engleski

Protocol for spectrophotometric determination of native and desialylated apo-transferrin molar absorption coefficients

Absorbance of a protein around 280 nm depends on its tryptophan (Trp), tyrosine (Tyr) and cystine (i.e. the disulfide bond) content. Molar absorption coefficient of a folded (native) protein can be determined by measuring absorbance of two protein solutions with identical concentrations, one containing only buffer and the other containing the same buffer and 6.0 M guanidine∙HCl. Since molar absorption coefficients of Trp, Tyr and cystine in 6.0 M guanidine∙HCl are known, molar absorption coefficient of an unfolded (denatured) protein at given wavelength can be calculated if the protein amino acid sequence and structure (i.e. the protein Trp, Tyr and cystine content) is known and the absorbances of the folded and unfolded protein are measured. The molar absorption coefficient of a folded protein at given wavelength is then equal to the product of a molar absorption coefficient of an unfolded protein and absorbance ratio of folded and unfolded protein at given wavelength. The required amount of the protein is 1 mg but approximately 60 % of folded protein can be recovered and used for further studies.

transferrin, absorbance, molar absorption coefficient, guanidine

nije evidentirano