PubMed Health⌕ Search

Biomedical subjects

M Waks

Publications and source records attributed to M Waks.

At least 37 records · Page 2Linked to original sources
PubMed1985

Proteins in membrane mimetic systems. Insertion of myelin basic protein into microemulsion droplets.

The insertion of myelin basic protein into microemulsion droplets of sodium bis (2-ethylhexyl) sulfosuccinate (AOT) has been studied by quasi-elastic light scattering. Measurements were made at both low and high molar ratios of water to surfactant, as a function of protein occupancy. The hydrodynamic radii of filled and empty droplets were experimentally evaluated. These were compared to values calculated using a water shell model of protein encapsulation, and excellent agreement was obtained. At low molar ratio of water to surfactant (w0 = 5.6), the hydrodynamic radius of filled droplets is significantly larger than the radius of empty ones. Under these conditions, about three empty (water-filled) droplets are required to build up a droplet of sufficient size to accommodate a single protein molecule. At maximum solubilization, which occurs at w0 = 5.6, a small fraction of droplets are found containing protein aggregates. In contrast, results at high values of w0 (22.4) reveal radii for empty and occupied droplets of comparable dimension, and the absence of aggregates. The results are discussed in terms of the model and the mechanism of interaction of this protein with the aqueous interfaces provided by these membrane-mimetic systems.

Animals↗
PubMed1984

A heme binding site on myelin basic protein: characterization, location, and significance.

Myelin basic protein (MBP), an extrinsic membrane protein from the myelin sheath, binds dicyanohemin. The binding generates absorption bands in the Soret region and quenches the fluorescence emitted by the sole tryptophan residue. The absorption titration curves in the Soret demonstrate that the binding is stoichiometric, one heme per protein, with a large value of the extinction coefficient (8 X 10(4) M-1 cm-1 at 420 nm). Fluorescence quenching titration curves indicate an identical stoichiometry and a low quenching efficiency of 20%. From the heme titration curve the association constant between dicyanohemin and MBP is estimated to be greater than or equal to 10 nM-1 in 50 mM 4-morpholinepropanesulfonic acid buffer, pH 7.0, at 20 degrees C. Digestion of MBP by Staphylococcus aureus V8 protease yields a peptide (38-118) whose heme binding properties are identical to those of MBP. In contrast, peptides obtained by digestion of MBP with cathepsin D do not exhibit any specific binding of dicyanohemin. The cleavage of the Phe-Phe (42-43) bond appears to be critical in this respect. A comparison of the sequence immediately preceding, including these residues with a probable heme binding site of a mitochondrial cytochrome b, reveals a high degree of homology. The possible significance of heme binding is discussed.

Amino Acids↗
PubMed1984

The thiol groups of the Folch-Pi protein from bovine white matter. Exposure, reactivity and significance.

The number and the reactivity of accessible thiol groups of the Folch-Pi apoprotein and proteolipid (50% of myelin proteins) were studied, by using a specific thiol-disulphide interchange reaction, in connection with the known solubility of this protein in organic and aqueous solvents. The high reactivity of 2,2'-dipyridyl disulphide towards thiol groups leads to the titration of 4.8 mol of SH groups/mol of protein (Mr 30000) in alkaline and acidic chloroform/methanol (2:1, v/v). Unlike previous findings, this value was consistently found from batch to batch and remained stable with time. In the proteolipid 1 mol of SH groups/mol was not accessible as compared with the apoprotein. In aqueous solvents, a similar number of 4.4 mol of SH groups/mol was also found. For the first time, kinetic studies carried out in chloroform/methanol discriminated between two classes of thiol groups. The reaction of 2 mol of SH groups/mol was characterized by apparent second-order rate constants whose values were 5-10-fold higher than those of the other class. Kinetic studies and cyanylation experiments in aqueous solvents also indicated the high reactivity of these thiol groups with Ellman's reagent. Together with kinetic results, studies on the stoichiometry of the interchange reaction of equimolar solutions of protein and disulphide indicate that these highly reactive thiol groups are near to each other in the amino acid sequence. The location of the thiol groups at the boundary between hydrophilic and hydrophobic domains of the Folch-Pi protein is suggested in connection with their possible structural and biological significance.

2,2'-Dipyridyl↗
PubMed1980

Modification of rat hemopexin properties upon heme binding.

Rat hemopexin and its complex with heme were found to have the same Stokes' radius, 3.9 nm, as determined by gel filtration. Therefore no polymerisation occurs as a result of heme binding. The conformational parameters calculated from circular dichroism spectra indicate that hemopexin and its complex consist of 20% beta sheet and mainly of disordered structure. No change of the secondary structure is therefore observed upon heme binding. Hemopexin reveals five bands by analytical electrofocusing with pI ranging from 5.5 to 5.95. This microheterogeneity is not due to sialic acid differences between the variants. Upon heme binding the pI of the variants decrease to lower values (from 4.8 to 5.25). This decrease in the pI value of hemopexin is thought to modify the tertiary structure through charge effects and may allow the binding of the heme-hemopexin complex to the hepatocytes.

Animals↗
PubMed1979

Is immunochemical determination of haptoglobin phenotype dependent?

Immunochemical methods have been used to determine the concentration of haptoglobins. The dependence on the phenotype was tested with highly purified Hp 2-1, Hp 2-2 and Hp 1-1, by immunonephelometry and radial immunodiffusion (RID). Measurements with three different instruments: automated immunonephelometer (AIP, Technicon), laser nephelometer (LN, Behring) and immunochemistry system (ICS, Beckman) were performed. For each type of apparatus antisera against a pool of haptoglobins were provided by the respective manufacturers. Some experiments were done with an antiserum to the haptoglobin heavy chain prepared in the laboratory. This study shows that haptoglobin determination depends neither on the physical geometry of the instruments or on the type of antiserum used in this work. In contrast, the data display a dependence on haptoglobin phenotype. When Hp 2-1, the most common phenotype, is taken as a standard, thd values obtained for Hp 2-2 are in good agreement with those obtained for Hp 2-1. However, the values obtained for Hp 1-1 are overestimated unless they are corrected by an experimental factor which has been determined in this study.

Haptoglobins↗
PubMed1979

Identification of haptoglobin in chicken serum and specificity of the chicken haptoglobin-hemoglobin complex formation.

1. The presence of haptoglobin in chicken serum has been demonstrated by three different techniques: gel filtration, cellulose acetate electrophoresis and fluorescence quenching. 2. Chicken haptoglobin shows a narrow species specificity; it binds only avian and reptilian but not mammalian hemoglobins. 3. Haptoglobin seems to have been subjected to profound changes during the course of evolution.

Animals↗
PubMed1977

Studies on hemoglobin tryptophanyl contact residues in the haptoglobin-hemoglobin complex.

Hemoglobin and apohemoglobin bind heptoglobin in the same molar ratio. Structural studies on haptoglobin-hemoglobin complex do not suggest any important structural changes in either protein upon binding. However, when apohemoglobin is bound to haptoglobin, a marked reduction in secondary structure, attributed to unfolding of globin chains, has been observed. Here we describe some properties of the haptoglobin-apohemoglobin (Hp-apoHb) complex, prepared by isoelectric focusing in the presence of an excess of haptoglobin. This complex does not exhibit the irreversibility of complexes obtained with hemoglobin in identical experimental conditions. The 'freezing' of the conformation of apohemoglobin upon binding to haptoglobin has been studied by fluorescence quenching experiments carried out in the presence of 8 M acrylamide. Changes in conformation of haptoglobin upon binding to apohemoglobin have been detected by titration of the exposed tryptophans using N-bromosuccinimide. Comparison of the additivity of exposed tryptophans in the complexes reveal that two tryptophans become inaccessible in the complex formation of haptoglobin with hemoglobin but not with apohemoglobin. These tryptophans, probably located on the alpha1beta2 contact interface of hemoglobin, have been tentatively identified as Trp-C3(37)beta.

Apoproteins↗
PubMed1977

[Immunochemical determination of human hemoglobin in biological fluids (author's transl)].

An assay of human hemoglobin by immunonephelometry-electroimmunodiffusion is proposed. Concentrations in the range from 5 to 180 mg% can be determined to within 2--5% accuracy. The immunonephelometric assay is independent of the presence of haptoglobin whereas the electroimmunodiffusion assay is not. This immunochemical assay, independent of the peroxydase activity of hemoglobin, can be carried out in any biological fluid (plasma, urine, gastric fluid).

Evaluation Studies as Topic↗
PubMed1977

Reconstitution of native human hemoglobin from separated globin chains and alloplex intermediates.

A complete experimental format is given for the reconstitution of human hemoglobin from the separated heme-free alpha- and beta-globin chains (alpha degrees, beta degrees) and hemin, by two alternative routes. Based on their oxygen binding properties, the reaction of the ferri-forms with reducing agent, and the response of the oxygen binding curves to pH variation and to the addition of the allosteric effector 2,3-diphosphoglycerate, the molecules are native. One reconstitution route uses direct addition of hemin to the separated globin chains with production of the separated subunits, which can then be recombined and reduced. This procedure occasions losses by precipitation in the heme-addition step except at high dilutions, and the yields are low. In the second pathway, either globin chain is mixed with the complementary untreated subunit to form the half-filled (with heme) intermediates, which combine stoichiometrically with hemin. No precipitation accompanies these reactions. For alpha-globin, the yield is about 50% because of incomplete combination with the heme-containing beta chain. For beta-globin, the yield is better than 70%. It is suggested that experiments intended to test either globin chain should use the second route in preparation for structural or functional comparisons with native hemoglobin.

Allosteric Site↗