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1 July 2004 Flexibility in Proteins: Tuning the Sensitivity to O2 Diffusion by Varying the Lifetime of a Phosphorescent Sensor in Horseradish Peroxidase
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Abstract

The heme in horseradish peroxidase (HRP) was replaced by phosphorescent Pt-mesoporphyrin IX (PtMP), which acted as a phosphorescent marker of oxygen quenching and allowed comparison with another probe, Pd-mesoporphyrin IX (Khajehpour et al. (2003) Proteins 53, 656–666). Benzohydroxamic acid (BHA), a competitive inhibitor of the enzyme, was also used to monitor its effects on phosphorescence quenching. With the addition of BHA, in the presence of oxygen, the phosphorescence intensity of the protein increased. In contrast, the addition of BHA, in the absence of oxygen, reduced the phosphorescence intensity of the protein. Kd = 18 μM when BHA binds to PtMP-HRP. The effect of BHA can be explained by two factors: (1) BHA reduces the accessibility of O2 to the protein interior and (2) BHA itself quenches the phosphorescence. Consistent with this, the oxygen quenching of the phosphorescence of PtMP-HRP gave a quenching constant of kq = 234 mm Hg−1 s−1 in the absence of BHA and kq = 28.7 mm Hg−1 s−1 in the presence of BHA. The quenching rate of BHA is 4000 s−1. The relative quantum yield of the phosphorescence of the Pt derivative is about six times that of the Pd derivative, whereas the phosphorescence lifetime is approximately eight times shorter. The high quantum yield and suitable lifetime make Pt-porphyrins appropriate as sensors of O2 diffusion and flexibility in heme proteins.

Janna Nibbs, Sergei A. Vinogradov, Jane M. Vanderkooi, and Bogumil Zelent "Flexibility in Proteins: Tuning the Sensitivity to O2 Diffusion by Varying the Lifetime of a Phosphorescent Sensor in Horseradish Peroxidase," Photochemistry and Photobiology 80(1), 36-40, (1 July 2004). https://doi.org/10.1562/2004-01-17-RA-046.1
Received: 13 January 2004; Accepted: 1 March 2004; Published: 1 July 2004
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