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Biomedical subjects

M L Bossi

Publications and source records attributed to M L Bossi.

8 recordsLinked to original sources
PubMed1987

Hydrophobic interaction between alkaline immobilines and ferritin during isoelectric focusing in immobilized pH gradients.

It has been found that three alkaline Immobilines (out of seven weak acids and bases used to generate immobilized pH gradients), having pK values of 6.2, 7.0 and 9.3, act as cross-linking agents, aggregating and precipitating out of solution ferritin and other large macromolecules (e.g., from serum and tissue extracts) present in body fluids and human biopsies. All the acidic Immobilines (pK 3.6, 4.4 and 4.6) and the basic species of pK 8.5 appear to be unreactive. The three precipitin Immobilines mimic cationic detergents, acting on the basis of two different principles at the opposite extremes, by ionic interaction at one end and by hydrophobic bonding at the other end of the molecule. The ionic type of interaction was clearly demonstrated, owing to its sensitivity to pH extremes and to progressively increasing ionic strength. The hydrophobic interaction in the region of the double bond (Immobilines are N-substituted acrylamido acids and bases) was deduced on the basis of the following observations: (a) oxidation of the double bond with introduction of a vicinal diol totally inhibited ferritin aggregation; (b) addition of SH groups to the double bond increased protein precipitation and (c) the protein-Immobiline aggregates were found to be sensitive to alkyl-substituted ureas (especially ethyl- and propylurea), which are known to bind to hydrophobic regions of proteins, and insensitive to urea, which is known to split only hydrogen bonds. Interestingly, neutral and zwitterionic detergents were unable to split the Immobiline-ferritin complexes, suggesting that their large micelles could not have access to the tightly packed Immobiline cross-linking region.

Chemical Phenomena↗
PubMed1987

Diffusion coefficients of proteins in carrier ampholyte versus immobiline gels.

The apparent diffusion coefficients of proteins in carrier ampholyte isoelectric focusing (CA-IEF) and in immobilized pH gradients (IPGs) are strongly dependent on the amount of buffering ions present in the system. However, whereas in CA-IEF increased levels of ampholytes facilitate diffusion, in IPGs they strongly quench it. It is concluded that a protein in an IPG matrix is isoelectric but not isoionic, in the sense that it forms a salt with the surrounding ions bound to the polyacrylamide matrix. This salt formation is beneficial as it greatly increases protein solubility at the pI. It is suggested that, when performing zymograms in situ, the IPG gel should contain at least twice the standard amount of Immobiline, so as to keep sharp enzyme bands even with prolonged incubation periods.

Animals↗
PubMed1987

Isoelectric focusing in immobilized pH gradients in the pH 10-11 range.

With the synthesis of a new, strongly basic Immobiline (pK 10.3 at 10 degrees C) it has been possible to formulate a new pH 10-11 recipe for focusing very alkaline proteins, not amenable to fractionation with conventional isoelectric focusing in carrier ampholyte buffers. In this formulation, water is added as an acidic Immobiline having pK = 14 and a unit molar concentration (or with a pK = 15.74 and standard 55.56 molarity) since around pH 11 its buffering power becomes significant. The gel contains a 'conductivity quencher', i.e. a density gradient incorporated in the matrix, with the dense region located on the cathodic side (pH 11) for (a) smoothing the voltage gradient on the separation cell and (b) reducing the anodic electrosmotic flow due to the net positive charge acquired by the matrix at pH 11 (1 mM excess protonated amino groups to act as counterions to the 1 mm OH- groups in the bulk water solution generated by the local value of pH 11). Excellent focusing is obtained for such alkaline proteins as lysozyme (pI 10.55), So-6 (a leaf protein, pI 10.49), cytochrome c (pI 10.45) and ribonuclease (pI 10.12).

Computer Simulation↗
PubMedDate not supplied

Casting immobilized pH gradients into cylindrical polyacrylamide gels.

A novel method is described for casting immobilized pH gradients in polyacrylamide gel rods of small diameter (2 mm), based on the principle of rotational centrifugation. The tubes are filled vertically with equal volumes of dense and light solution (250 microliter each) titrated to the extremes of the desired pH gradient, and then tilted at 2.5 degrees to the level. After 5 min at rest, to allow for sliding of the two menisci to equilibrium position, the glass tubes are rotated for 3 min at 180 rpm, followed by an additional 3 min at 180 rpm by reversing the sense of rotation. A homogeneous linear gradient is thus produced. The rotating platform is then raised to 90 degrees and the gels allowed to polymerize under standard conditions. Formation of linear and reproducible pH gradients is ensured by using stabilizing density gradients of low viscosity (0-5% glycerol, having a maximal ratio viscosity/density of 1.1).

Acrylic Resins↗