Refolding of the Immunoglobulin Light Chain1

Abstract

The kinetics of the refolding reactions of type λ Bence Jones proteins from 4 M GuHCl were studied by CD, ultraviolet absorption, and fluorescence spectrophotometry. The kinetics were complex and consisted of at least three phases, an undetectable fast phase, a detectable fast phase, and a slow phase. The slow phase followed first-order kinetics and the three experimental methods used gave similar rate constants for all the Bence Jones proteins (about 3 × 10⁻³ s⁻¹). The refolding reaction of V_L fragment was too fast to be measured in the present experiments. The refolding process of C_L fragment was very similar to those of Bence Jones proteins except that the detectable fast phase was less significant. The rate constant of the slow phase observed for the C_L fragment was similar to those of the slow phase observed for Bence Jones proteins. The activation energy of the slow phase was the same for a Bence Jones protein and its C_L fragment. These results indicate that the refolding kinetics of the C_L domain are very similar to those of isolated C_L fragment and that refolding of the V_L domain precedes refolding of the C_L domain, even though both domains have similar immunoglobulin folds. However, the results of refolding experiments on Bence Jones proteins, and VL and CL fragments in the presence of ANS, as well as other lines of evidence, indicate that the refolding kinetics of the Bence Jones protein molecule cannot be expressed as simple sum of the refolding reactions of isolated V_L and C_L fragments.