K. Das

Photophysics of hypericin and hypocrellin A in complex with subcellular components: interactions with human serum albumin.

Abstract— Time‐resolved fluorescence and absorption measurements are performed on hypericin complexed with human serum albumin, HSA (1:4, 1:1 and ∼5:1 hypericin: HSA complexes). Detailed comparisons with hypocrellin A/HSA complexes (1:4 and 1:1) are made. Our results are consistent with the conclusions of previous studies indicating that hypericin binds to HSA by means of a specific hydrogen‐bonded interaction between its carbon‐yl oxygen and the N1‐H of the tryptophan residue in the HA subdomain of HSA. (They also indicate that some hypericin binds nonspecifically to the surface of the protein.) A single‐exponential rotational diffusion time of 31 ns is measured for hypericin bound to HSA, indicating that it is very rigidly held. Energy transfer from the tryptophan residue of HSA to hypericin is very efficient and is characterized by a critical distance of 94 Å, from which we estimate a time constant for energy transfer of ∼3 × 10–15 s. Although it is tightly bound to HSA, hypericin is still capable of executing excited‐state intramolecular proton (or hydrogen atom) transfer in the ∼5:1 complex, albeit to a lesser extent than when it is free in solution. It appears that the proton transfer process is completely impeded in the 1:1 complex. The implications of these results for hypericin (and hypocrellin A) are discussed in terms of the mechanism of intramolecular excited‐state proton transfer, the mode of binding to HSA and the light‐induced antiviral and antitumor activity.

The leghemoglobin proximal heme pocket directs oxygen dissociation and stabilizes bound heme

Sperm whale myoglobin (Mb) and soybean leghemoglobin (Lba) are two small, monomeric hemoglobins that share a common globin fold but differ widely in many other aspects. Lba has a much higher affinity for most ligands, and the two proteins use different distal and proximal heme pocket regulatory mechanisms to control ligand binding. Removal of the constraint provided by covalent attachment of the proximal histidine to the F‐helices of these proteins decreases oxygen affinity in Lba and increases oxygen affinity in Mb, mainly because of changes in oxygen dissociation rate constants. Hence, Mb and Lba use covalent constraints in opposite ways to regulate ligand binding. Swapping the F‐helices of the two proteins brings about similar effects, highlighting the importance of this helix in proximal heme pocket regulation of ligand binding. The F7 residue in Mb is capable of weaving a hydrogen‐bonding network that holds the proximal histidine in a fixed orientation. On the contrary, the F7 residue in Lba lacks this property and allows the proximal histidine to assume a conformation favorable for higher ligand binding affinity. Geminate recombination studies indicate that heme iron reactivity on picosecond timescales is not the dominant cause for the effects observed in each mutation. Results also indicate that in Lba the proximal and distal pocket mutations probably influence ligand binding independently. These results are discussed in the context of current hypotheses for proximal heme pocket structure and function. Proteins 2002;46:268–277.

A comparison of the excited-state processes of nearly symmetrical perylene quinones: hypocrellin A and hypomycin B

Abstract The excited-state photophysics of two naturally occurring nearly symmetrical perylene quinones are discussed: hypocrellin A and hypomycin B. Hypocrellin A has a hydroxyl group peri to a carbonyl group on either end of its long molecular axis in addition to a hydroxyl group on its seven-membered ring. On the other hand, hypomycin B is unique among this class of known naturally occurring perylene quinones in that it possesses only one hydroxyl group, which is peri to a carbonyl group. These quinones are investigated in different nonionic micellar environments. For hypocrellin A and hypomycin B, a micelle concentration 10 times in excess of that used for hypericin in a previous study, i.e. 100 times the critical micelle concentration, must be employed to obviate aggregation. Under such conditions, the p K a of the peri hydroxyl groups of hypocrellin A have been determined to be 8.9. The p K a of the protonated carbonyl groups could not be measured. A comparable value is estimated for hypomycin B. The differing solubilities and behaviors of hypericin and hypocrellin in micellar environments are briefly discussed in the context of their biological activity. The excited-state processes in hypocrellin A and hypomycin B are compared on a time scale of several hundreds of picoseconds. No deuterium isotope effect is observed for hypomycin B. This result is discussed in the light of the previous assignment of the primary photoprocess in hypocrellin A to hydrogen atom transfer.

Fluorescence Properties of Recombinant Tropomyosin Containing Tryptophan, 5-Hydroxytryptophan and 7-Azatryptophan

Tropomyosin mutants containing either tryptophan (122W), 5‐hydroxytryptophan (50H122W) or 7‐azatryp‐tophan (7N122W) have been expressed in Escherichia coli and their fluorescence properties studied. The fluorescent amino acids were located at position 122 of the tropomyosin primary sequence, corresponding to a solvent‐exposed position c of the coiled‐coil heptapeptide repeat. The emission spectrum of the probe in each mutant is blue‐shifted slightly with respect to that of the probe in water. The fluorescence anisotropy decays are single exponential, with a time constant of 2–3 ns while the fluorescence lifetimes of the probes incorporated into the proteins, in water, are nonexponential. Because tryptophan in water has an intrinsic nonexponential fluorescence decay, it is not surprising that the fluorescence decay of 122W is well described by a triple exponential. The fluorescence decays in water of the nonnatural amino acids 5‐hydroxytryptophan and 7‐azatryptophan (when emission is collected from the entire band) are single exponential. Incorporation into tropomyosin induces triple‐exponential fluorescence decay in 5‐hydroxytryptophan and double‐exponential fluorescence decay in 7‐azatryptophan. The range of lifetimes observed for 5‐hy‐droxyindole and 5‐hydroxytryptophan at high pH and in the nonaqueous solvents were used as a base with which to interpret the lifetimes observed for the 50H122W and indicate that the chromophore exists in several solvent environments in both its protonated and unprotonated forms in 50H122W.

Does Solvent Influence the Ground-state Tautomeric Population of Hypericin?¶

Abstract Nuclear magnetic resonance measurements indicate that hypericin exists in the same “normal” tautomeric form irrespective of whether the solvent is dimethyl sulfoxide or tetrahydrofuran. This result is discussed in the context of previous experimental and theoretical work. It is concluded that solvent perturbations cannot induce tautomerization in hypericin.