BSA Concentrations Research Articles

Characterization of bovine serum albumin adsorption on chromium and AISI 304 stainless steel, consequences for the Pseudomonas fragi K1 adhesion

Adsorption of BSA on the surface of chromium and 304 stainless steel, has been characterized by Contact Angle Measurements, X-ray Photoelectron Spectroscopy (XPS) and Infrared Reflection Absorption Spectroscopy (IRRAS). Bacterial adhesion has been tested and compared on these two materials before and after pre-conditioning the surface with BSA. Chromium and stainless steel surfaces, when covered by a natural oxide layer, exhibit different energetic characteristics as shown by their γ s - and γ s LW respective values. These data vary upon immersion in BSA solutions, tending towards common values for duration of immersions. After immersion in BSA solutions, the evolution of the N 1s XPS signal, specific for the BSA, suggests that the surface is nearly saturated in a few minutes. Longer times of immersion only lead to a re-ordering of the adsorbed layer. Immersion tests in dilute BSA solutions (0.01 g/l) enabled us to make clear a higher reactivity of chromium towards the protein compared to stainless steel. These differences are cancelled at higher BSA concentrations (1 g/l). IRRAS spectra of BSA adsorbed on the two substrates demonstrated the appearance of amide I and amide II bands with small shifts and intensity variations supporting orientation changes of the protein when the concentration or immersion time varies. A model for the building up of the BSA layer is proposed, which accounts for these data. Chromium and stainless steel surfaces, also have different behaviours towards adhesion of Pseudomonas fragi K1, whereas surfaces that are pre-conditioned by BSA behave in a similar way. The overall number of adherent bacteria is decreased on stainless steel, whereas it is hardly affected on chromium. On both surfaces, the fraction of viable cells is increased.

Steroidogenesis in cumulus cells of bovine cumulus–oocyte-complexes matured in vitro with BSA and different concentrations of steroids

The present in vitro experiments were designed to evaluate the ability of bovine cumulus–oocyte-complexes (COCs) to produce steroids and also to evaluate the modulatory effects of added estradiol, progesterone and testosterone on the steroidogenic activity of COCs. Considerable estradiol accumulation was observed in the control maturation medium for in vitro maturation of bovine COCs during the 24 h of maturation ( P<0.05). When testosterone was added to the medium at various concentrations, a slight estradiol accumulation occurred, which, however, was lower ( P<0.05) than that observed in the control medium. Slight estradiol accumulation was observed in maturation medium containing progesterone at concentrations of 2.5, 5.0 and 10.0 μg/ml, but these increases were less ( P<0.05) than those observed in the control medium. However, in the presence of 1.0 μg/ml progesterone, estradiol accumulation was equal to that of the control medium ( P>0.05). Progesterone accumulation ( P<0.05) was observed in the control medium for in vitro maturation of bovine COCs. When estradiol was added to the maturation medium, progesterone accumulation was observed, but was significant ( P<0.05) only when the medium was supplemented with the lesser concentrations of estradiol utilized in the experiment (1.0 μg/ml). The results demonstrated that (1) cumulus cells of bovine COCs are able to secrete estradiol and progesterone in culture systems for in vitro maturation, and this steroidogenesis is modulated by the steroids progesterone, testosterone and estradiol, and (2) the addition of estradiol to the in vitro maturation medium of bovine oocytes should be reviewed, since cumulus cells of COCs have been demonstrated to secrete estradiol in the maturation medium.

Determinação das Condições Ótimas para Análises de PCR-RAPD em Atta sexdens rubropilosa Forel (Hymenoptera: Formicidae)

PCR-RAPD has been widely used for genetic analysis in several organisms. However, due to complex interactions among the components of the PCR reaction it is unlikely that a single amplification condition can be suitable for all situations. In order to determine the optimum conditions for using PCR-RAPD in taxonomical analyses of the genus Atta, the following factors were tested: concentrations of MgCl2, DNA, BSA, cycling programs and methods of DNA extraction, using Fractional Factorial Design and Central Composite Design. DNA extraction methods had little influence on PCR-RAPD reactions, while the cycling programs showed the largest effect. The MgCl2 concentration was less important than the amount of DNA used in the reaction. Using cycling program P2, a significant improvement in the number of DNA fragments was achieved when MgCl2 concentration was increased to 3.0 mM and the BSA and DNA concentrations were reduced from 0.1% to 0.05% and from 2 to 1 ng/ml, respectively. The optimum conditions for PCR-RAPD with Atta specimens obtained in this study were: 25 ml reaction with 10 mM Tris-HCl (pH 9.0), 3.0 mM MgCl2, 50 mM KCl, 100 mM of each dNTP, 0.2 mM of primer, 1.0 U of Taq polymerase, 25 ng template DNA (extracted by Cheung's method) and BSA 0.05%, using the amplification program which consisted of 3 min at 94oC, 3 min at 35oC, followed by 40 cycles of 1 min at 94oC, 1 min at 36oC and 2 min at 72oC, and a 5 m final extension at 72oC.

An innovative synthetic tissue-mimicking material for high-intensity focused ultrasound

A dosimetry study of the high-temperature and pressure regimes involved in high-intensity focused ultrasound (HIFU) requires experiments on biological tissues because no synthetic tissue-mimicking phantom is available. Unfortunately, the development of coagulative lesions cannot be observed in real-time in opaque tissues. Furthermore, the natural heterogeneous structure of tissue complicates direct comparison with numerical models. In this study, a new optically transparent phantom is evaluated. It is principally composed of a polyacrylamide gel, and includes a thermally sensitive indicator protein that becomes optically diffusive when denatured. Various tests were undertaken to characterize the acoustical, thermal, and optical properties of this material for a range of protein concentrations. The attenuation coefficient can be usefully modified by adjusting the quantity of embedded proteins to permit some selection of acoustic regime. It is also possible to emphasize cavitation activity at lower BSA concentrations, or thermal effects at higher concentrations. This new phantom adequately matches tissue for most of the measured parameters and facilitates the study of the HIFU bioeffects. [Work supported by ONR Grant No. N00014-96-1-0630 and NIH Grant No. HL64208-02.]

Recent developments in the characterization of water interacting with proteins by 17O NMR

Abstract Transverse and longitudinal 17 O-water relaxation rates were detected in different samples of BSA solutions after one-quantum and triple-quantum-filtered NMR sequences. Another contribution other than quadrupolar relaxation was found for transverse relaxation, which did not change significantly with the concentration and hence could not correspond to agglomeration of proteins. This was interpreted as chemical exchange between different types of 17 O-water in fast motion; probably free water and water weakly bound to the proteins. At lower BSA concentrations, two peaks were detected for water; this was in agreement with this hypothesis. The interactions between BSA and lactic acid were also studied. It was shown that at a sufficient concentration of lactic acid, the number of strongly bound water molecules detected diminishes. On the other hand, the weakly bound water properties do not change significantly.

The quantitative 19F-imaging of albumin at 1.5 T: a potential in-vivo tool

19F-MR-imaging has been used to quantitate albumin concentration in a phantom at 1.5 T. The experimentally derived relationship between albumin concentration and the T1 relaxation time of a fluorinated marker, tetrafluorosuccinic acid (TFSA) was used to calculate the albumin concentration from a quantitative 19F T1 map acquired using a gradient echo sequence. There was close correlation between calculated and actual BSA concentrations (r = 0.99, SE = 0.15). The potentially interfering effect of paramagnetic species on T1 relaxation times was also investigated. Relaxivity data show that albumin concentration measurements should be performed prior to any contrast agent administration.

Albumin overload induces apoptosis in LLC-PK1cells

The degree of albuminuria is a well-known adverse prognostic indicator in human glomerular diseases. However, the mechanisms by which albuminuria by itself contributes to tubulointerstitial injury and progression of renal disease remain unclear. We tested the hypothesis that apoptosis may represent one of the mechanisms by which tubule epithelial cells are damaged after albumin overload in vitro. Cultured LLC-PK(1) proximal tubule cells were incubated with varying concentrations of BSA. This resulted in a dose- and duration-dependent induction of apoptosis, as evidenced by internucleosomal DNA cleavage (DNA laddering and nick-end labeling), externalization of plasma membrane phosphatidylserine (annexin labeling), and characteristic morphological changes (cell shrinkage and nuclear condensation). Albumin overload also resulted in a dose-dependent upregulation of Fas and Fas-associated protein with death domain (FADD), and activation of caspase 8. Incubation with the caspase 8 inhibitor IETD ameliorated the albumin-induced apoptosis. Collectively, our results indicate that albumin overload induces apoptosis of cultured LLC-PK(1) cells, mediated at least in part by the Fas-FADD-caspase 8 pathway.

Single-stranded nucleic acid-binding protein, Pur alpha, interacts with RNA homologous to 18S ribosomal RNA and inhibits translation in vitro.

Pur alpha is a highly conserved, eukaryotic sequence-specific DNA- and RNA-binding protein involved in diverse cellular and viral functions including transcription, replication, and cell growth. Pur alpha exerts its activity in part by interacting with other viral and cellular proteins. One such protein is the human immunodeficiency virus (HIV) type I regulatory protein Tat. Earlier studies have demonstrated that this interaction is mediated by Pur alpha-associated RNA (PARNA) and that RNA immunopurified from mammalian expressed Pur alpha was capable of reconstituting the interaction between these two proteins. In the current study, we characterize four RNA species which were immunopurified with Pur alpha. Northern blot analysis with one of the PARNAs revealed a highly abundant signal of approximately 2.0 kilobases (kb) present in all cell lines tested. Sequence analysis of each of the four PARNA clones revealed a high homology to different regions of the human 18S ribosomal RNA sequence. Based on this homology, we investigated the influence of Pur alpha on translation. Luciferase assays were performed after coupled in vitro transcription/translation reactions with a vector containing a luciferase reporter construct and increasing concentrations of BSA, GST, and GST-Pur alpha. Inclusion of GST-Pur alpha in these reactions resulted in a dose-dependent inhibition of luciferase activity. Similar inhibition was observed with in vitro translation reactions performed with in vitro transcribed luciferase RNA and increasing concentrations of GST-Pur alpha. In control experiments, inclusion of increasing concentrations of GST-Pur alpha with luciferase protein resulted in no effect on luciferase activity. Taken together, these data demonstrate that Pur alpha inhibits translation reactions in vitro. Moreover, this Pur alpha-mediated inhibition of translation can be abrogated by HIV-1 Tat protein.

Maillard reactions by α-oxoaldehydes: detection of glyoxal-modified proteins

Proteins can be chemically modified by sugars by glycation, or the Maillard reaction. The Maillard reaction produces irreversible adducts on proteins that are collectively known as advanced glycation end products, or AGEs. Recent studies indicate that several α-dicarbonyl compounds, including glyoxal (GXL), are precursors of AGEs in vivo. We developed antibodies against a GXL-modified protein (GXL-AGE) and purified a mixture of GXL-AGE-specific antibodies by chromatography on GXL-modified bovine serum albumin (BSA-GXL) coupled to EAH-Sepharose. This preparation was then processed on a human serum albumin-carboxymethyllysine (HSA-CML)–NHS-Sepharose to remove CML-specific antibodies. We used the resulting purified antibody in a competitive ELISA to probe GXL-AGEs in vitro and in vivo. We found increasingly greater antibody binding with increasing concentrations of GXL-modified BSA, but the antibody failed to react with either free CML or protein-bound CML. Incubation experiments with BSA revealed that glyceraldehyde, ribose and threose could be precursors of GXL-AGEs as well. Experiments in which GXL was incubated with N-α-acetyl amino acids showed that the antibody reacts mostly with lysine modifications. The GXL-derived lysine–lysine crosslinking structure, GOLD was found to be one of the antigenic epitopes for the antibody. Analysis of human plasma proteins revealed significantly higher levels of GXL-AGE antigens in type II diabetic subjects compared with normal controls ( P<0.0001). We also found GXL-AGEs in human lens proteins. Bovine aortic endothelial cells cultured for 7 days with 30 mM glucose did not accumulate intracellular GXL-AGEs. These studies underscore the importance of GXL for extracellular AGE formation (except in lens where it is likely to be formed intracellularly) and suggest that changes associated with age and diabetes might be prevented by alteration of GXL-AGE formation.

Effects of Pasture Allowance on the Yield and Composition of Milk from Cows of Different β-Lactoglobulin Phenotypes

The objective of this study was to determine whether the differences in the composition of milk from cows of different β-lactoglobulin (β-LG) phenotypes are affected by the amount of pasture available and, hence, pasture dry matter intake. Twenty-two Friesian cows of each of the AA and BB variants of the β-LG phenotype were subjected to ad libitum grazing or restricted grazing in crossover experiments during spring (early lactation, ∼60 d in milk) and summer (mid to late lactation, ∼180 d in milk). Milk samples were collected from each cow at the end of each 8-d treatment period and analyzed for composition. Cows of the AA variant of the β-LG phenotype had higher concentrations of whey protein and β-LG, but lower concentrations of casein (CN), α-CN, κ-CN (summer only), and BSA, than cows of the BB variant. Compared with cows with a restricted allowance, cows grazing ad libitum had higher milk yields and concentrations of protein, casein, whey protein, and all individual proteins except BSA and immunoglobulin. There were no interactions between effects of pasture allowance and phenotype on milk yield or composition. The data show that having adequate pasture for grazing cows is important not only to maximize milk yield, but also to optimize concentrations of protein and casein, and hence the manufacturing potential of milk. Further, the differences in composition of milk from cows of differing β-LG phenotypes persisted during short-term restrictions in pasture allowance, and between spring and summer.

Generation of Complement Fragment C5a in Milk is Variable among Cows

The appearance of chemotactic fragments of complement at sites of infection is an important component of innate immunity. The contribution of C5a, the most biologically active complement fragment, to the recruitment of phagocytes in milk is not well defined, in particular the amount of C5a that is released in normal milk before inflammation. The generation of C5a in normal milk upon activation of complement by invading bacteria depends on the amount of available C5 and on the activity of the C3/C5-convertase of the alternative pathway. Concentrations of C5 were measured in one fore and one rear uninfected quarter of 19 Holstein cows. Values were consistent within cows, but widely dispersed among cows (0.19 to 1.94% blood concentration). C5 concentrations in milk were loosely related to concentrations in blood. By comparison, the range of milk concentrations of C3 (1.4 to 4.4%, mean 2.46±0.63% of blood concentration) was narrower. Two groups of six cows with high milk concentrations of C5 (cows H5: mean = 1.31%) and six cows with low milk concentrations of C5 (cow L5: mean = 0.21%) were constituted for further analysis of complement activation. There was a positive correlation between concentrations in milk of BSA and C5, but not between concentrations of BSA and C3. The activities of the C3- and C5-convertases were assessed through the deposition on complement activating bacteria (Streptococcus agalactiae) of C3 and C5 fragments, respectively. The deposition of C3 was 1.7-fold higher, and the deposition of C5 was 2.75-fold higher in milk from H5 cows than in milk of L5 cows. Higher concentrations of C5 and better functioning of C5-convertase were mirrored by a much higher concentration of C5a in milk from H5cows (12.30ng/ml) than in milk of L5 cows (0.76ng/ml) after activation of complement with zymosan. These results indicate that cows differed widely in their capacity to generate C5a in milk before inflammation, and that milk C5 concentrations were a primary limiting factor for C5a generation. Cows with the lowest milk concentrations of C5 are likely unable to use the complement system for initial recruitment of leukocytes.

Fertilizing Potential of Mouse Spermatozoa Cryopreserved in a Medium Containing Whole Eggs

Experiments were conducted to improve survival of mouse spermatozoa through the cryopreservation process. In the first experiment, percentages of motile spermatozoa and fertilizing capacities of spermatozoa were evaluated when mouse spermatozoa were cryopreserved using three previously reported cryopreservation media: (1) 18% raffinose in 3% skim milk; (2) Tes/Tris medium containing 25% egg yolk and 1.25% glycerol; and (3) PBS containing 18% raffinose and 1.75% glycerol, each at three different cooling rates (−3, −10, and −50°C/min). Spermatozoa frozen in the skim milk/raffinose medium exhibited the highest percentage of motile spermatozoa (39%) when cells were frozen at −10°C/min (P < 0.05). The second experiment evaluated the effects of modifying the Tes/Tris/egg yolk medium, comparing different concentrations of egg yolk, BSA, and sodium dodecyl sulfate. Reducing egg yolk from 25% of the medium volume to 5%, increased percentages of motile spermatozoa after cryopreservation from 29 to 36% (P < 0.05). Addition of 1% BSA and sodium dodecyl sulfate to medium containing 5% egg yolk further improved percentages of motile spermatozoa after freezing. In the final experiment, 20% whole egg was substituted for 5% egg yolk and 1% BSA used in previous experiments and resulted in percentages of motile spermatozoa (51%) equal to that of the skim milk–raffinose medium. However, fertility rates were higher (68%) than for spermatozoa frozen in the skim milk–raffinose medium (P < 0.05) and were comparable to the fertility rates of fresh spermatozoa (77%; P > 0.05). In conclusion, freezing mouse spermatozoa in a medium containing 20% whole egg, 0.035% sodium dodecyl sulfate, and 1.25% glycerol using a cooling rate of −10°C/min preserves the motility and fertilization capacity of mouse spermatozoa.

Effects of atropine on the concentration and composition of milk protein in dairy cows

AbstractThe objective of this study was to measure the effects of varying doses of atropine on the concentration and composition of milk protein and on blood α-amino N levels. Four treatments were administered to each of 12 cows over 12 days in a replicated Latin square experiment. There were at least 2 days between each of 4 treatment days. Treatments were: control (C; saline); low dose (L; 30mg atropine/kg LWT); medium dose (M; 40mg atropine/kg LWT); and 2 x low dose (2L). All treatments were administered via subcutaneous injection immediately after the morning milking; the second dose of the 2L treatment was given two hours later. Milk was sampled from each cow at the morning milking (time 0 h). Cows were then milked again 2, 6 and 10 h after treatment, and milk samples again collected. Blood samples were drawn from the coccygeal vein of each cow after each milking. Atropine decreased milk secretion at 6 h for the 2L treatment and 10 h for all treatments. Atropine reduced concentrations of milk protein and casein at 2 h and 6 h, but not at 10 h. Concentrations of whey proteins and of α-casein were depressed by atropine only at 6 h post-treatment, while a reduction in α-lactalbumin due to atropine was observed at 6 and 10 h post-treatment. In contrast, atropine increased concentrations of IgG and BSA at 6 h and 10 h post-treatment. Atropine also increased the ratio of casein:total protein at 6 h after injection. There was no difference between the effects of the low and medium doses of atropine, but the double low dose induced effects which were greater than for the single doses. Effects of a single dose of atropine were greatest for most milk proteins at 6 h post treatment; thus this would be the most useful milk sampling time for future experiments. Atropine did not significantly affect α-amino N concentrations in whole blood, although there was a trend for a reduction for all treatments at 2 h after treatment. Atropine may be useful for reducing milk protein concentrations and circulating levels of certain blood amino acids to base levels, during studies designed to elucidate the effects of perturbations in the blood amino acid profile on milk protein composition.

Effect of Peptide Loading and Surfactant Concentration on the Characteristics of Physically Crosslinked Hydrogel

The purpose of this study was to investigate the effect of varying drug load and concentration of a surfactant (sodium lauryl sulfate [SLS]) on the release characteristics of a model peptide (bovine serum albumin [BSA]), and study the net effects of the swelling properties of the hydrogel matrix [poly(vinyl alcohol) (PVA)]. The PVA hydrogel was prepared by a freeze-thaw process in the absence of a chemical crosslinking agent. The effect of protein loading on drug release was examined at three levels (0.65, 1.3, and 2%), whereas the effect of SLS was studied at four levels (0, 0.07, 0.13, and 0.26%). The baseline time for reaching equilibrium swelling was 48 hr for the hydrogel containing 0.65% BSA, and the equilibrium swelling time decreased significantly as the protein load was increased to 2%. The net effect of increased BSA concentrations resulted in faster BSA dissolution from the hydrogel matrix. The equilibrium-swelling ratio decreased from 21 to 10% when SLS was added to the PVA solution, which resulted in a reduction in the extent of equilibrium swelling; however, the time to reach equilibrium swelling was increased. The investigation provided a mechanistic basis toward the development of a hydrogel formulation by altering the concentration of two fundamental components, i.e., drug and surfactant, within the delivery system.

Cholesterol Efflux-Mediated Signal Transduction in Mammalian Sperm: Cholesterol Release Signals an Increase in Protein Tyrosine Phosphorylation during Mouse Sperm Capacitation

We previously demonstrated that mouse sperm capacitation is accompanied by a time-dependent increase in protein tyrosine phosphorylation that is dependent on the presence of BSA, Ca2+, and NaHCO3, all three of which are also required for this maturational event. We also demonstrated that activation of protein kinase A (PK-A) is upstream of this capacitation-associated increase in protein tyrosine phosphorylation. BSA is hypothesized to modulate capacitation through the removal of cholesterol from the sperm plasma membrane. In this report, we demonstrate that incubation of mouse sperm medium containing BSA results in a release of cholesterol from the sperm plasma membrane to the medium; release of this sterol does not occur in medium devoid of BSA. We next determined whether cholesterol release leads to changes in protein tyrosine phosphorylation. Blocking the action of BSA by adding exogenous cholesterol-SO−4 to the BSA-containing medium inhibits the increase in protein tyrosine phosphorylation as well as capacitation. This inhibitory effect is overcome by (1) the addition of increasing concentrations of BSA at a given concentration of cholesterol-SO−4 and (2) the addition of dibutyryl cAMP plus IBMX. High-density lipoprotein (HDL), another cholesterol binding protein, also supports the capacitation-associated increase in protein tyrosine phosphorylation through a cAMP-dependent pathway, whereas proteins that do not interact with cholesterol have no effect. HDL also supports sperm capacitation, as assessed by fertilization in vitro. Finally, we previously demonstrated that HCO−3 is necessary for the capacitation-associated increase in protein tyrosine phosphorylation and demonstrate here, by examining the effectiveness of HCO−3 or BSA addition to sperm on protein tyrosine phosphorylation, that the HCO−3 effect is downstream of the site of BSA action. Taken together, these data demonstrate that cholesterol release is associated with the activation of a transmembrane signal transduction pathway involving PK-A and protein tyrosine phosphorylation, leading to functional maturation of the sperm.

Bovine immunoglobulin G, beta-lactoglobulin, alpha-lactalbumin and serum albumin in colostrum and milk during the early post partum period.

Colostrum and milk samples from 60 Holstein-Friesian cows were analysed for concentrations and yields of immunoglobulin G (IgG), beta-lactoglobulin (beta-lg), alpha-lactalbumin (alpha-la) and serum albumin (BSA) throughout the first 16 milkings post partum (8 d of lactation) using a single radial immunodiffusion assay. Concentrations (mg/ml, means +/- SD) at first milking were IgG 59.8 +/- 28.5, beta-lg 14.3 +/- 4.6, alpha-la 2.04 +/- 0.6, BSA 1.21 +/- 0.44. Large variations were recorded for IgG concentrations (15.3-176.2 mg/ml) and yields (0.2-925 g). Cows in their first lactation produced significantly lower concentrations and yields of colostral IgG than cows in later lactations. A colostral yield of IgG below the 100 g required to prevent calf hypo-gamma-globulinaemia was found in 18.3% of the cows. The concentrations of IgG, beta-lg and BSA dropped abruptly in subsequent milkings and alpha-la concentration decreased slowly. The mean IgG concentration was < 2 mg/ml after eight milkings and < 1 mg/ml after fifteen milkings. However, IgG concentration did not differ significantly, at the 1% level, during milkings 11-15. The results were tabulated to make it possible to calculate the excess of whey proteins that would be obtained if early milks were illegally added to the milk supply.

Direct observation of the self-association of dilute proteins in the presence of inert macromolecules at high concentration via tracer sedimentation equilibrium: theory, experiment, and biological significance.

The technique of tracer sedimentation equilibrium [Rivas, G., et al. (1994) Biochemistry, 2341-2348 (1); Rivas, G., et al. (1996) J. Mol. Recognit. 9, 31-38 (2)] is utilized, together with an extension of the theory of sedimentation equilibrium of highly nonideal solutions [Chatelier and Minton, (1987) Biopolymers 26, 1097-1113 (3)], to characterize the thermodynamic activity and/or the state of association of a dilute, labeled macromolecular solute in the presence of an arbitary concentration of a second, unlabeled macromolecular solute. Experiments are performed on solutions of labeled fibrinogen (0.25-1 g/L) in bovine serum albumin (0-100 g/L) in the presence and absence of divalent cations (Ca(2+), Mg(2+)), and on solutions of labeled tubulin (0.2-0.6 g/L) in dextran (0-100 g/L). It is found that in the absence of the divalent cations, the large dependence of the thermodynamic activity of fibrinogen on BSA concentration is well accounted for by a simple model for steric repulsion. In the presence of the cations and sufficiently large concentrations of BSA (>30 g/L), fibrinogen appears to self-associate to a weight-average molar mass approximately twice that of monomeric fibrinogen. Tubulin appears to self-associate to an extent that increases monotonically with increasing dextran concentration, reaching a weight-average molar mass almost 3 times that of the alphabeta dimer in the presence of 100 g/L dextran. Possible biological ramifications are discussed.

Development of monoclonal ELISAs for azinphos-methyl. 2. Assay optimization and water sample analysis.

Two enzyme-linked immunosorbent assays (ELISA) for the insecticide azinphos-methyl have been optimized and characterized. Both ELISAs are based on monoclonal antibodies produced from an immunogen with a hapten containing a phthalimido moiety and on protein conjugates of heterologous ligands containing a 1,2,3-benzotriazine group. Assay I was performed in the conjugate-coated ELISA format and assay II in the antibody-coated format. Several physicochemical factors (ionic strength, pH, incubation times, and Tween 20 and BSA concentrations) that influence assay performance were studied and optimized. Regarding specificity, both monoclonal immunoassays highly cross-reacted with azinphos-ethyl and phosmet. Finally, both assays were applied to the analysis of azinphos-methyl in spiked real water samples. For assay I the sensitivity, estimated as the I(50) value, was 0.40 nM, with a practical working range between 0.10 and 1.75 ng/mL and a limit of detection of 0.05 ng/mL. For assay II the sensitivity was 1.01 nM, with a practical working range between 0.32 and 2.54 ng/mL and a limit of detection of 0.08 ng/mL.

Electrochemical studies of the interaction of tetraphenylporphyrin tetrasulfonate (TPPS) with albumin

An electrochemical investigation of the interaction of TPPS with BSA on a Hg electrode is reported for the first time. The addition of BSA to TPPS solution results in a decrease of both the reduction and the oxidation current with no change of the peak potentials. In presence of BSA, no new peaks appear, and the standard rate constant ks is not significantly changed. The reaction of TPPS with BSA yields a kind of supramolecular complex TPPS-BSA, which is electrochemically non-active. The equilibrium constant for the complex has been calculated. The decrease of the peak current can be used to determine BSA concentrations.

A multi point conductivity measurement system for characterisation of protein foams

Protein foams play an important role in both food and biotechnological processes. A sound understanding of foaming properties of proteins relevant to such processes is useful e.g. to allow adequate control of unwanted foams and appropriate choice of protein-physical system when foams of certain characteristics are required. In general, measurements of changes in foam volume (volumetric method) are used for foam characterisation. However, recently there has been increased interest in the use of measurement methods based on conductivity and capacitance. Simple relative techniques based on electrical conductivity measurements provide information on both foamability and foam stability. A multi point conductivity measurement system has been designed and used for characterisation of model protein foams (0.1 and 1.0 mg ml −1 Bovine serum albumin, BSA). The solution of BSA was sparged with nitrogen or carbon dioxide gas at constant flow rate (90 cm 3 min −1) via a stainless steel sinter (0.5 or 2.0 μm in pore size). A comparison of foaming properties determined by volumetric and conductimetric techniques is provided. Both methods show that more stable foams are obtained for solutions at higher BSA concentrations. At all BSA concentrations, higher foamability and stability are achieved with a smaller sinter pore size. When nitrogen rather than carbon dioxide is used as a dispersed phase, higher foamability and foam stability are obtained. The conductivity measurements indicate that foamability is dependent on gas type, whereas, volumetric measurements do not show such differences.