Reactivity of Singlet Oxygen Toward Proteins: The Effect of Structure in Basic Pancreatic Trypsin Inhibitor and in Ribonuclease A

The reactions of singlet oxygen, ¹O₂, with large peptides have been described previously. It was found that even in these systems, which in their native form are generally not supposed to possess a stable structure in solution, the polypeptide does impede the access of ¹O₂ to the amino acids that react readily with ¹O₂. Here we describe the ¹0₂ reaction with two proteins of well-defined structure. The quenching of ¹O₂ by bovine pancreatic trypsin inhibitor (BPTI) and by ribonuclease A (RNase A) was compared to that of a solution at the same concentration as those of its constituent amino acids that react readily with ¹O₂. The proteins were studied in their native form, when partly denatured by splitting their S-S bonds and when fully denatured. It was found that while in the native form the quenching rate constant was seven times lower in BPTI (2.2 vs 15.2 times 10⁷WM^-1 s^-1) and three times lower in RNase A (11.0 vs 32 times 10⁷M^-l s^-1) than in a mixture of its constituent amino acid residues, it increased upon denaturation reaching in the fully denatured state the value of the corresponding amino acid mixture. More striking is the effect of the protein structure when comparing the fraction of the encounters between ¹O₂ and protein, which cause damage to the protein, as reflected in the decrease of its biological activity. This decrease is assumed to be due to the chemical (oxidative) reactions of ¹O₂ in the protein. In the exceptionally stable BPTI the fraction of such encounters was 0.05 and in RNase A it was 0.2, whereas for the amino acid tryptophan in solution, 0.7 of the collisions with ¹O₂ led to a chemical reaction.     

QUENCHING OF TRYPTOPHAN PHOSPHORESCENCE IN ALCOHOL DEHYDROGENASE FROM HORSE LIVER and ITS TEMPERATURE DEPENDENCE

Abstract— The phosphorescence of alcohol dehydrogenase from horse liver (LADH) can be observed at room temperature. The quenching of this long-lived light emission, which comes from a tryptophan residue well buried within the interior of the enzyme structure, was measured. The rate constants for the quenching by the small oxygen molecule and by the I ^-1ion were found to be 1.4 → 10⁸ M ^-1 s^-1 and 10⁸ M ^-1 s^-1, respectively, at room temperature. The temperature dependence of the quenching yields an activation energy of about 14 kcal/mol. This activation energy and the meaning of the accompanying large pre-exponential factor in the Arrhenius equation, A = 10¹⁸ M ^-l s^-1, are discussed in terms of a model in which the quencher threads its way through the protein network.     

REACTIVITY OF SINGLET OXYGEN TOWARD LARGE PEPTIDES

Abstract— The reactions of singlet oxygen, ¹O₂, with amino acids and their derivatives have been studied previously. It was found that only five amino acid residues interact readily with ¹O₂. Here we describe its reactions with the large peptides melittin, neuropeptide Y (NPY) and insulin in their native and in their denatured forms. The singlet oxygen quenching by a polypeptide was compared with that of a solution at the same concentration as those of its constituent amino acids, which are known to react efficiently with ¹O₂. It was found that the quenching rate by such a mixture exceeded that of the polypeptides in their native form. The ratio of the rate constants for NPY to that of the corresponding amino acid mixture in solution was 0.75. For melittin in its monomeric form it was 0.83 and for a tetramer of melittin (at high ionic strength) it was 0.70. For native insulin the ratio of the rate constants was 0.55. For oxidized insulin with its -S-S- bridges opened the figure became 0.80. However, the quenching by all the polypeptides in their fully denatured form (in the presence of 6 M urea) equalled that of the corresponding amino acid mixtures. Although polypeptides are generally supposed not to possess a stable secondary structure in solution the effects are explained by shielding of some of the reactive amino acid residues in the chain by temporary folding or incipient secondary structures of the native polypeptide.
It is shown that the kinetics for a homogeneous solution of quenchers applies also to measurements in a polypeptide solution where the quenchers are localized along the polypeptide backbone and thus form clusters in solution.     

FLUORESCENCE LIFETIME STUDY OF THE DENATURATION OF RIBONUCLEASE-A

Abstract. Fluorescence quantum yield and lifetime measurements of the tyrosine residues in ribonuclease-A (RNase) were used to study the conformational changes involved in the denaturation of the enzyme. Measurements were done on RNase and on selectively acetylated RNase in the native, the partly denatured (reductive cleavage of S-S bridges or treatment with 8 M urea) and in the fully denatured state. The data were interpreted to mean that the opening of the S-S bridges causes large parts of the enzyme chain to unfold while leaving a hydrophobic region; including one of the tyrosine residues, intact. The biological activity of RNase is destroyed by this unfolding. Urea apparently does penetrate the protein coil but does not greatly affect the RNase structure since some of its biological activity is still retained. The opening of the S-S bridges in the presence of urea destroys the native conformation (and biological activity) completely leaving the protein in the form of an uncoiled polypeptide chain. It is suggested which parts of the protein structure might be affected by partial denaturation.     

PHOTOCHEMISTRY OF REDUCED LUMIFLAVIN IN ITS ACIDIC FORM

Abstract— The photooxidation of reduced lumiflavin in its acidic form, LfH 3 ⁺, takes place in two consecutive steps. Upon illumination of LfH 3 ⁺ in its absorption band at 313 nm the semiquinone, LfH 3 ⁺, is formed. Two LfH 2 ⁺ ions are consumed for every LfH 2 ⁺ formed. Illumination of the semiquinone in its absorption band at 495 nm causes further oxidation so that the oxidized LfH⁺ ion is formed. In this reaction one LfH 3 ⁺ ion is photolyzed for every LfH⁺ formed. In addition, a hydrogen atom is released in the photooxidation of LfH 2 ⁺. Mechanisms for the two photoreactions are proposed.     

Reactivity of singlet oxygen with tryptophan residues and with melittin in liposome systems

The reactivity of singlet oxygen, 1O2, with amino acids, polypeptides and proteins has been studied extensively in solution, in micelles and also in vesicles. Here we attempt to examine its reactivity with N-acetyltryptophan amide (NATA), with a tryptophan residue with a long aliphatic chain attached, Trp(CH2)16, and with melittin--a small membrane protein--in a solution containing liposomes. In such a heterogeneous system the sensitizer and/or the tryptophan residue can be located in the ambient D2O, in the liposome membrane or at the membrane-solution interface. The sensitizer meso-tetra(N-methyl-4 pyridyl)porphine tetratosylate (mTPTT) is located in the aqueous phase while hematoporphyrin (HP) is embedded in the membrane. The quenching of 1O2 by the tryptophan residues and by melittin in solution, when using either of the sensitizers, was compared with the data in the liposome-containing system. It was found that the location of the sensitizer and of the quencher in the liposome membrane or in the surrounding solution greatly affects the quenching rate constants of 1O2.     

CHARGE TRANSFER COMPLEXES BETWEEN POLYENES AND AROMATIC ACCEPTORS

Abstract. Both the cone pair electron donating ability of a polyene Schiff base and the strength of the bond formed indicate a weakening of the complex formed with increase in the conjugated double bond system.     

PRIMARY PHOTOCHEMICAL PRODUCTS OF DITHIOGLYCOLIC ACID IN AQUEOUS SOLUTION

Abstract— The photochemistry of dithioglycolic acid at 254 nm was investigated in deaerated aqueous solutions in the pH range 1.4–7.3. Initial yields of the primary photochemical products H₂S,–SH and aldehyde-(probably glyoxylic acid) were determined. The complex pH dependence of these simultaneously formed first stable products is interpreted in terms of the ground-state ionic equilibria, and in addition pH-dependent processes occurring in the excited state and labile intermediate sequence.
It is suggested that the photochemical mechanism involves two parallel pathways: one a hydrolytic splitting of S - S leading to H₂S formation (unaffected by the presence of O₂ or isopropanol), the other a free radical mechanism via C–S breakage, which is affected by the presence of O₂ or isopropanol.     

REACTIVITY OF SINGLET OXYGEN TOWARD AMINO ACIDS AND PEPTIDES

Quenching of singlet oxygen (¹O₂) in D₂O-ethanol by the amino acids tryptophan, tyrosine, histidine, methionine, cysteine and their derivatives was measured by exciting the sensitizers rose bengal or meso-tetra (N-methyl-4-pyridyl)porphyrin tetratosylate in the presence of oxygen and the above quenchers in solution. In our polar solvent, containing 75% D₂O on a molar basis it was found that (1) substitution of the aromatic ring in indole, phenol and imidazole by the electron-donating methyl group increases the total (i.e. nonreactive and reactive) quenching rate constant by a factor of five to eight. Free or blocked amino and carboxyl groups removed by two methylene groups from the ring counteract the above increase in the rate constant. The reactive quenching of singlet oxygen, which leads to oxidative destruction of the aromatic ring, correlates with the above substitution effects. It has been proposed that the quenching process takes place by formation of an exciplex between ¹O₂ and the quencher. Thus our results indicate that the better an electron donor the amino acid residue is the more pronounced is the charge transfer contribution in the exciplex formed with ¹O₂ and the more likely it is to lead to charge separation and hence to a chemical reaction. (2) Oligopeptides in solution or peptide bonds linked to the amino acid residue have only a minor effect on singlet oxygen. It can therefore be expected that the polypeptide chains per se in the protein network will not interact significantly with the single oxygen molecules present. The quenching of the latter should, to a first approximation, depend only on the presence of the above reactive amino acid residues and to their accessibility to ¹O₂ as well as on the effective dielectric constant within the protein structure.