Effects of substituted metal-free porphyrins in apo-horseradish peroxidase

Abstract

The peroxidase-like catalytic activities of some synthetic porphyrins following substitution in apo-horseradish peroxidase (apo-HRP) were studied by UV-vis and fluorescence spectrophotometries in buffer potassium phosphate solution at pH 7. The insertion of these synthetic porphyrins in apo-HRP is very difficult, due to steric hindrances between coordinated metals in porphyrins and the side-chains of amino acids in native protein. However, because of planar coordination following insertion in an active site of apo-HRP tetrapyridylporphyrin(TPyP), a metal-free porphyrin, adsorbs free metal ions from the environment and shows higher peroxidase-like catalytic activity than other new synthetic porphyrins. The results indicate that the metal-free porphyrins with optimum Reinheitzahl (RZ) show higher catalytic activity relative to others. The fluorescence emission of TPyP only, and of apo-HRP-TPyP, displayed significantly different wavelengths for the maximum aromatic fluorescence intensity. The λmaxfor apo-HRP-TPyP was around 447 nm, while for TPyP, alone, it increased to 463 nm, which showed that the aromatic groups' surroundings changed upon substitution of porphyrins in a low polar environment.