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The carcinogenic hydrocarbon 3.4-benzopyrene is soluble in aqueous solutions of different proteins. The solubilities are easily determined by the fluorimetric method. The fluorescence o. the hydrocarbon in the protein solutions is not quenched by molecular oxygen. Nevertheless only in presence of air (oxygen) an irreversible decrease of the fluorescence intensity occurs under irradiation with UV-light of wavelength 366 mμ, which is considerably faster than under nitrogen or in solutions of the hydrocarbon in ethanol or aqueous caffeine.
In the systems investigad, a correlation was found between the half-life period of the reaction and the SH-group activities. The participation of protein-SH-Groups in the 3.4-benzopyrene photoreaction is demonstrated by ampèrometric Ag⊕-titrations.
The influence of protein denaturation and inhibiting additives on the photoreaction are investigated by the fluorimetric method.
Irradiation- and oxygen-dependence of the reaction are analogous to the observations of photodynamic action and skin cancer induction by 3.4-benzopyrene.
By 366 mµ irradiation of β-lactoglobuline solutions containing 3.4-benzopyrene the heatdenaturation characteristics of the protein are changed. The same changes are produced without 3.4-benzopyrene by UV-light of the wavelength 280 mµ. Treatment of the β-lactoglobuline solutions with an amount of cigarette smoke, which certainly does not contain 3.4-benzopyrene in sufficient concentration, acts in the same direction.
Along with the changes in the protein properties the typical fluorescence of 3.4-benzopyrene vanishes. The hydrocarbon does not act as a catalyst in photodynamic action, but is chemically altered as well as the protein, at least in the system under investigation.
Diluted aqueous solutions of some proteins (bovine serum albumin, β-Lactoglobubin, Peroxidase) show weak phosphorescence lasting over several minutes after they have been irradiated with light in the range 3500-4200 A. Addition of Eosin after the irradiation amplifies in some cases the intensity of luminescence to a value of about hundred. If Eosin is present at the irradiation process the excitation to phosphorescence is possible with light of the wavelength 5460 A.
After denaturation processes which destroy the configuration of proteins (Urea, Guanidine-HCI. detergents, heat at higher pH) the ability of phosphorescence disappears altogether; likewise after blocking the SH-groups by benzochinone or a total oxidation or reduction of the SS-groups which causes an complete unfolding of the peptide chain.
In solutions of bovine serum-albumin irradiated with 3650 Å at room temperature and afterwards frozen to -178°C no radicals could be observed by measurements of electron-spin-resonance but they were detectable if the irradiation took place in the presence of H2O2.
The reactions Xanthinoxidase-Xanthine-O2, Peroxidase-H2O2 and bovine serum-albumin-H2O2-Fe (II) EDTA are accompanied by chemiluminescence. By comparison with the behaviour of oxidised serum-albumin it could be shown that the chemical reaction produces an excited state of the native protein.
The observations lead to the conclusion that the weak phosphorescence of long duration originates from a triplet-state which is sufficiently populated only as the consequence of cooperative phenomena attending the undisturbed α-Helix-structure of the protein.