Average Isoelectric Point Research Articles

Explorando el impacto del extracto de bellota en la calidad y sabor de las hamburguesas de carne de res

Protein microparticles are of interest in the development of low-fat foods as stabilizing agents and, for their production, it is important to consider the influence of the processing conditions on stability and size of particles. In the present study, the characteristics of microparticles of soy protein isolate (SPI) obtained by microfluidization (MF) (110 MPa and 3 MF cycles) of aggregates obtained by heat treatment (95 °C for 15 min) and different pH values (3 , 5, 7 and 9). The results of particle size, zeta potential and turbiscan stability index (TSI) showed that the least stable microparticles in suspension were obtained at pH 5, due to their proximity to the average isoelectric point of soy proteins (4.5). In contrast, the most stable microparticles in suspension were obtained at pH 9, presenting small particle sizes (398.35 ± 4.19nm) and a low TSI (0.8 ± 0.08). By means of atomic force microscopy (AFM) it was possible to observe the breakdown of the aggregates for the formation of microparticles during the microparticulation process. Surface roughness and image texture analyses allowed evaluating the effect of MF on the surface of the microparticles. The most relevant descriptor for the characterization of microparticlers was the second angular moment whereas contrast, correlation, inverse difference moment, entropy, and fractal dimension did not correlate with the characteristics of the images in the whole range of operating conditions given that their calculation algorithms are not sensitive enough to detect the small variations in roughness of the samples. The best conditions for production of microparticels were 3 cycles of MF and pH 9 which gave place to homogenous and stable systems.

Obtention and evaluation of physico-chemical and techno-functional properties of macauba (Acrocomia aculeata) kernel protein isolate

The consumption of plant proteins is increasing worldwide. These proteins have an important role in human nutrition as well as in the technological properties of foods. Thus, there is a great interest in exploring new sources of plant proteins, such as macauba (Acrocomia aculeata), which is a promising tropical palm tree, native to Brazil, whose fruits are rich in oil, proteins and dietary fiber. Hence, the objective of this work was to obtain and evaluate the physico-chemical and techno-functional properties of the macauba kernel protein isolate (MKPI). Defatted macauba kernel flour was obtained and used to produce the MKPI by isoelectric precipitation. Then, the proximate composition, amino acid profile, and physico-chemical and techno-functional properties of the MKPI were determined. The MKPI stood out for its high protein content (94.9%) and high levels of arginine (16.21%) and glutamate (20.84%). The MKPI average isoelectric point was at pH 4.9 and its proteins showed low solubility in the pH range from 4.0 to 6.0. Moreover, the hydrophobicity of MKPI proteins was higher at pH 3.5 than at pH 7.0, and they had higher oil holding capacity (153.77%) than water holding capacity (97.29%). Regarding the MKPI emulsifying and gelling properties, emulsions with 0.5% and 1.0% of MKPI remained stable during storage and the minimum gelling concentration was 14%. Thus, the MKPI has a great potential to be produced and used by the food industry due to its nutritional and techno-functional properties.

Nucleotide and protein researches on anaerobic fungi during four decades.

Anaerobic fungi habitat in the gastrointestinal tract of foregut fermenters or hindgut fermenters and degrade fibrous plant biomass through the hydrolysis reactions with a wide variety of cellulolytic enzymes and physical penetration through fiber matrix with their rhizoids. To date, seventeen genera have been described in family Neocallimasticaceae, class Neocallimastigomycetes, phylum Neocallimastigomycota and one genus has been described in phylum Neocallimastigomycota. In National Center for Biotechnology Information (NCBI) database (DB), 23,830 nucleotide sequences and 59,512 protein sequences have been deposited and most of them were originated from Piromyces, Neocallimastix and Anaeromyces. Most of protein sequences (44,025) were acquired with PacBio next generation sequencing system. The whole genome sequences of Anaeromyces robustus, Neocallimastix californiae, Pecoramyces ruminantium, Piromyces finnis and Piromyces sp. E2 are available in Joint Genome Institute (JGI) database. According to the results of protein prediction, average Isoelectric points (pIs) were ranged from 5.88 (Anaeromyces) to 6.57 (Piromyces) and average molecular weights were ranged from 38.7 kDa (Orpinomyces) to 56.6 kDa (Piromyces). In Carbohydrate-Active enZYmes (CAZY) database, glycoside hydrolases (36), carbohydrate binding module (11), carbohydrate esterases (8), glycosyltransferase (5) and polysaccharide lyases (3) from anaerobic fungi were registered. During four decades, 1,031 research articles about anaerobic fungi were published and 444 and 719 articles were available in PubMed (PM) and PubMed Central (PMC) DB.

Acid-shifted isoelectric point profiles of the proteins in a hypersaline microbial mat: an adaptation to life at high salt concentrations?

In a metagenomic analysis of a stratified hypersaline (9 % salt) microbial mat in Guerrero Negro, Mexico, Kunin et al. (Mol Systems Biol 4:198, 2008) detected a significantly acid-shifted proteome, and concluded that adaptation by enriching proteins with acidic amino acids is more widespread than previously assumed. We here reevaluate these data and conclusions by comparing the isoelectric point profiles of the Guerrero Negro microbial mats (average isoelectric point 6.8) with those of the proteins encoded by the genomes of prokaryotes adapted to different salt concentrations ranges and belonging to different phylogenetic and physiological groups. Average isoelectric points below 6.8 were found not only in the proteomes of the moderately halophilic aerobic bacteria Halomonas elongata and Chromohalobacter salexigens, but even in common types of marine bacteria of the genera Alteromonas and Aliivibrio. We did not find clear evidence that the isoelectric point profile of the Guerrero Negro microbial mat can be considered to be the result of species-independent molecular convergence of the members of the microbial community determined by the salinity of the overlaying brine.

Effect of charge on microvascular permeability in early experimental sepsis in the rat

A key feature of sepsis is hypovolemia due to increased microvascular permeability. It has been suggested that the negative charge of albumin and of the endothelial glycocalyx is important for maintenance of the normally low permeability for albumin. Here we tested the hypothesis that charge effects contribute to the increased permeability in sepsis. Transcapillary escape rate (TER) and initial distribution volume for 125I-labeled bovine serum albumin (BSA, isoelectric point pH 4.6) and for 131I-labeled charge modified BSA (cBSA, average isoelectric point, pH 7.1) was measured 3h after sepsis was induced by cecal ligation and incision (CLI) (n=11) and in control animals (n=12). The importance of charge for permeability in sepsis was estimated by comparing the ratio between TER for cBSA and TER for BSA during control conditions to that after CLI. Plasma concentration of the glycocalyx component glycosaminoglycans (GAGs) was measured in separate control and CLI animals. The initial distribution volume for BSA and cBSA in control animals was 38±3ml/kg and 47±4mL/kg and decreased by 17% and 19%, respectively, following CLI. TER for BSA increased from 16.7±4.1% in the controls to 20.1±1.9% following CLI. Corresponding values for cBSA were 26.7±5.6% and 29.8±3.5%, respectively. The ratio between TER for cBSA and TER for BSA was 1.62±0.1 in the control group and 1.49±0.1 following CLI (p<0.05). Plasma GAG concentrations were higher in CLI animals than in the control group. We conclude that CLI induce hypovolemia secondary to increased microvascular permeability. Negative charge contributes to the normally low permeability of albumin and the importance of charge is decreased in early experimental sepsis. The observed charge effects are associated with CLI-induced breakdown of the glycocalyx.

Different IGH Rearrangement and Somatic Hypermutation Patterns in Hairy-Cell Leukemia, Hairy-Cell Leukemia Variant and Splenic Marginal Zone Lymphoma.

Hairy cell leukemia (HCL) and its variant form (HCL-v) are rare B-cell disorders, with different and distinct morphology, immunophenotype, clinical behaviour and response to treatment. HCL-v patients do not respond to purine analogues and have a median survival of seven years compared to over 20 years in HCL, a disease potentially curable with purine analogues in up to 80% of cases. Despite these differences, the resemblance between these two malignancies and splenic marginal zone lymphoma (SMZL) has led to the suggestion that they all may share a common clonal progenitor that homes within the marginal zone of the splenic white pulp. Since the study of immunoglobulin heavy chain gene (IGH) rearrangements provides crucial information regarding the differentiation stages at which particular B-cells are transformed, we have investigated the configuration of the IGH genes in a series of well defined HCL (n=17), HCL-v (n=28) and SMZL (n=82) samples. VDJH rearrangements were PCR-amplified and sequenced using an automated 3130xL sequencer (ABI). The sequences obtained were compared to the closest germline segments using the IMGT database (http://imgt.cines.fr) in order to study the level of somatic hypermutation as well as gene segment usage and CDR3 composition in IGH rearrangements. We were able to amplify a functional VDJH rearrangement in all 127 cases. A significant proportion of SMZL (31/82; 38%) and HCL-v (7/28; 25%) cases were found to be unmutated (range: 0–1.8%). In contrast, all but one HCL cases 16/17 (94%) showed more than 2% deviation from the germline sequence. This suggests that HCL-v is more similar to SMZL than HCL with respect to IGH mutational status. There was a significant over-representation of the VH1 family in the SMZL cases (38%; p<0.01). Regarding specific VH gene segment usage, VH1-02 and V4-34 were significantly over-represented in the SMZL group (28% and 12%, respectively; p<0.001). This was especially striking in the unmutated SMZL cases, the majority (20/31; 64%) using either V4-34 or V1-02. Regarding specific VH segment usage, those SMZL cases carrying a VH1-02 rearrangement showed a similar CDR3 length and composition, with an average isoelectric point of 10.5, and 43% of these cases associated with DH3-03 usage. Within the HCL-v group, there was a significant over-representation of the VH4 family (39%; p<0.001). VH4-34 and VH4-59 were significantly over-represented (18% and 11%, respectively; p<0.001), and mainly restricted to the unmutated cases, 71% of them using VH4-34 or VH4-59. In contrast, there was no significant over-representation of any VH family or gene segment in the typical HCL cases.CONCLUSIONS: Our findings show that in SMZL specific VDJH stereotypes can be identified, likely as a consequence of antigen interaction, which are not present in HCL or HCL-v. Furthermore, HCL-v shares more similarities with SMZL, in terms of IGH rearrangement and somatic hypermutation patterns, than with typical HCL and suggest that different processes occur in the pathogenesis of these three disorders. The clinical implications of these findings are being addressed.

Adsorption of Pathogenic Prion Protein to Quartz Sand

Management responses to prion diseases of cattle, deer, and elk create a significant need for safe and effective disposal of infected carcasses and other materials. Furthermore, soil may contribute to the horizontal transmission of sheep scrapie and cervid chronic wasting disease by serving as an environmental reservoirforthe infectious agent. As an initial step toward understanding prion mobility in porous materials such as soil and landfilled waste, the influence of pH and ionic strength (l) on pathogenic prion protein (PrPsc) properties (viz. aggregation state and zeta-potential) and adsorption to quartz sand was investigated. The apparent average isoelectric point of PrPsc aggregates was 4.6. PrPsc aggregate size was largest between pH 4 and 6, and increased with increasing l at pH 7. Adsorption to quartz sand was maximal near the apparent isoelectric point of PrPsc aggregates and decreased as pH either declined or increased. PrPsc adsorption increased as suspension l increased, and reached an apparent plateau at l approximately 0.1 M. While trends with pH and l in PrPsc attachment to quartz surfaces were consistent with predictions based on Born-DLVO theory, non-DLVO forces appeared to contribute to adsorption at pH 7 and 9 (l = 10 mM). Our findings suggest that disposal strategies that elevate pH (e.g., burial in lime or fly ash), may increase PrPsc mobility. Similarly, PrPsc mobility may increase as a landfill ages, due to increases in pH and decreases in l of the leachate.

Analysis of curated and predicted plastid subproteomes of Arabidopsis. Subcellular compartmentalization leads to distinctive proteome properties.

Carefully curated proteomes of the inner envelope membrane, the thylakoid membrane, and the thylakoid lumen of chloroplasts from Arabidopsis were assembled based on published, well-documented localizations. These curated proteomes were evaluated for distribution of physical-chemical parameters, with the goal of extracting parameters for improved subcellular prediction and subsequent identification of additional (low abundant) components of each membrane system. The assembly of rigorously curated subcellular proteomes is in itself also important as a parts list for plant and systems biology. Transmembrane and subcellular prediction strategies were evaluated using the curated data sets. The three curated proteomes differ strongly in average isoelectric point and protein size, as well as transmembrane distribution. Removal of the cleavable, N-terminal transit peptide sequences greatly affected isoelectric point and size distribution. Unexpectedly, the Cys content was much lower for the thylakoid proteomes than for the inner envelope. This likely relates to the role of the thylakoid membrane in light-driven electron transport and helps to avoid unwanted oxidation-reduction reactions. A rule of thumb for discriminating between the predicted integral inner envelope membrane and integral thylakoid membrane proteins is suggested. Using a combination of predictors and experimentally derived parameters, four plastid subproteomes were predicted from the fully annotated Arabidopsis genome. These predicted subproteomes were analyzed for their properties and compared to the curated proteomes. The sensitivity and accuracy of the prediction strategies are discussed. Data can be extracted from the new plastid proteome database (http://ppdb.tc.cornell.edu).

Global analysis of the Brucella melitensis proteome: Identification of proteins expressed in laboratory-grown culture.

Brucella melitensis is a facultative intracellular bacterial pathogen that causes brucellosis, a zoonotic disease primarily infecting sheep and goats, characterized by undulant fever, arthritic pain and other neurological disorders in humans. A comprehensive proteomic study of strain 16M was conducted to identify and characterize the proteins expressed in laboratory-grown culture. Using overlapping narrow range immobilized pH gradient strips for two-dimensional gel electrophoresis, 883 protein spots were detected between pH 3.5 and 11. The average isoelectric point and molecular weight values of the detected spots were 5.22 and 46.5 kDa, respectively. Of the 883 observed protein spots, 440 have been identified by matrix-assisted laser desorption/ionization-mass spectrometry. These proteins represent 187 discrete open reading frames (ORFs) or 6% of the predicted 3197 ORFs contained in the genome. The corresponding ORFs of the identified proteins are distributed evenly between each of the two circular B. melitensis chromosomes, indicating that both replicons are functionally active. The presented proteome map lists those protein spots identified to date in this study. This map may serve as a baseline reference for future proteomic studies aimed at the definition of biochemical pathways associated with stress responses, host specificity, pathogenicity and virulence. It will also assist in characterization of global proteomic effects in gene-knockout mutants. Ultimately, it may aid in our overall understanding of the cell biology of B. melitensis, an important bacterial pathogen.

Element of a validation method for MU-B3 monoclonal antibody using an imaging capillary isoelectric focusing system.

This paper presents an imaging capillary isoelectric focusing (CIEF) assay for the determination of the identity, stability, and isoform distribution of a murine monoclonal antibody (MU-B3). The experiments were conducted using a Convergent Bioscience iCE280 instrument. The optimum carrier ampholyte composition that gave the best peak separation was found to be 25% Pharmalyte pH 3-10 and 75% Pharmalyte pH 5-8. The antibody gave a highly reproducible CIEF profile with three major peaks having average isoelectric point (pI) values of 6.83, 6.99, and 7.11. Intraday and interday reproducibility of pI values was found to be within RSD of 0.5%. The CIEF profile was also the same, with an alternate column cartridge and alternate batches of methyl cellulose. A plot of peak areas versus MU-B3 concentration was linear (R2 = 0.995) up to a concentration of 0.5 mg/mL in the sample solution. Peak area measurements were reproducible to within 7% RSD. The CIEF profiles of two other antibodies were distinctly different from the profile of MU-B3, showing that the assay is specific. After a sample of MU-B3 was subjected to heat stress by exposure to heat at 55 degrees C for 4 h, its CIEF profile was altered with extra peaks appearing at lower pI values, indicating that the assay could be used to monitor stability. The result of the heat stress experiment was also confirmed with a parallel slab-gel IEF analysis of the antibody sample before and after application of the heat stress. The results of this work suggest that imaging CIEF can be used for product testing under a quality control environment. The assay can be used for pI profiling of proteins and for monitoring structural changes (deamidation, glycosylation, etc.) during the manufacturing process and upon storage.

Homologous nuclear-encoded mitochondrial and cytosolic isoproteins. A review of structure, biosynthesis and genes.

Mitochondrial and cytosolic proteins may be expected to differ in specific traits due to their different intracellular location. However, the identification of these differences between mitochondrial and cytosolic proteins is complicated by the heterogeneity of the two protein groups. These difficulties have been overcome by comparing traits of homologous genes, which are derived from a common ancestor gene, and their gene products. An earlier report [Hartmann, C., Christen, P. & Jaussi, R. (1991) Nature 352, 762-763] describing a positive net charge difference between the mature parts of nuclear-encoded mitochondrial proteins and their homologous cytosolic isoproteins, could be corroborated by extending the data collection. New data were gathered from computer databases and published studies. The average isoelectric points of the mitochondrial and cytosolic isoproteins are 7.5 and 6.5, respectively. Depending on the type of protein, the observed difference results from differences in the number of basic and/or acidic amino acid residues in the isoproteins. Probably both the conditions required for mitochondrial protein import and the local conditions within the organelle furthered the evolution of basic protein structures. The contribution of the mitochondrial targeting peptide to the positive charge of precursors of nuclear-encoded mitochondrial proteins is largest when the value of the isoelectric point of the mature protein is small. This mutual dependence of the charge of the targeting peptide and the mature protein part supports the notion that positive charge is essential for mitochondrial protein import. Several traits other than electric charge, i.e. codon usage, chromosome location, structural organization or regulation of the genes, do not show specific differences between the sets of the heterotopic isoproteins. There is no preference of gene location for any of the gene sets; only rarely are the genes for a mitochondrial and a cytosolic isoprotein located on the same chromosome. A variant of the 3' splice-site consensus exists in genes of nuclear-encoded mitochondrial proteins. This is most likely a consequence of the evolution of the genes in separate lineages before endosymbiosis led to the formation of mitochondria. Some of the original mRNA group II intron self-splicing functions of the endosymbiont seem to persist in part of the cytosolic splicing machinery and apparently require a specific consensus sequence [Juretic, N., Jaussi, R., Mattes, U. & Christen, P. (1987) Nucleic Acids Res.15, 10083-10086].

Study of the adsorption of F(ab') 2 onto polystyrene latex beads

An experimental investigation on the adsorption of F(ab') 2 from rabbit IgG onto polystyrene (PS) latex beads is described. All adsorption isotherms were of high affinity and showed well-defined plateaus. Maximum protein adsorption was found around the average isoelectric point (IEP) of the dissolved protein. According to the findings, the F(ab') 2 adsorption on the polystyrene surface is strongly irreversible with respect to ionic strength changes. The pH changes, however, exert a certain effect on the adsorption-desorption process of F(ab') 2 on negatively charged polystyrene surfaces. In order to determine the role played by the electrostatic forces in the F(ab') 2 adsorption onto negatively charged latex particles, an electrokinetic study of the protein-latex complexes has also been carried out. The isoelectric pH of the F(ab') 2-PS complexes is always smaller than the IEP of the dissolved F(ab') 2, indicating that the PS surface charge must partly compensate the positive charge on the protein. Finally, a comprehensive study on the colloidal stability of the sensitized latex beads was performed.

Purification and characterization of zyxin, an 82,000-dalton component of adherens junctions.

We describe here the purification and characterization of a recently identified adherens junction protein that has an apparent molecular mass of 82 kDa on sodium dodecyl sulfate-polyacrylamide gels (Beckerle, M. C. (1986) J. Cell Biol. 103, 1679-1687). The 82-kDa protein was isolated from avian smooth muscle by a low ionic strength alkaline pH extraction followed by ammonium sulfate fractionation. Sequential chromatographic separation using DEAE-cellulose, phenyl-Sepharose CL-4B, and hydroxylapatite resins results in a purified 82-kDa protein. The 82-kDa protein has a Stokes radius of 5.6 nm and a relative sedimentation coefficient of 3.0 S. The calculated native molecular mass of the protein based on its hydrodynamic properties is 69 kDa, and the derived frictional ratio (f/fo) is 2.1. The protein does not focus discretely by isoelectric-focusing-sodium dodecyl sulfate-polyacrylamide gel electrophoresis; there are numerous isoelectric point variants in the range of 6.4-7.2, with the average isoelectric point being 6.9. The 82-kDa protein is phosphorylated in vivo and appears to be a cytoplasmic component of adherens junctions. The properties of the 82-kDa protein distinguish it from other known adherens junction proteins of this molecular mass. In fibroblasts, the 82-kDa protein is found in adhesion plaques as well as along actin-containing stress fibers near where they terminate at sites of cell-substratum adhesion. It is also found in the cell-cell adherens junctions of pigmented retinal epithelial cells and the dense plaques of smooth muscle cells. Since the 82-kDa protein is found at both cell-substratum and cell-cell adherens junctions, we propose to call it zyxin, meaning a joining, to indicate that it is found at regions where extracellular ligands are structurally and functionally joined to the cytoskeleton.

Storage protein accumulation during zygotic and somatic embryo development in Picea abies (Norway spruce)

Total protein was extracted from zygotic embryos and from somatic embryos of Picea abies (L.) Karst. (Norway spruce) cultured in vitro at different times during their development. An analysis by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and 2‐dimensional gel electrophoresis of the protein extracts showed that protein composition and the temporal changes in protein abundance were very similar in the two embryo types. Both zygotic and somatic embryos accumulated storage proteins in abundance during their maturation phase of growth; the somatic embryos when cultured on medium containing 90 mM sucrose and 7.6 μM ABA. The major storage proteins are composed of polypeptides with molecular masses of about 22, 28, 33 and 42 kDa and they are identical in both embryo types according to their molecular mass and average isoelectric points. These proteins are also the most abundant proteins in the female gametophytic tissue of the mature seed.

Characterization of a human B‐lymphocyte carcinoma cross‐reacting antigen (BLCa) in B lymphocytes identified by two murine monoclonal antibodies

The murine monoclonal antibody (MAb) MA6 is selectively reactive against a large variety of human B lymphocytes including those in early stages of B-cell differentiation such as committed progenitors of B lymphocytes, pre-B lymphocytes, Burkitt lymphoma cells, and those at later stages of differentiation such as peripheral blood B lymphocytes and myeloma cells. The major antigen identified by this antibody on such B lymphocytes (BLCa) is a 55-kDa glycoprotein or a group of similar glycoproteins, with the MA6-reactive determinant localized on the carbohydrate moiety. On isoelectric focusing, this antigen exhibits a degree of charge microheterogeneity, migrating as a diffused band with an average isoelectric point at pH 5.7. BLCa was also identified by another murine MAb, MA5. The antigenic determinant recognized by this antibody is also localized on the carbohydrate moiety of the molecule, but, unlike the MA6-reactive determinant, it is shared by other glycoproteins from different types of cells.

Heterogeneity and structure of brain tubulins from cold-adapted Antarctic fishes. Comparison to brain tubulins from a temperate fish and a mammal.

Tubulins purified from brain tissue of Antarctic fishes assemble in vitro to form microtubules at the low temperatures experienced by these extreme psychrophiles (Williams, R. C., Jr., Correia, J. J., and DeVries, A. L. (1985) Biochemistry 24, 2790-2798). We have initiated studies to determine the structural requirements for assembly of Antarctic fish tubulins at low temperatures. As a first step we have compared the heterogeneity, structures, amino acid compositions, and net charge of brain tubulins purified from three Antarctic fishes (Notothenia gibberifrons, Notothenia coriiceps neglecta, and Chaenocephalus aceratus), from the temperate channel catfish (Ictalurus punctatus), and from a mammal (the cow). Each preparation contained the alpha- and beta-tubulins and was free of microtubule-associated proteins. When examined by isoelectric focusing and by two-dimensional electrophoresis, brain tubulins from the Antarctic fishes were found to be highly heterogeneous; each was resolved into approximately 20 isoelectric variants. The distributions of the isotubulins from the cold-adapted fishes were similar but differed significantly from those of tubulins from catfish and cow. The average isoelectric points of the alpha- and beta-tubulins from the Antarctic fishes were more basic than the isoelectric points of the corresponding tubulins from bovine brain. Peptide mapping confirmed that tubulins from the Antarctic fishes and the mammal differed in structure. The amino acid compositions of fish and mammalian tubulins were similar, but Antarctic fish tubulins apparently contained fewer Glx residues than did catfish or bovine tubulins. Finally, native tubulins from an Antarctic fish and the cow differed slightly in net negative charge. Thus, brain tubulins from the cold-adapted fishes differ structurally from the tubulins of a temperate fish and of a mammal.

Altered isoelectric focusing of α 2-macroglobulin from plasma of patients with diabetes mellitus

Plasma from both type I and type II patients with diabetes mellitus displayed elevated α 2-macroglobulin trypsin-binding activity relative to normals. Column isoelectric focusing indicated that the average isoelectric point (pI) of the major form of crude α 2-macroglobulin was 6.6, 5.9, and 6.2 for normal, type I and type II subjects, respectively. Focused crude diabetic α 2-macroglobulin displayed significantly less recovered trypsin-binding activity relative to controls, particularly above a pI value of 6.0. Following preincubation with trypsin, the crude diabetic α 2-macroglobulin displayed a single isoelectric form (pI value of 5.3–5.5) and a substantial increase in recovered activity which were both comparable to normal α 2-macroglobulin. Purified α 2-macroglobulin from both normal and diabetic subjects displayed similar focusing profiles having a distribution of three forms with a pI value of 5.3–5.4 for the principal form. Trypsin-preincubated samples of purified α 2-macroglobulin displayed a single form with a pI value of 5.5–6.0. Preincubation of purified normal or diabetic α 2-macroglobulin with a plasma preparation devoid of α 2-macroglobulin from normal or diabetic plasma resulted in significantly lowered recovery of activity. All of the above studies suggest that the altered focusing properties observed for crude diabetic α 2-macroglobulin are due to components of the plasma rather than to α 2-macroglobulin itself.

The preparation and use of fluorescent-protein conjugates for microvascular research

A procedure is described for making large quantities (100 ml) of fluorochrome-labeled albumin. Chromatographic techniques are described for the purification of commercial albumin (BSA) and the purification of albumin from serum. We report experimentally determined optimal conditions for the covalent attachment of fluorescent dyes (rhodamine isothiocyanate (RITC) and fluorescein isothiocyanate (FITC)) to albumin. Subsequent removal of all unreacted fluorescent material (UFM) was achieved using charcoal adsorption. We observed no loss of protein following charcoal treatment. The final protein conjugate was analyzed by polyacrylamide gel electrophoresis, gel chromatography, and isoelectric focusing. The conjugates were determined to be free of UFM and homogeneous with respect to molecular weight. However, FITC conjugation lowered the average isoelectric point of albumin by 0.1 to 0.3 pH units. Illustrations of combining fluorescence microscopy with FITC-BSA and RITC-BSA to view microvascular phenomena in skeletal muscle and the heart are given. Knowledge of the biochemical characteristics of the fluorochrome employed is important for proper interpretation of experimental results using this technique.

Identification and characterization of gp TFA-1, a guinea pig T cell surface antigen associated with T cell function.

Monoclonal antibodies (Ab) were produced that specifically recognized guinea pig T cells. FACS analysis revealed that Ab 188 bound to the majority of peripheral T lymphocytes of strain 2 and strain 13 guinea pigs and to a minor population of thymocytes. It failed to react with the Ia-bearing guinea pig B cell leukemia line EN-L2C, with macrophages, bone marrow cells, erythrocytes, or thrombocytes. Treatment of T cells with Ab 188 and complement prevented T cell activation. Culturing primed T cells with antigen- or mitogen-pulsed syngeneic or with allogeneic macrophages in the continuous presence of Ab 188 produced a marked, dose-dependent inhibition of T cell proliferation. The antigen defined by Ab 188 was therefore designated guinea pig T lymphocyte function-associated antigen-1, gp TFA-1. The magnitude of inhibition by Ab 188 varied between 65 and 85% whereas three other antibodies to guinea pig T cells had no inhibitory effect on T cell proliferation. Time course experiments revealed that gp TFA-1 is critically involved in an early phase of T cell activation. Maximal inhibition was achieved only if the antibody was present from the beginning of the cell culture; the addition of antibody after 24 hr of culture no longer had an inhibitory effect. Ab 188 did not induce T cell mitogenesis. Two-dimensional analysis (one-dimensional, IEF; two-dimensional, SDS-PAGE) of immunoprecipitates obtained from NP40 lysates of [35S]methionine-labeled T cell blasts indicated that a molecule was specifically precipitated that consisted of two noncovalently associated polypeptide chains with apparent m.w. of 43,000 and 38,000. Both subunits displayed extensive charge heterogeneity focusing at an average isoelectric point of 5.0 and 6.5, respectively. The gp TFA-1 molecule exhibits striking similarities in its functional and structural properties to recently described clonotypically expressed T cell glycoproteins, which were shown to be involved in antigen recognition by T cells in the murine and human systems.

Flocculation of slaughterhouse effluents with aluminium salts

Abstract The use of aluminium salts to remove suspended solids and a proportion of the soluble material from slaughterhouse effluents was examined. Acidification prior to the addition of 40 mg/1 aluminium is recommended so that the final pH is approximately 5.0. Removal efficiency is greatest at a pH just above the average isoelectric point of the effluent proteins.