Adding Sodium Dodecyl Sulfate Research Articles

The effect of surface tension in capillary viscometry of biological materials.

Surface tension has a measurable effect on the rate of flow in capillary viscometers of aqueous solutions of biological materials. Changing the surface tension of saline from 70 to 33 dyn/cm by adding sodium dodecyl sulfate reduces its flow time in C

Human Carbonic Anhydrase B: LOCATION OF TYROSINE RESIDUES THAT REACT WITH TETRANITROMETHANE

Abstract As shown previously by J. A. Verpoorte and C. Lindblow (J. Biol. Chem., 243, 5993 (1968)) 3 of the 8 tyrosine residues in human carbonic anhydrase B can be nitrated with tetranitromethane. The present studies involve separation of tryptic peptides from the nitrated protein, with sequence determinations to locate the positions of the nitrated residues in the total sequence of the protein. The most rapidly nitrated residue is at position 20 from the amino-terminal end; another that reacts more slowly is in the region of still undetermined total sequence (residues 92 to 152 inclusive) and a third, at position 88, reacts still more slowly. No evidence was found for reactivity of the 5 other tyrosine residues, two of which are at positions 7 and 51, one is in the undetermined region, and two are in the carboxy-terminal half of the molecule. Great difficulty was initially found in separating the peptides containing nitrated tyrosine residues from other peptides in the tryptic hydrolysate. This difficulty, probably due primarily to aggregation, was overcome by adding sodium dodecyl sulfate before fractionations of the peptides on Sephadex G-50 and then on G-25. Subsequent chromatography removed the detergent and effected final purification. The procedure may prove useful in other cases where separation of peptides is difficult.

Ultracentrifugal analysis of staphylococcal alpha toxin.

Ultracentrifugal examination of staphylococcal alpha toxin at different stages of purification showed the presence of a major component having a sedimentation coefficient of 2.8S, present to the extent of more than 90% of the sample, and identifiable with active toxin. Several minor components having S(20,w) values of 11.5S, 8.5S, and 2.0S were detected. The 11.5S component presumably is identical with a toxin aggregate studied earlier and designated 12S; the 8.5S component appears to be delta toxin. A sedimentation equilibrium study of more highly purified material gave 32,700 as the best estimate of molecular weight of alpha toxin. Lowering the pH of the partially purified alpha toxin from 10.2 to 5.3 resulted in a small increase in S(20,w) of the 11.5S component and in the disappearance of the 8.5S component, whereas the S(20,w), molecular weight, and hemolytic activity of the toxin remained constant. Exposure of toxin to pH 3.5 irreversibly reduced the S(20,w) to 2.0S, the molecular weight to about 16,000, and caused irreversible inactivation. Raising the pH of acid-inactivated toxin and adding sodium dodecyl sulfate to 1% increased the S(20,w) to near its normal value (2.7S) but did not restore activity.

COMPOSITION OF THE SHEATH OF SPHAEROTILUS NATANS.

Romano, Antonio H. (University of Cincinnati, Cincinnati, Ohio) and Joyce P. Peloquin. Composition of the sheath of Sphaerotilus natans. J. Bacteriol. 86:252-258. 1963.-The sheath of Sphaerotilus natans was isolated and subjected to chemical analysis. Isolation of the sheaths was accomplished by incubating cells in the presence of lysozyme and ethylenediaminetetraacetic acid in tris(hydroxymethyl)aminomethane buffer and adding sodium dodecyl sulfate subsequently. Under these conditions, there was complete dissolution of cells. The sheaths, which were left intact by this treatment, were recovered by centrifugation, washed exhaustively, lyophilized, and subjected to analysis. Hydrolysis of the sheath material with 2 n HCl at 100 C resulted in the liberation of reducing sugars amounting to 36% of the dry weight. Amino sugar accounted for 11% of the dry weight. Paper chromatography of hydrolysates showed the presence of glucose and hexosamine. Tests for muramic acid were negative. In addition to carbohydrate, 27% protein and 5.2% lipid were found to be present. Fractionation studies indicated that essentially all of the polysaccharide was associated with a trichloroacetic acid-soluble fraction. The sheath is therefore considered to be a protein-polysaccharide-lipid complex, which is chemically and anatomically distinct from the cell wall and the slime layer. It is hypothesized that this unique structure may be related to the microcapsule found in many gram-negative bacteria, and may represent a structural specialization of this more common structure.