Demonstration That Phosphorescent 6‐bromo‐2‐naphthyl Sulfate Can Be Used to Probe Heme Accessibility in Heme Proteins

Abstract

The phosphorescence properties of 6-bromo-2-naphthyl sulfate (BNS) in aqueous solution were studied. The phosphorescence lifetime is several hundred microseconds and is self-quenched. Although a fluorescent photoproduct is formed from BNS, it does not interfere with the decay properties of triplet-state BNS and its utility as a probe of the accessibility of the heme group in heme proteins. Quenching of BNS phosphorescence does not occur for the non-heme protein lysozyme and apomyoglobin but occurs by a dynamic mechanism with a quenching constant of 1-2 x 10(9) M-1 s-1 for cytochrome c and myoglobin and with a quenching constant of 6.2 x 10(9) M-1 s-1 for protoporphyrin IX. The phosphorescence of an inclusion complex of 1-bromonaphthalene and beta-cyclodextrin is not quenched by heme-containing proteins. The temperature and viscosity dependencies of the rate with which BNS phosphorescence is quenched by microperoxidase-11 are consistent with unit quenching efficiency. These results indicate that quenching of BNS phosphorescence occurs only upon contact with the quencher, and the quenching constant can be used to assess the degree of accessibility of the heme group.

Accessibility of oxygen with respect to the heme pocket in horseradish peroxidase

“…As we pointed out in the introduction, understanding the mechanism by which small molecules penetrate proteins and access reactive centers is of importance. Our quenching studies coupled with the results of Schuh and associates,48–50 Barboy and Feitelson,30, 31 Papp et al,45 and Brunet et al,63 indicate that the heme group in HRP is much more protected than the heme in myoglobin and metal‐substituted hemoglobin. The remarkable finding is that the addition of the substrate analog drastically affects the penetration of oxygen, as indicated by the magnitude of the oxygen quenching rate constant.…”

“…In this experiment, BNS is excited and quenching by the porphyrin compound is examined. This reaction can indicate the exposure of heme to solvent,48 because only when the heme is exposed can it quench the BNS phosphorescence. Phosphorescence quenching profiles of BNS as a function of porphyrin‐containing substances are given in Figure 6.…”

“…We have also employed 6‐bromo‐2‐naphthyl sulfate (BNS) as an external phosphorescent probe of heme accessibility. Schuh and collaborators introduced the use of this molecule 48–50. BNS is about the same size as the aromatic substrates of HRP and can be used to determine whether larger molecules can access the heme pocket.…”

Optical thermometer based on the stability of a phosphorescent 6-bromo-2-naphthol/α-cyclodextrin2 ternary complex

“…The laser-induced phosphorescence intensities of the BN(CD) 2 complex and 6-bromo-2-naphthyl sulfate (BNS) in deaerated solution are similar. 22 Since similar phosphorescence intensity of BNS is produced by both laser and broad bandpass-filtered, 7 J flashlamp excitation, it should also be possible to produce adequate phosphorescence from the BN(CD) 2 complex using a simple, relatively inexpensive flashlamp excitation source. 3) The instrumentation required to produce and detect long-lived phosphorescence is relatively low in cost.…”