phosphate buffer at pH 7.3 in equilibrium with AR and OL bound to BSA (about 30 microM) inside resealed human red cell ghosts at low molar ratio (v). Data were obtained at 0 degree C, 10 degrees C, 23 degrees C, and 38 degrees C for v between 0.012 and 1.5. Regression analyses of the data suggest that BSA has three equivalent binding sites for AR and OL at the four temperatures. The equilibrium dissociation constants (Kd) were the same in potassium and in sodium buffers. They were calculated for AR and OL on basis of three equivalent binding sites per mol BSA. Kd values increase with temperature and the AR values are, on average, approximately 5-fold greater than those of OL within the investigated temperature range. At 23 degrees C, Kd values for three equivalent sites are 15.60 +/- 0.73 nM and 2.89 +/- 0.14 nM corresponding to -44.2 +/- 0.15 kJ/mol and -48.3 +/- 0.1 kJ/mol in free energies of binding AR and OL, respectively. The difference, 4.1 kJ/mol, fits fairly well with the theoretical difference in hydrophobic effects of the two aliphatic chains, 3.7 kJ/mol.(ABSTRACT TRUNCATED AT 250 WORDS)

A method to study protein denaturation by measurements of apparent molar volumes

Protein denaturation was studied by measurements of apparent molar volumes of bovine serum albumin (BSA)-water-urea systems at 25°C. The viscosity measurements carried out on aqueous BSA solutions at various fixed concentrations as a function of temperature have shown that the denaturation process can best be followed at 60gkg −1 BSA solution. The apparent molar volumes of urea in water and in 60gkg −1 BSA solution were measured at 25°C and the differences between the two were interpreted in terms of structural changes taking place during denaturation of BSA by urea. A simple method was proposed to calculate the volume change per mole of protein upon denaturation. The sample calculation for BSA has shown that there is a volume contraction of 1854 cm 3 per mol BSA when the urea concentration reaches 13 M.

A fluorescamine-based assay for the degree of glycation in bovine serum albumin

The fluorescence resulting from the reaction of a protein with fluorescamine is an indication of the number of free amino groups in that protein, consequently glycated proteins which have fewer free amino groups will show less fluorescence. With the appropriate standards, the difference in fluorescence of glycated and non-glycated proteins can be translated into the number of glycated sites in a protein. Glucose (0·22 m final concentration) was incubated with bovine serum albumin (BSA) (0·1 mg/ml) in a total volume of 50 ml of 0·1 m phosphate buffer pH 7·2 at 37°C for a period of 35 days. In order to assess the effect of metal catalyzed free radical damage to the protein, the incubation was also carried out in the presence of a metal chelating agent EGTA (0·38 mg/ml). As expected, the fluorescence of both mixtures decreased with time, as more lysyl groups became glycated. Calculations based on the standard curve of t-BOC-lysine showed that approximately 18 mol of glucose were incorporated per mol of BSA during the incubation in the presence of EGTA and 23 mol in the absence of EGTA. This difference is explained by the metal catalyzed free radical fragmentation of BSA which exposes more N-terminal amino groups for reaction with glucose.

The binding of palmitoyl-CoA to bovine serum albumin

Bovine serum albumin (BSA) is routinely utilized in vitro to prevent the adverse detergent effects of long-chain acyl-CoA esters (i.e., palmitoyl-CoA) in enzyme assays. Determination of substrate saturation kinetics in the presence of albumin would only be valid if the relationship between bound and free substrate concentrations was known. To elucidate the relationship between bound and free palmitoyl-CoA concentrations in the presence of BSA, several different techniques including equilibrium dialysis, equilibrium partitioning, fluorescence polarization and direct fluorescence enhancement were investigated. Direct fluorescence enhancement using a custom synthesized fluorescent probe, 16-(9-anthroyloxy)palmitoyl-CoA (AP-CoA), was the best approach to this question. Measurement of the relationship between mol palmitoyl-CoA bound per mol of BSA ( ν ) versus −log[free palmitoyl-CoA] revealed that the binding of palmitoyl-CoA to BSA, like palmitate was nonlinear, suggesting the presence of more than one class of acyl-CoA binding sites. Computer analyses of the binding data gave a best fit to the 2,4 two-class Scatchard model, suggesting the presence of two high-affinity primary binding sites ( k 1 (1.55 ± 0.46) · 10 −6 M −) and four lower affinity secondary binding sites ( k 2 = (1.90 ± 0.09) · 10 −8 M −1). Further analyses using the six parameter stoichiometric (stepwise) ligand binding model supports the existence of six binding sites with the higher affinities associated with the binding of the first mole of palmytoyl-CoA and weaker binding occurring after the first two sites are occupied. The association constants from this model of multiple binding diminish sequentially (i.e., K 1 > K 2 > K 3 > … ⩾ K 6), suggesting that each mol of long-chain acyl-coA binds to BSA with decreasing affinities.

Efficient coupling of glycopeptides to proteins with a heterobifunctional reagent

A heterobifunctional linking reagent containing a masked aldehydo group and acyl hydrazide was synthesized for coupling of glycopeptides and other amino-containing compounds to proteins. After conversion to acyl azide, the reagent reacts with the amino group of a glycopeptide, and the modified glycopeptide is deacetalized with a weak acid to unmask the aldehydo group, which is then conjugated to bovine serum albumin (BSA) by reductive alkylation with pyridine-borane. The overall reaction scheme proceeds under relatively mild conditions. When the protein amino group was in a large excess (greater than 6-fold) of the aldehyde reagent, the efficiency of conjugation was as high as 88% even at submicromole levels. As a test case for application of this reagent, 6-aminohexyl beta-D-galactopyranoside (Gal-AH) was attached to the linking reagent and conjugated to BSA at various aldehyde-to-protein molar ratios ranging from 25 to 200. The level of O-galactosyl residue incorporated into BSA by this reagent far exceeded that observed in a similar reductive alkylation involving S-galactoside reagents [Lee, R. T., & Lee, Y. C. (1980) Biochemistry 19, 156-163]. By use of the present conjugating procedure, as many as 112 mol of Gal-AH residues were incorporated per mole of BSA, which represents near total modification of the amino groups. Some binding characteristics of the new BSA derivatives were studied in the mammalian hepatic galactose/N-acetylgalactosamine specific lectin system along with other types of BSA derivatives (containing S-galactosyl residues). In general, the behavior of the new derivatives was similar to that of other types. For instance, the affinity increased exponentially at low sugar substitution levels (up to 30 mol of galactosyl residues/mol of BSA), and the slope of exponential increase and affinity at a given sugar substitution level was similar to those of other types.

The kinetics of phosphoinositide hydrolysis in rat basophilic leukemia (RBL-2H3) cells varies with the type of IgE receptor cross-linking agent used.

We have re-examined, by high pressure liquid chromatographic (HPLC) procedures, the hydrolysis of 3H-labeled inositol phospholipids in rat basophilic leukemia (RBL-2H3) cells. Previous studies showed no clear correlation between the release of any particular inositol metabolite and the calcium signal in these cells. Paradoxically no responses were observed when the cells were stimulated with the antigen, aggregated ovalbumin, in the absence of external Ca2+. We report here that in the absence of external Ca2+ aggregation of the IgE receptor by agents other than aggregated ovalbumin causes the release of small amounts of [3H]inositol phosphates and a small increase in levels of cytosol Ca2+ (approximately 25 nM). The response, however, varied with the type of stimulant used. Within seconds after addition of 24 mol of dinitrophenol conjugated with 1 mol of bovine serum albumin to cells primed with dinitrophenol-specific IgE there was a small burst in release of [3H]inositol 1,4,5-triphosphate, [3H]inositol 1,3,4,5-tetrakisphosphate, and [3H]inositol 1,3,4-trisphosphate which was followed by a gradual rise in inositol 1,3,4-trisphosphate, inositol bisphosphate, and inositol monophosphate. Eventually, all inositol phosphates reached different steady state levels which were maintained for at least 40 min. In contrast, the initial response to oligomeric IgE, which aggregates receptors at a relatively slow rate, was muted although the subsequent development of the response was the same. The levels of inositol pentakisphosphate and hexakisphosphate remained unchanged. These and other studies with cell extracts support the conclusion that inositol 1,4,5-trisphosphate, a putative messenger for release of intracellular Ca2+, was converted to inositol 1,3,4,5-tetrakisphosphate and thence to inositol 1,3,4-trisphosphate. Both trisphosphate metabolites were dephosphorylated in sequential fashion by phosphatase enzymes in the cytosolic and membrane fractions. However, the appearance of several isomers of inositol monophosphates and bisphosphates suggested that degradation proceeded through multiple pathways in the intact cell.