Fractionation solyubilizovannogo in 0.5% SDS-Na protein with a subsequent release in the crystalline form reached a three-stage low-temperature fraction (-50C) methanol precipitation, reducing the concentration of detergent, respectively, 2.5 and 5 times. The final stage of purification of the BR was separation of protein from low molecular weight impurities by gel permeation chromatography, for which the BR-containing fraction was passed through the column twice with dextran Sephadex G-200 equilibrated with 0.09 M Tris-borate buffer (pH 8.35) with 0.1% SDS-Na and 2.5 mM ETDA (Fig. 6). According to the developed method able to obtain 8-10 mg of 2H-labeled BR 1 g of bacterial biomass, which satisfies the homogeneity requirements for the reconstruction of the membranes and confirmed electrophoresis in 12.5% polyacrylamide gel with 0.1% SDS-Na, apomembran regeneration from trans-retinal and reversed-phase HPLC of methyl esters of N-DNS-amino acids. A little way out was not an obstacle BR for subsequent mass spectrometric analysis, But here it must be stressed that to ensure a high yield of protein is necessary to accumulate more biomass raw materials.
An important step was the hydrolysis experiment, deuterium-labeled bacteriorhodopsin, which had to be carried out under conditions to prevent isotopic exchange of hydrogen by deuterium and conservation of tryptophan residues in the protein. We considered two alternatives – acid and alkaline hydrolysis. Acid hydrolysis of protein under standard conditions (6N. HCl or H 8. H2SO4, 1100S, 24 h) leads to complete destruction of tryptophan and the partial destruction of serine, threonine and other amino acids in the protein.