Flexibility in proteins: tuning the sensitivity to O2 diffusion by varying the lifetime of a phosphorescent sensor in horseradish peroxidase.

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. K(d) = 18 microM when BHA binds to PtMP-HRP. The effect of BHA can be explained by two factors: (1) BHA reduces the accessibility of O(2) 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 k(q) = 234 mm Hg(-1) s(-1) in the absence of BHA and k(q) = 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 O(2) diffusion and flexibility in heme proteins.

Nitrosation of cysteine and reduced glutathione by nitrite at physiological pH.

Unlike the formation of nitrosothiols by nitrous acid, our study revealed that NO2- effectively reacted with L-cysteine or reduced glutathione (GSH) at pH 7.0 and 7.4, to form orange-pink products of S-nitrosocysteine (CySNO) or S-nitrosoglutathione (GSNO). The reactions were in a concentration-dependent manner. These products exhibited not only peak absorbances at around 340 and 540 nm, but also unique colors and patterns of mobility on cellulose thin layer chromatographic plates. In comparison, the S-nitrosation of dithiothreitol was noted exclusively under acidic pH. In addition, the S-nitrosation of hemoglobin (Hb) by either peroxynitrite (PN) or NO2- at pH 6.0 was detected via Western blot. The half-life of degradation of CySNO in NO2- solution was significantly shorter than that of GSNO at a wide range of pH. In the absence of NO2-, degradation of GSNO was facilitated by incubation with L-cysteine, but not L-serine. In the signaling process involving NO -->PN --> NO2- --> CySNO/GSNO --> NO, L-cysteine may function as a NO-carrier to reach shorter-distance targets, and also an "activator" to release NO from GSNO. Furthermore, L-cysteine may play a vital role in reducing (severe) oxidative stress.

Further study on S-nitrosation by nitrite.

At neutral pH, S-nitrosoglutathione was formed by the reaction of reduced glutathione and sodium nitrite. The degradation of S-nitrosoglutathione, presumably by transnitrosation/denitrosation, was catalyzed by L-cysteine, or CoA-SH. Additionally, from the crude extract of rat brain, one protein with a large molecular mass was nitrosolated with nitrite, and was split into duplet peptides noted in Western blot. Furthermore, the incubation of nitrite with IgG may generate the intermediates of active nitrogen/oxygen species and lead to significant production of gas bubbles.