ROLE OF THE INTERSUBUNIT DISULFIDE BOND IN THE UNFOLDING PATHWAY OF DIMERIC RED KIDNEY BEAN PURPLE ACID-PHOSPHATASE

Quantitative equilibrium denaturation studies on oligomeric proteins h ave the potential to provide information on the role of subunit intera ctions in protein function and structure. We studied the equilibrium d enaturation of red kidney bean purple acid phosphatase (KBPAP), a homo dimer with a single disulfide bond between the two subunits, with an o bjective to understand the role of the intersubunit disulfide bond in KBPAP structure. Binding of 8-anilino-1-naphthalenesulfonic acid, enzy matic activity, size-exclusion chromatography, tryptophan fluorescence and circular dichroism studies revealed that the protein undergoes un folding through at least three intermediates. Susceptibility of KBPAP for denaturation increases on reduction of the disulfide and aggregati on was the predominant product of denaturation. In terms of stability, an intersubunit disulfide bond contributes to 25% of the overall stab ility of the dimer.