Reactive Dyes as a Spectral Probe for Protein Folding

Abstract

A single blue dye was covalently attached to the active site of ribonuclease by reaction of the protein with Procion Blue MX-R, C.I. 61205 reactive blue 4, for 30 minutes at pH 8.5 and 35 degrees. This attachment resulted in the loss of over 95% of the catalytic activity of the enzyme without disruption of its folded structure as judged by size exclusion chromatography and by circular dichroic measurements. Addition of increasing concentrations of the denaturant guanidinium chloride to the protein-dye conjugate first generated visible difference spectra characteristic of the free dye followed by spectra at higher denaturant concentrations characteristic for displacement of the conjugated dye from the active site by the competitive inhibitor 2′-CMP. Formation of the latter difference spectra coincided with the denaturation of the protein-dye conjugate as judged by far ultraviolet circular dichroic measurements. The kinetics of refolding the denatured protein-dye conjugate were observed at 650 nm in 2 M guanidinium chloride maintained at pH 6 and 10 degrees. The decrease in absorbance fit well with a single first order reaction having a half-time of 1080 seconds. This value is within a factor of two of the half-time reported for the refolding of unconjugated ribonuclease observed using either tyrosine absorbance or fluorescence measurements.