muramidase and 3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate

All existing protocols for protein separation by two-dimensional (2-D) gel electrophoresis require the full reduction, denaturation, and alkylation as a precondition for an efficient and meaningful separation of such proteins. Existing literature provides a strong evidence to suggest that full reduction and denaturation can be achieved in a relatively short time; the same thing, however, can not be said for the alkylation process, which the present study shows that more than 6 h are required for a complete alkylation. We have used matrix assisted laser desorption/ionisation-time of flight-mass spectrometry (MALDI-TOF-MS) to monitor protein alkylation by iodoacetamide over the period 0-24 h at pH 9. The present, fast and specific MS method provided clear indication on the extent and speed of alkylation which reached approximately 70% in the first 2 min, yet the remaining 30% resisted complete alkylation up to 6 h. The use of sodium dodecyl sulfate (SDS) during the alkylation step resulted in a strong quenching of this reaction, whereas 2% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) exerted a much reduced inhibition. The implications of the present measurements on 2-D gel analysis in particular and proteomics in general are discussed.

Topics: Alkylation; Animals; Binding Sites; Cattle; Chickens; Cholic Acids; Cysteine; Electrophoresis, Gel, Two-Dimensional; Iodoacetamide; Kinetics; Lactalbumin; Lysine; Muramidase; Peptide Mapping; Proteins; Sodium Dodecyl Sulfate; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

Three proteins have been subjected to hydrophobic-interaction chromatography in the presence of submicellar concentrations of the surfactant (3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS). At several concentrations of CHAPS below the critical micelle concentration, CHAPS increased the retention of lysozyme and pancreatic trypsin inhibitor, but decreased that of ribonuclease A. The dependence of retention on CHAPS concentration was substantially different for the three proteins, i.e., the surfactant showed selectivity in its interactions with the proteins. In the solvents that were used for chromatography the surface tension decreases monotonically with increasing CHAPS concentration. Since different proteins may either be eluted or retained by the addition of CHAPS, our findings are inconsistent with the idea that retention is a simple function of surface tension. It also appears unlikely that the selectivity we have observed can be accounted for by a formulation of retention as a function of surface tension. As an alternative interpretation, we present a scheme of multiple equilibria and their kinetic components as a basis for formulating the dependence of retention on surfactant concentration.

Topics: Ammonium Sulfate; Cholic Acids; Chromatography; Micelles; Muramidase; Proteins; Ribonuclease, Pancreatic; Surface Tension; Surface-Active Agents; Trypsin Inhibitors