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Biomedical subjects

P G Righetti

Publications and source records attributed to P G Righetti.

At least 19 recordsLinked to original sources
PubMed1995

Detection of traces of a trisulphide derivative in the preparation of a recombinant truncated interleukin-6 mutein.

A new mutein of interleukin-6, called delta 22-IL-6 Cys 3,4, characterized by the deletion of the first 22 amino acids at the N-terminal end and by the substitution of the first two cysteines (Cys23 and Cys29) with serine residues, was produced in Escherichia coli and was found to maintain the structural and functional properties of the human native form. A partially purified preparation still showed in isoelectric focusing a minor acidic component (pI 6.10) and a more basic component (pI 6.70), the native form having a pI of 6.56. This preparation was further fractionated in a multi-compartment electrolyser with isoelectric membranes, which allowed the collection of the more alkaline species for characterization. Mass spectra of the pI 6.70 form gave an additional mass of 32 atomic mass units (amu), suggesting the addition of two oxygen atoms (a potential oxidation of two methionine residues to sulphoxide). However, the five methionine residues in this higher pI form were identified after enzymatic hydrolysis and peptide mapping and were found to be in a reduced state. In addition, the pI 6.70 form was quickly converted into the native form by mild reductive treatment. On digestion and fingerprinting, the peptide from residues 50 to 65 of the pI 6.70 species (containing the only two cysteine residues of the molecule) exhibited a more hydrophobic behaviour in reversed-phase high-performance liquid chromatography and retained a mass increase of 32 amu. These experimental findings more likely suggest the addition of an extra sulphur atom to the only disulphide bridge to give an unusual protein trisulphide molecule.

Amino Acid Sequence↗
PubMed1995

CAG triplet analysis in families with androgen insensitivity syndrome by capillary electrophoresis in polymer networks.

The potential use of capillary zone electrophoresis in polymer networks (linear polymers above the entanglement threshold, added to the background electrolyte for sieving purposes) for analysis of DNA fragments amplified by a polymerase chain reaction, is shown. In typical runs, the capillary is filled with Tris-borate-EDTA buffer, at pH 8.3, containing 6% linear polyacrylamide as a dynamic sieving matrix. Such formulations allow replenishing the capillary with fresh sieving solution when resolution decays after prolonged use (typically > 30 injections per capillary are obtained). The DNA fragments are detected by their intrinsic absorbance at 254 nm. This system has been applied to the analysis of CAG triplet polymorphism in families carrying the androgen insensitivity syndrome. While easy separation is obtained for fragments 139 base pairs (bp) and 160 bp (in families carrying a difference of 7 CAG repeats) even more difficult cases (such as those of families exhibiting fragments of 136 and 139 bp, thus differing by only one CAG repeat) are resolved with precision and diagnostic value.

Androgen-Insensitivity Syndrome↗
PubMed1995

Separation and quantitation of reverse transcriptase polymerase chain reaction fragments of basic fibroblast growth factor by capillary electrophoresis in polymer networks.

In human ovarian carcinomas (epithelial and endometrial tumors) the presence of mRNA coding for the basic fibroblast growth factor (bFGF) was previously demonstrated by reverse transcription-polymerase chain reaction (PCR). For quantitation purposes, competitive PCR is adopted, using a competing fragment a sequence of a highly homologous bovine bFGF. However, separation and detection of the PCR products by slab gel electrophoresis and ethidium bromide staining gives poor quantitative data due to the nonstoichiometric binding of the dye. Thus, the only possible quantitation that can be obtained is via autoradiography with 32P-labeled primers. We report here a capillary electrophoresis protocol, in 6% linear, liquid polyacrylamide as a sieving system, able to fully resolve and quantify the undigested (354 bp) bovine and the digested (295 bp and 59 bp) human fragments, with peak ratios in good agreement with the autoradiographic data.

Adrenal Cortex↗
PubMed1994

Preparative isoelectric focusing in multicompartment electrolyzers: novel, hydrolytically stable and hydrophilic isoelectric membranes.

Preparative isoelectric focusing in multicompartment electrolyzers is based on the production of isoelectric membranes of precise isoelectric point, able to buffer at their pI value and to titrate proteins tangent to or crossing the membranes. Up to the present, such membranes have been based on polyacrylamide chemistry; acrylamide, however, is neither stable in acidic nor basic environments. We describe here novel membranes, produced with a unique monomer, N-acryloylaminoethoxyethanol (AAEE). Poly(AAEE) membranes are extremely stable to alkaline hydrolysis (500 times more stable than polyacrylamide) and even more hydrophilic than the latter matrix. This allows production of highly reproducible membranes (these do not change their pI with time, since no acrylic acid is produced by hydrolysis upon storage) which do not adsorb proteins by hydrophobic interaction.

Acrylic Resins↗
PubMed1994

Detection of point mutations by capillary electrophoresis in liquid polymers in temporal thermal gradients.

A new and fast method is described for detection of point mutations in polymerase-chain reaction (PCR)-amplified DNA, based on capillary electrophoresis in sieving liquid polymers in presence of temporal thermal gradients. The background electrolyte contains a constant amount of denaturing agent (e.g., 6 M urea) and the DNA fragments are injected in a constant-temperature plateau below the melting temperature (Tm). After loading, a temperature ramp is activated (typically from 0.2 to 0.6 degrees C/min, according to the melting profiles of the DNA duplexes under investigation) with resultant branching of homo- and heteroduplexes at different times along the migration path. In the case of individuals heterozygous for a point mutation, the expected four-band pattern is obtained. The temperature gradient is not produced externally, via circulating coolant and a thermostat, but is generated internally by using a dedicated computer program able to calculate the precise inner temperature under given electric conditions. The method is applied to the identification of three point mutations located in exon 17b (R1066H, R 1066C, F1052V) and of two polymorphisms located in exon 14a (V868V, T854T) of the cystic fibrosis transmembrane conductance regulator (CFTR) gene.

Adenine↗
PubMed1994

Focusing of alkaline proteases (subtilisins) in pH 10-12 immobilized gradients.

Isoelectric focusing in very alkaline immobilized pH gradients (IPG) was adopted for checking the purity and assessing the pI value of two strongly alkaline proteases: Savinase and Durazym. The first enzyme (known to be the most alkaline) contains 5 Asp, 5 Glu, 7 His, 7 Tyr, 5 Lys, no Cys and 8 Arg residues and should have a theoretical pI of 9.7. Yet, when focused in a pH 9-11 IPG interval, it was lost in the cathodic compartment. After repeated attempts at creating even more alkaline pH intervals, a pH 10-12 IPG range was finally optimized and proved successful in focusing both enzymes midway between the two electrodic compartments. The pI of Savinase was measured as 11.15 +/- 0.15; that of Durazym as 10.95 +/- 0.20 and that of the pI marker cytochrome c as 10.6 +/- 0.17. Both enzymes (and a number of minor components in each preparation) were proven to be active by an in situ zymogram consisting of a casein/agar overlay. The discrepancy between theoretical and experimental pI values could not be fully reconciled: when correcting for pK values of amino acids in proteins at 10 degrees C, instead of the tabulated values at 25 degrees C, the pI should increase to a value of 10. Differential UV spectra showed that ca. 1/2 Tyr are buried in the protein interior and are thus unable to contribute to surface charge. This further increases the pI value by 0.3 pH units to a value of 10.3, still quite removed from the experimentally assessed pI value (in the gel, at 10 degrees C) of 11.15.

Amino Acid Sequence↗
PubMed1992

Preincubation with cysteine prevents modification of sulfhydryl groups in proteins by unreacted acrylamide in a gel.

It has been found that the double bond of free, unreacted acrylamide in a gel can react with a free -SH group of proteins, forming a cysteinyl-S-propionamide adduct. When beta-lactoglobulin was incubated at concentration levels lower than those of free acrylamide, left after polymerizing a 5% T, 4% C gel (barely 12 mM), under anaerobic conditions in 0.1 M borate, pH 9.5, for 1 h and then the tryptic digests analyzed by high performance liquid chromatography (HPLC), two new peptides were detected. The two new peaks were recovered and sequenced by the Edman degradation procedure. They correspond to the sequence from Leu-149 to Ile-162. Residue No. 160 was found to be a cysteinyl-S-propionamide reaction product. Interestingly, only this residue, out of a total of 5 Cys residues, had reacted. No other amino acids (including -NH2 terminus and free -NH2 in Lys) reacted with free acrylamide. The addition of free acrylamide to the -SH group could be completely inhibited if: (i) the gel was extensively washed prior to sample application, or (ii) the gel was incubated for 1 h in 100 mM free Cys.

Acrylamide↗
PubMed1992

The Immobiline family: from "vacuum" to "plenum" chemistry.

We list here a total of 17 acrylamido acids and bases as potential buffers and titrants for isoelectric focusing separations in immobilized pH gradients. The chemistry of these compounds is reviewed and general guidelines are given for their proper use. In particular, it is shown that the most delicate compounds are the basic species, since they can undergo several degradation pathways, including: (i) spontaneous hydrolysis to acrylic acid and a diamine; (ii) spontaneous autopolymerization to oligomers and n-mers; (iii) oxidation to N-oxides during the persulfate polymerization step. A hydrophobicity scale has been constructed, by partitioning the deprotonated species in water/1-octanol phases. A scale of resistance to alkaline hydrolysis for the basic acrylamido buffers is also given, followed by general consideration on the structure/stability relationship of these chemicals.

Acids↗
PubMed1992

Immobilized pH 2.5-11 gradients for two-dimensional electrophoresis.

Extremely wide immobilized pH gradients, pH 2.5-11, for isoelectric separation of complex protein mixtures are described. These pH gradients are theoretically and practically the maximum that can be achieved at present with the available acrylamido buffers and titrants. Conditions are described for reducing conductivity and electroendosmosis in extreme pH ranges. Furthermore, new conditions are described for the separation of proteins in the second dimension. Using this protocol, nearly all the possible cellular products can be separated in one single two-dimensional map.

Electric Conductivity↗
PubMed1992

Modified silver staining for immobilized pH gradients.

Silver development of gels containing an immobilized pH gradient has proved difficult so far because the bonded buffers (especially the tertiary amino acrylamido derivatives) tend to absorb silver ions with a resultant heavy background of increasing darkness from the anode to the cathode. We report a variant of silver staining in which thiosulfate is used twice: (i) prior to silver impregnation, at the millimolar level, to enhance sensitivity, and (ii) during development, at the micromolar level, to decrease the background.

Hydrogen-Ion Concentration↗
PubMed1992

Steady-state heat transfer and thermal zone spreading in gel isoelectric focusing.

The zone spreading caused by a transverse pH profile due to a temperature gradient through the thickness of a gel slab is estimated. The temperature difference (delta T) between the upper and lower gel surfaces can be calculated as a function of the electric power dissipated in the gel and the gel dimensions. It is found that when delta T is only 1 degree C the zone spreading due to this thermal excursion is as high as 0.5 mm. Thus, an admissible delta T is found to be equal to 0.2 degrees C, since this corresponds to a thermal zone spreading of only 0.1 mm, i.e. the same order of magnitude as the spatial resolution of a laser scanner. A thermal zone spreading of 0.1 mm is compatible with a resolving power of 0.01 pH unit, the current limit of conventional isoelectric focusing in amphoteric buffers. A requirement for the thickness of a gel slab is formulated: e.g., at 40 W applied power (over a gel surface area of 25 x 11 cm), a thermal zone spreading limited to 0.1 mm can only be obtained with a gel thickness of approximately 170 microns.

Hot Temperature↗
PubMed1992

Purification of recombinant human growth hormone by isoelectric focusing in a multicompartment electrolyzer with Immobiline membranes.

Recombinant human growth hormone (r-hGH) expressed in Escherichia coli, was 70-80% purified by a combination of ion-exchange chromatography and metal ion affinity chromatography. For the last purification step, a multicompartment electrolyzer was used, containing three compartments delimited by isoelectric membranes and two additional anodic and cathodic chambers. The central compartment was situated between two membranes having isoelectric points (pI) of 5.08 (anodic) and of 5.16 (cathodic), i.e. equidistant from the pI value of hGH (pI 5.12). r-hGH was isoelectric between these two membranes and could not leave the central chamber, while more acidic and more cathodic impurities collected in the two lateral chambers under the influence of the electric field. The r-hGH, thus purified, exhibited a single band by isoelectric focusing (IEF) in immobilized pH gradients (IPG) and gave recoveries greater than 90%. The problem of isoelectric precipitation in a practically ion-free environment was alleviated by focusing in 30% glycerol added with 1% neutral detergent (Nonidet-P40). The latter was eliminated by passage through a Q-Sepharose column after collecting the pI 5.12 band from the electrolyzer. Also the pre-hormone (pre-hGH) can be purified in a similar manner (30% glycerol, 1% Nonidet P-40) between two membranes having pIs 4.77 (anodic) and 4.87 (cathodic) (pre-hGH pI 4.82). This paper demonstrates the possibility of purifying by a focusing process also poorly soluble proteins at the pI.

Growth Hormone↗
PubMed1991

Biomedical relevance of two-dimensional protein mapping.

State-of-the-art and future perspectives are discussed for the application of two-dimensional protein maps to basic medical research and routine clinical chemistry problems. Despite the technical advances that allow effective processing of a large number of samples and the refinement of devices and procedures for image analysis, at present two-dimensional maps are mostly confined to research purposes, i.e. to the inventory of normal constituents of body fluids and tissues on the one hand, and to qualitative-quantitative alterations of some protein spots in a number of instances (genetic, degenerative, infectious or xenobiotic diseases) on the other. It is hoped that in some instances a single primarily affected component will be able to be identified and then specifically tested (for instance by immunological means) as a diagnostic marker, but complex pathological patterns would still require the analysis of a large number of peptides at the resolution level only afforded by two dimensions. Further simplification of the protocols, for example with ready-made gels, and data reduction systems might then allow the application of the technique to be extended to general clinical laboratories.

Genetic Diseases, Inborn↗
PubMed1991

Isoelectric focusing in immobilized pH gradients: applications in clinical chemistry and forensic analysis.

The applications of isoelectric focusing in immobilized pH gradients in clinical chemistry and forensic analysis are reviewed. Strong emphasis is given to the separation of serum proteins, in particular alpha 1-acidic glycoprotein, acid phosphatase, alkaline phosphatase, alpha 1-antitrypsin, apolipoproteins, complement component, factor B, factor XIIIB, group-specific component, lecithin:cholesterol acyltransferase, phosphoglucomutase, prealbumin, protein C and transferrin. The analysis of human parotid salivary proteins is discussed and an assessment is given of the state of the art in thalassaemia screening.

Blood Proteins↗
PubMed1991

Sodium dodecyl sulphate electrophoresis of urinary proteins.

The analysis of urinary proteins and their identification are discussed, particularly in regard to the technique of sodium dodecyl sulphate electrophoresis in polyacrylamide gradient gels. Urine collection, storage and preparation are evaluated, especially in regard to problems connected with concentration and dialysis of such samples. The instrumental approach to sodium dodecyl sulphate polyacrylamide gel electrophoresis represented by the Phast System appears to be particularly valuable in routine clinical analysis of urine specimens, since no sample pretreatment is required. The following types of proteinurias are evaluated: (a) orthostatic proteinurias; (b) post-renal proteinurias; (c) Bence-Jones proteinuria; (d) lower and upper urinary tract infection (cystitis and pyelonephritis) and (e) diabetes mellitus proteinurias.

Diabetes Mellitus↗
PubMed1991

Analysis of acrylamido-buffers for isoelectric focusing by capillary zone electrophoresis.

Immobilized pH gradients use a series of weak acrylamido acids and bases (Immobiline) to create a pH gradient along the separation axis. These buffers can be degraded in water by two mechanisms: (i) hydrolysis of the amido bond, with generation of free acrylic acid and either an amino acid or a diamine; (ii) autopolymerization to oligomers and/or n-mers. In order to check for these degradation products, different capillary zone electrophoresis systems for analysis of all Immobilines have been devised. The acidic compounds are resolved in 100 mM acetate, pH 4.0, whereas the alkaline Immobilines are separated in 50 mM phosphate buffer, pH 7.7 (or pH 7.2 for the weaker species). Polymers of alkaline Immobilines are resolved in 50 mM phosphate buffer, pH 2.5, in 1% Ficoll-400. All Immobilines are detected underivatized, by their adsorption at 214 or 254 nm. A calibration curve has been constructed for quantification of acrylic acid contamination. As little as 1 mol% of acrylic acid contamination in Immobiline solutions can be detected, with a sensitivity limit below 0.2 mM (at the injection port).

Acrylamides↗