ON T H E KINETICS OF TYROSINE DESTRUCTION AND DOPA FORMATION BY ULTRA-VIOLET RAYS L. EARLE ARNOW Laboratory of Physiological Chemistry, University of Minnesota, M i n n e a p o l i s , Minnesota Received September 22, 1037
One of the products produced when I-tyrosine is destroyed by ultraviolet radiant energy is dopa (3,4-dihydroxyphenylalanine)(2). In the experiments described in t h i s paper, tyrosine and dopa have been determined by methods previously reported by the author (1). The only significant modification in the analytical procedure consisted in the use of a compensation cup' in the dopa determinations. This was necessary because the irradiated solutions developed a brown color. This color was precipitated by the reagents used in determining tyrosine, and did not interfere with the determination of this compound. A Victor therapy quartz-mercury arc (air-cooled)' was used as a source of ultra-violet radiant energy. This instrument, as used, operated with a voltage drop of 6.5 t o 70 volts across the quartz-mercury tube. All solutions were irradiated a t a distance of 30 cm. from the light source. In a preliminary experiment 20 cc. of tyrosine solution, containing 22.1 micromoles of tyrosine, wits placed in a test tube. The tube was loosely stoppered and immersed in a boiling water bath for 6 hours. Since no dopa was Eormed by this procedure, the solutions exposed to the lamp were not cooled. In general, the temperature of the solutions undergoing irradiation quickly rose to about 48-50°C. and remained practically unchangrd until the lamp was turned off. The kinetics of the reactions were investigated by placing 20-cc. portions (containing 22.1 micromoles of tyrosine in distilled water) of tyrosine solution in well-corked clear quartz test tubes (25-cc. capacity) and exposing these solutions to the radiant energy for varying lengths of time. KINETICS O F TYROSINE DESTRUCTION
The results of tyrosine analyses are given in table 1. T represents micromoles of tyrosine present in the 20 cc. of solution, t represents time, and For a description of the use of the compensation cup see J. F. McClendon (A Manual of Biochemistry, p. 305. John Wiley and Sons, New York (1934)). This lamp was made available t o the author through the courtesy of Dr. W . K. Stenstrom and the University of Minnesota Hospitals. 415
416
L. JZARLE ARNOW
D represents micromoles of dopa present i n 20 cc. of solution. If log T is plotted graphically as a function of t, a straight line results. The differential equation relating T and t is thus
in which k1 is the velocity constant of the reaction (t is measured in hours). The integrated form of this expression is
T
=
Toe-k1t
where T o refers to the amount of tyrosine initially present in 20 cc of solution. The value of k l , calculated from the data, is 0 . 0 i l . Calculated values of tyrosine are given in table 1. TABLE 1
____ f
Destruction of tyrosine and formation of dopa by ultra-violet rays -___ D D T T (AxALYTICAt)
-
hours
nicrorsoles i n 83 cc.
0 1
22.1 20.1 19.4 18.1 16.4 14.4 8.8 7.2
2 3 4 6 12 15.8 -
__-____
1 I II
’
11
(CALCUtATED)
(ANALYTICAL)
mraomoles in $0 cc.
mirrornolea i n 90 cc.
nimomores i n EO cc.
(CALCULATED)
20.1 19.2 17.9 16.6 14.6 9.4 7.2
0.4 0.7 1.2 1.9 2.2 2 .6 2.8
0.5 0.7 1.2 1.6 2.1 2.5 2.8
I _ _ _ _
’
___-_
The above equations indicate that the destruction of tyrosine in these experiments occurred as a first-order reaction. Verification of this concluqion was obtained by irradiating tyrosine solutions of different initial concentrations for 4 hours. The results are given in table 2. These data indicate that the fraction of tyrosine still present in a given time interval n as independent of the initial concentration, a necessary condition of first-order reactions. Thc absorption spectrum of tyrosine in aqueous solution is given in the International Critical Tables (3). The molecular extinction coefficients of tyrosine vary from about 5900 at 2800 A. U. to about 1000 at 2460 .4. U. hIcAlister’s (4)data indicate that there are four strong mercury emission bands in this region. a t 2804, 2652, 2536, and 2483 4.E., respectively. The emission bands at 2967 A. U. and 3022 A. U. are relatively of much less importance, because the inolecular extinction coefficients of tyrosine at these wave lengths are relatively low. Calculations using these figures show that practically all the radiant energy between 2460 A. U. and 2800 A. U. is absorbed by the tyrobine solutions undergoing irradiation. This
DESTRUCTION OF TYROSINE BY ULTRA-VIOLET RAYS
417
fact suggests that the mechanism of tyrosine destruction may be of the type indicated by the following equations, in which X represents an active intermediate :
X
T+hv--+X
(1)
X+T
(2)
+ T -+ D + other products
(3)
If reaction 2 is rapid as compared with reaction 3, this series of reactions would result in a first-order destruction of tyrosine. In the above argument it is tacitly assumed that an unlimited supply of oxygen is available, a condition which is met by the experimental procedure, since a large excess of oxygen is always present. No tyrosine is destroyed in a 4-hour period when tyrosine solutions are irradiated in vacuo. TABLE 2 Destruction of tyrosine in a 4-hour period T
TO ~
mimonolea in 80 cc.
5.5
11.1 22.1
I
T -
To
micromoles in 80 cc.
4.1 8.3 16.4
0.75 0.75
0.74
KINETICS OF DOPA FORMATION
The rate of dopa formation should be given by the equation
where D refers to the micromoles of dopa formed in the irradiated solution, and where a is a constant. If a is 1, each moleccle of tyrosine which is destroyed results in the formation of a molecule of dopa; if a is less than 1, it represents the fraction of destroyed tyrosine molecules which form dopa. One isolated experiment which illustrates that dopa is destroyed by the radiant energy was performed. Twenty cubic centimeters of dopa solution (containing 5 07 micromoles of dopa) was exposed to the lamp for 4.5 hours. Analysis showed that 29 per cent of the dopa had been destroyed. No tyrosine had been formed. If it is assumed that the destruction of dopa is a first-order reaction, D - d_ = kzD dt
418
L. EARLE ARNOW
where D represents micromoles of dopa present a t time 1, and kz is the velocity confitant. If the above assumptions are made, the rate of change of dopa in irradiated tyrosine solutions is given by the expression, dD = alclT
dt
- knD
Integrating this expression,
The only unknowns in this equation are a and k 2 . If data are taken from several points on the dopa-time curye, and the resulting equations are solved for a and k z , the following expression results:
This equation appears to approximate the experimental data fairly well. Calculated and analytical values for dopa are compared in table 1. This equation can be interpreted to indicate that one-third of the tyrosine molecules which are destroyed are converted to dopa; and that the destruction of dopa is of the first order, the velocity constant having a value of 0.074. The author wishes to thank Dr. Robert S. Livingston for his kindness in assisting the author in the interpretation of the data presented in this paper. REFERENCES (1) ARNOW,L. E.: Science 86, 176 (1937); J. Biol. Chem. 118, 531 (1937). (2) ARNOW,L. E.: J. Biol. Chem. 120, 151 (1937). (3) International Critical Tables, Vol. V, p. 373. McGraw-Hill Book CO., New York (1929). (4) MCALISTER, E. D.: Smithsonian Inst. Pub., Misc. Collections 87, 1 (1933).
