Coomassie brilliant blue G-250 dye-binding technique for determination of autolytic protein breakdown in Euglena gracilis and comparison to other methods of autolysis measurement.
The Coomassie brilliant blue G-250 dye-binding technique of M. M. Bradford (1976, Anal. Biochem. 72, 248-254) for protein quantification has been used to measure autolytic protein breakdown in cell-free extracts of Euglena gracilis. Specific autolysis rates were calculated from the difference between initial and actual absorbances at different incubation times of Coomassie brilliant blue-stained protein. They were found to depend linearly on time and initial protein concentration. Calibration against another method of protein determination is necessary due to different color yields with various protein mixtures. The high sensitivity and reproducibility of this method permit determination of specific autolysis rates below 0.1% mg-1 h-1 over a pH range between 3 and 8, without protein precipitation or pH adjustment, and in the presence of high amounts of amino acids and/or small peptides. Results obtained by this method are comparable to those of other autolysis measurements and to proteolytic activity determination by azocaseinolysis. Proteolytic autolysis has been observed in both the soluble and the particulate fractions of E. gracilis cell-free extracts, but displays different pH optima and specific activities in these fractions, as is also the case for azocaseinolysis. The method described is easy to perform, inexpensive, time saving, and should be applicable to other biological systems as well.
D000818:Animals; D001329:Autolysis; D002364:Caseins; D002474:Cell-Free System; D003124:Colorimetry / Q000379:methods*; D010450:Endopeptidases / Q000032:analysis; D005056:Euglena gracilis / Q000032:analysis*; D006863:Hydrogen-Ion Concentration; D011506:Proteins / Q000032:analysis*; D012394:Rosaniline Dyes
D002364:Caseins; D011506:Proteins; D012394:Rosaniline Dyes; C026018:azocasein; D010450:Endopeptidases; C004692:coomassie Brilliant Blue