Absorption Spectra of Proteins and Amino Acids After Ultraviolet Irradiation

Irradiation of proteins with ultraviolet light alters many of their physical and chemical properties ( 16) including the ultraviolet absorption spectrum.The present work is an attempt to explain the changes in this spectrum in chemical terms. MethodsEgg albumin was purified according to the method of Kekwick and Cannan (9) except that it was lyophilized after the third recrystallization.The lyophilized preparation, which contained 14.3 per cent nitrogen, was dissolved in 0.05 M phosphate buffer, pH 7.3, to give a 1 per cent solution.The substances tested, in concentrations of 0.001 to 0.01 M, were dissolved in the buffer.The solutions were irradiated for 24 hours, with constant stirring, with a General Electric 30 watt mercury resonance lamp which has predominant radiation at 2537 A. The dosage, as determined by a Hanovia ultraviolet meter, was approximately 1.7 X 10' ergs per sq.mm.Absorption spectra were measured in the Beckman quartz spectrophotometer.The results are expressed as extinction coefficients ((log lo/l)/cd) where log lo/l is the optical density, c is the concentration in gm. per 100 ml. for E or moles per liter for E, and d is the thickness of the cell in cm. RESULTS AND DISCUSSIONThe absorption spectrum of egg albumin before and also after various periods of irradiation is shown in Fig. 1.The observations are similar to those of previous authors (1,12,18,21).It should be noted that the extinction coefficient increases over the whole range of wave-lengths, but especially in the region of the original minimum around 2500 A and also above 3000 A where the unirradiated protein is almost non-absorbing.Since Lerner and Barnum (11) have shown that the absorption of ultraviolet light by protein can be accounted for quantitatively in terms of the