Analysis of cibacron blue f3g-a interaction with therapeutic proteins by maldi-tof mass spectrometry

Abstract

The antraquinone dye Cibacron blue F3G-A is widely used as a ligand in affinity chromatography systems. Cibacron blue F3G-A is able to bind most types of proteins. In spite of the extensive application, the binding mechanisms between Cibacron blue F3G-A and proteins remain poorly understood. The formation of the complexes between Cibacron blue F3G-A and two therapeutic proteins, recombinant human interferon-a2b and recombinant human growth hormone, was found earlier by applying gel-permeation chromatography in combination with the absorption difference spectroscopy. Kd of the complexes Cibacron blue F3G-A – interferon-a2b and Cibacron blue F3G-A – growth hormone were found to be equal to 0.9-1.5 µM and 5.6 µM, respectively, at pH 6.9 and 25 oC. To determine probable dye binding sites in interferon-a2b and growth hormone molecules, time resolved limited proteolysis and MALDI-TOF MS analysis of obtained peptide mixtures was used. Trypsin was chosen for the digestion of proteins. The addition of the dye influenced mass spectra. The occurrence of new peaks indicating peptide-dye complexes was observed. Analysis of peptide maps revealed that peptides A17HR19 and L20HQLAFDTYQEFEEAYIPK38 of human growth hormone, and R14TLMLLAQMR23 and D33RHDFGFPQEEFGNQFQK50 of human interferon-a2b exhibit affinity to Cibacron blue F3G-A.