Stability and kinetics of chondroitinase ABC in macromolecular crowded environment

Abstract

Chondroitinase ABC (ChABC) has been exploited to digest upregulated chondroitin sulphate in nerve injury and intervertebral disc hernia. Yet, frequent administration and high dosage are suggested due to possible thermal instability issue but patient may risk immunoreaction due to accumulation of foreign proteins. Whereas the macromolecular crowdedness of extracellular environment can stabilise the protein structure, we attempted to clarify the stability issue by using 300 mg/ml Ficoll 70 as crowding agent to mimic the macromolecular crowded extracellular environment. Without Ficoll, both subtypes of ChABC (ChABC I & II) lost their activity at 37 oC by 60 % to 70 % in 30 min and the activity loss was rescued by the crowded environment. Structural diagnostic UV absorption scan showed continuous change in the absorption spectrum of both subtypes attributing activity loss to structural destabilisation. We then tested if the macromolecular crowdedness affects the digestion efficiency. The activities of ChABCs varied with crowdedness implying that the crowdedness of the application site affects the dosage needed. The Vmax, maximum reaction rate, of ChABC I dropped by 58% in crowded environment but only 5% for ChABC II. This reveals differential effects of crowdedness on two subtypes. Our data suggested crowdedness 1) stabilises protein structure of ChABCs at physiological temperature and 2) differentially alters activity of two subtypes. Therefore, The dosage needed could be less than that previously reported.