Photodestruction QuantumYield of Adenine in Aqueous A Physical Chemistry Experiment Mercedes Rivera Cayey University College, Cayey. PR 00633 Most undergraduate physical chemistry textbooks include photochemistry topics in their chemical kinetics or snectrosconv .-chapters. . However,. the physical . - chemistry currlculum is so extensive that sometimes it is not possible to discuss imnortanttopics like this in a two-semester course. A :partial soiution to this problem is t o incorporate a photochemistry experiment in the p-chem laboratory course. An examination of nine laboratory manuals reveals a lack of experiments dealing with the subject. The experiments described in these manuals discuss the determination of the quantum yield for a photochemical reaction, which is defined as the number of molecules of reactant consumed or product formed for each photon of light absorbed. The followine reactions are studied: ~hotobrominationof cinnamic acid i n carbon tetrachloride (i) and photohydrolysis of monochloroacetic acid (2). Many students show dissatisfaction with physical chemistry because the course has been taught as an abstract suhject and is often unrelated to the life sciences. For the laboratory course, we can select experiments designed to illustrate the principles of physical chemistry, to teach basic lahoratory procedures, and at the same time to be relevant to the lifesciences. In the present experiment, the photodestrurtinn quantum yield of adenine, one of the main components and light-absorbing centers of deoxyribonucleic acid (DNA) is determined in aqurous solution. The damaging effects of UV light on living organisms have been explained in terms of photoinduced base lesions in nucleic acids. These lesions, if unrepaired, could lead to lethal, mutagenic, or tumorgenic effec-ts (3.4). The study of the photochemistry of nucleic acid constituents has concentrated mainly on the photoreactivity of pyrimidine hases (5). For the latter the major photoproducts that have been identified are the cyclohutane-type dimers, and most of the evidence indicates that thymine dimers are responnihle for a majority of the lethal and mutagenic acid of 11V light radiation ( 3 , 5 , 6 ) . Although the photochemical information on the purine bases is scarce, the importance of the role of these bases in the interaction of lieht with nucleic acids has also been reported on aspecific adenine recognized (7).~orscGke(8,9) photoproduct formed in oligodeoxyadenylic acid chains, a result that suggests that a similar photoproduct may exist in irradiated DNA. Later, Bose et al. (10) reported evidence for the formation of an adeninethymine photoadduct in the deoxydinucleoside monophosphate and presented evidence for the formation of this photoproduct in UV-irradiated DNA. A kinetic study (11) of the UV decomposition of several purines in the presence of oxygen or nitrogen showed decomposition rates of zero order with respect to the purine concentration after an initial induction period. For adenine, decomposition was faster under oxygen as compared with reactions performed under nitrogen. Kland and Johnson
suggested oxidative breakdown with the amino group being the point of attack RNH,% R N H O H ROH ~ 02
H2O
The isolation of bypoxanthine in the photochemical oxidation of adenine was presented as further evidence in favor of this step. Photodestruction yields for purine free base in aqueous solutions have been reported by Arce et al. (12). For degassed solutions a yield bf 0.005 was obtained, whereas under aerobic conditions this yield was reduced to 0.001. The reducine effect of oxveen on the vield was explained in terms of t h e participation of the triplet state of purine in the destruction mechanism. This paper describes the determination of photodestruction ouantum vields of adenine in deoxveenated and aerobic aqueous solutkw a t room temperatu;; Although this experiment was carried out with a C a y 14, the measurements could he performed using a standard UV-visible scanning s~ectro~hotometer. This experiment requires onlv a lowpress& mercury lamp and 'elatively inexpensive and commonly available chemicals. Another benefit of this experim e n t is that topics such as excited states (~ablonskidiagram), photobiology, and isosbestic points can be introduced in the laboratorv discussion. A wavelengthat which more than one absorbing species have identical molar absorptivities is called the isoshestic. point (13). If the sum of the concentrations of two species in solution is held constant, there will be no change in absorbance a t this wavelength as the ratio of the species is varied. Thus, the isosbestic point is a crossine-over point in a soectrumat a wavelength where the absoibance-is independent of the ratio of the two concentrations of the absorbing.spe. cies. The existence of one or more isosbestic points in a system provides information regarding the number of species present. The presenceof twoor more isoshestic points is very strone evidence that there are two and onlv two species iuvo1;ed in the system, since i t would be highiy improbable that another species would have equal molar ahsorptivities a t these wavelengths (13). This criterion does not apply if a third eauilibrium c o m ~ o n e nis t nresent that has zero absorbance in the wavelength region ihvestigated (14). Experimental
Sample Preparation Sulutims of known concentration of adeninr (Aldrirh 99aoJ ranging from 3.7 X 10 1.04.5 X 10 5 M .were prepared by usmgamndard weighing and volumetric techniques. An aliquot of 3.0 mL of the solution was introduced into a 1-cm2quartz optical cell with quartzto-Pyrex graded seal and stoppered with a rubber septum cap. Samples were deoxygenated by bubbling nitrogen through the solution for 15min (prior to irradiation).Aerobic sampleswere prepared by bubbling Oz through the solution during the irradiations. Volume 66 Number 12 December 1989
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1rrad;ation Conditions The solutions were irradiated at room temperature with a lowpressure Hg photochemical immersion lamp (Ace Glass) set at 1.5 em from a metal cell holder mounted in an optical bench. A lamp of this type can he purchased for about $400-500. The distance hetween the cell and the light source should be kept fixed since this affects the incident light intensity on the samples. Actinometry The incident light intensity on the samples was measured by ferrioxalate actinometry developed by Parker (15) and Hatchard (16). The preparation of solutions and the experimental details concerning the use of the chemical actinometer are well described in the literature ( I n . A 1-cm2quartz optical cell containing 3.0 mL of actinometer solution was irradiated for approximately 60 s. The intensity (photons-') of the light beam was determined hy
where N(FeZt) is the number of Fez+ ions formed (17) during a certain irradiation interval t, $(Fez+)is the quantum yield of Fez+ formation, which has a value of 1.24 in the wavelength region of 254 to 366 nm, and 0.869 is the fraction of UV irradiation of 254 nm wavelength (mercury resonance line) produced by the lamp. The procedure followed to obtain this value, which is an average of at least three determinations, is described below. The total actinometric response to the light emitted by the mercury lamp was determined by irradiating ferrioxalate solutions in a quartz cell. This value was compared with the incident light intensity obtained when ferrioxalate solutions are phatolyzed in a Pyrex cell, which cuts light of wavelength less than 300 nm. The value of the fraction of UV irradiation of the particular light source should be determined in advance and given to the students.
WAVELENGTH (nm)
Figure 1. UVabsorption spectraof deoxygenatedadenine sample. A: unphotoiyred sample; B and C: after 5 and 10 min of photolysis, respectively.
Quantum Yield Determination The experiment waa performed in duplicate runs for both deoxy. genated and aen,bic samples. The U V rpectrum of samples was recorded in the wavelength regionof 210- :ldOnm prior t
