To better understand protein folding and unfolding mechanisms in heme proteins, we are investigating the conformational stability of horseradish peroxidase (HRP). HRP is a member of the heme-containing plant peroxidases that catalyze the oxidation of a variety of organic and inorganic compounds. The x-ray crystal structure of HRP isoenzyme C reveals two bound Ca²⁺ ions that are suspected of being required for catalysis, structural support, and overall stability of the protein. Our specific aim is to investigate the effects of different anions on the structural stability of Ca²⁺-bound and Ca²⁺-depleted HRP. The structural stability of HRP at acidic pH in the presence of various anions (chloride and phosphate) is measured using absorbance spectroscopy. The unfolding of HRP is indicated by the shift in the heme Soret band from 403 nm to 370 nm. Interestingly, spectral changes in the heme Soret band at acidic pH as a function of time depends on the anion present in solution. Upon dissolving Ca²⁺-bound HRP in water adjusted to pH 3 with HCl, the Soret band remained unchanged after 30 minutes (λ_max = 403 nm). In 50 mM phosphate buffer adjusted to pH 3 with HCl, λ_max blue-shifted from 409 nm to 370 nm within 30 minutes. This suggests phosphate destabilizes HRP at acidic pH by removing Ca²⁺ from HRP. To confirm the role Ca²⁺ plays in stabilizing HRP, Ca²⁺-depleted HRP is prepared by reversibly unfolding HRP and adding EDTA to complex protein-bound Ca²⁺. Experiments measuring the structural stability of Ca²⁺-depleted HRP at acidic pH in the presence of various anions (chloride and phosphate) will be reported.