Spectroscopic determination of protein concentrations from proteinase K digests.
A method to determine protein concentrations and absorptivities based on absorbance measurements of proteinase K digests has been developed. Molar absorptivities of proteinase K digests at 56 degrees C can be predicted by using the following equation: epsilon (M)(280)=5318 x (No.of Trp) + 1227 x (No.of Tyr) + 133 x (No.of Cys-Cys). Protein concentration in the digest can be determined by dividing the corrected digest solution absorbance by the calculated epsilon(M)(280). The absorptivity of a native protein can then be calculated by dividing the absorbance of the intact protein solution by the concentration value obtained for the digest solution. Precision of the experimental data is within +/-3%, and the error of the method does not exceed 4.5%. The accuracy of determination does not depend on the size of the protein, Trp/Tyr ratio, presence or absence of certain chromophores, or other structural factors. The method requires amounts of protein routinely used for absorbance measurements.
Kats
Michael
M
Pharmaceutical Research Institute, Bristol-Myers Squibb Company, Syracuse, NY 13221, USA. katsm@bms.com
St Germain
Michael E
ME
D000818:Animals; D002417:Cattle; D019286:Endopeptidase K / Q000378:metabolism*; D006801:Humans; D010446:Peptide Fragments / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D011506:Proteins / Q000737:chemistry / Q000378:metabolism*; D012680:Sensitivity and Specificity; D013056:Spectrophotometry, Ultraviolet / Q000379:methods*; D013997:Time Factors
D010446:Peptide Fragments; D011506:Proteins; D019286:Endopeptidase K