Radiat. Phys. Chem. Vol. 20, No. 4, pp. 241-245, 1982 0146-5724/82/100241-05503.00/0 Printed in Great Britain. Pergamon Press Ltd.

RIBOFLAVIN AS A NEW VERSATILE SOLUTE FOR THE DETERMINATION OF OH

RADICAL RATE CONSTANTS BY THE COMPETITION KINETIC TECHNIQUE

KAMAL KISHORE, P. N. MOORTHY and K. N. RAO Chemistry Division, Bhabha Atomic Research Centre, Trombay, Bombay 400085, India

(Received 20 April 1982)

AbstraetwRiboflavin (RF) has been used as a standard for the determination of OH radical rate constants by the competition kinetic method. In oxygenated solutions its radiolytic decomposition was found to closely correspond to G(-RF)= (l/2)Go.. Its rate constant was first evaluated by employing ethanol as the standard and taking kOH+C2H~OH = 1.85 X 109 dm3mol-ts -1. The kOH+RV value

+ 10 3 1 I was found to be constant (= 1.2 _ 0.05 × 10 dm mol- s- ) over the pH range 1-11. Using this value the rate constant for reaction of a number of other solutes have been determined and compared with literature values. The advantages of riboflavin over other stanclard solutes are discussed.

INTRODUCTION RATE CONSTANTS for reaction with OH radicals have been determined in the past by either follow- ing the disappearance of OH cl) or the formation of a product transient ~2) in pulse radiolysis experi- ments or by the competit ion kinetics method in both pulsed ~3) and steady state <4) experiments. In the latter method a standard or reference solute whose rate constant for reaction with OH radicals is known, is used to compete with the unknown compound of interest.

The first method is limited by the fact that OH has a very weak absorption t2~ in the far UV region and interference from product transients makes it very difficult to follow its disappearance. Also if the compound (for which OH rate constant is to be determined) has more than one site where OH can attack and can, therefore, give more than one product transient, then following the formation of one of the product transients will not give the rate constant for reaction with the compound as a whole. In such cases the competit ion kinetics method gives more reliable results. In this con- nection, steady radiolysis methods are much sim- pler than pulsed methods as they do not require sophisticated equipments. However , it is essential that the radiation chemistry of the standard solute is understood before it is employed in steady state radiolytic competit ion kinetics methods. In the past, compounds like thymine ~5), PNDA (para nitrosodimethylaniline) c4> and Safranine-T t6) had been used as standards. In these cases the bleach-

ing of the optical absorption of the standard at a particular wavelength was followed to estimate G(-S) at different values of [X]I[S] where X is the unknown and S the standard compound. As thymine absorbs in the UV region it can be used only in those cases where the other solute has no absorption in this region this rules out many organic compounds which generally absorb stron- gly in the UV. The disadvantage with PNDA is that it can be used only at pH 9 while the use of Safranine-T is limited to the pH range 0.5-5.5. Even in this range Safranine-T has a pKa at ~ 2 and its rate constant for reaction with OH is different at pH's below and above this value. Also it cannot be used at concentrations higher than 2 x l 0 - S m o l d m -3 because of aggregation and hence uncertainity in the determination of its concentration by light absorption. To overcome these difficulties we have tried to develop an alternative standard. The compound chosen is riboflavin. It has the following merits: Its concen- tration can be accurately measured by its ab- sorption at 445 nm in the pH range 2-8. There is a bleaching of the 445 nm band under conditions such that only OH radicals react (i.e. aerated solu- tion). The radiolysis products do not absorb at this wavelength. In air saturated solutions the radioly- sis mechanism is unambiguous and G ( - R F ) = (1/2)Goa. It has no pKo ~7) in the range 0.25-10 and hence can be used over a wide pH range. Also higher concentrations (8-10 x 10 -5 mol dm -3) can be employed. The results of using riboflavin as a

RPC Vol. 20, No. 4---A 241

242 KAMAL KISHORE et al.

standard for estimation of OH radical rate con- stants are presented in this paper.

EXPERIMENTAL

Riboflavin used in this study was from Sigma Chemical Company and was used as such. No impurities were detectable by TLC. Other vitamins used were the purest available grade obtained from either Sigma Chemical Company or Calbiochem. All other chemicals were either BDH "Analar" or Merck "G. R. " grade. Triple distilled water was used to make all the solutions. Absorbance of the solutions was measured with a Beckman DU spec- trophotometer. Polarographic measurements were car- ried out using a Bruker Model E310 Modular Polaro- graph. A ~°Co y-source with a dose rate of 4x 1020 eV dm 3 min-t was used for irradiating the solutions. H202 in irradiated solutions of riboflavin was estimated by Gupta's method (s) as iodide method cannot be used due to strong light absorption by riboflavin at 350 nm. In Gupta's method absorbance of the Ferric Xylenol orange complex is measured at 540 nm, where riboflavin has no absorption. TLC of irradiated riboflavin solutions was carried out using the method of Treadwell et al. (9) The products were detected by keeping the developed plate under UV lamp (A = 365 nm). These were scraped and digested with hot water and their absorption spectrum was recorded. The products had negligibly small ab- sorption at 445 nm. In most of the cases the total dose used was 8.6 x 102° eV dm -3 and with this dose riboflavin decomposition was less than 20%. The estimation of G(-RF) was always done by adjusting the solution pH to 7. At all the doses employed, G(-RF) remained constant for air saturated riboflavin solution in the concentration range 4 × 10 _5 to 1.2 × 10 -4 mol dm -3.

RESULTS AND DISCUSSION

The standard solute used in competition kinetics method for estimation of OH rate constant should satisfy the following criteria.

(1) The compound should be available in pure form and it should be possible to estimate it ac- curately without any intereference from its radiolysis products. (2) Its radiation chemistry should be well understood, i.e. it should be pos- sible to correlate its decomposition and the for- mation of radiolysis products with the yields of primary species. The decomposition or the for- mation of the product which is followed, should be due to reaction with OH radicals only. The stan- dard should be inert towards other radiolytic spe- cies or these should be preferentially scavenged by some other solute such as oxygen. (3) G(-S) should be constant over the concentration range of the standard to be used. (4) It is desirable that the standard has no pK~ over a large pH range; otherwise there may be a change in the radiolysis mechanism below and above the pKo. (5) The compound for which OH rate constant is to be determined and its radiolysis products should not interefere in the estimation of the standard.

We found that riboflavin satisfied all these cri- teria. It is available in pure form and it can be accurately estimated by measuring its absorbance at 445 nm in the pH range 2-8, even in irradiated solutions. This has been confirmed independently both by polarography and by TLC. By the latter technique we have also confirmed that its radioly- tic products (formed in air saturated solutions) do not absorb at 445 nm. If the compound for which OH rate constant is to be determined and its radiolysis products do not absorb at 445 nm or if appropriate correction can be applied for their absorbance, then we can easily follow the decom- position of riboflavin resulting from its reaction with OH radicals, even in presence of other solutes.

It is already known ,o~ that semiriboflavin radi- cals formed by the reaction of eaq- with riboflavin and the final reduction product leucoriboflavin"" can be easily oxidized back to riboflavin by oxygen. Hence in air saturated solutions even if some of the e~q and H react with riboflavin, they will not lead to decomposition of riboflavin. This means that in air saturated solutions the decom- position or decolouration of riboflavin is entirely due to OH radicals. G( -RF) in neutral air saturated solutions has been estimated to be about 1.2, which is close to (1/2)GoH indicating that 2 OH radicals are required to decompose one riboflavin molecule. G(H202) in air saturated solu- tions was found to be equal to GH2o2 + 1/2 (Geaq + GH). These stoichiometries were observed at all pHs in the range 0-11 (Table 1). In N20 saturated neutral solutions of riboflavin, G(H202) was found to cor- respond to the molecular yield. These results in- dicate that the riboflavin radical formed by OH reaction does not react with H202.

The plots of (1/2G(-RF)) vs ([S]/[RF]), for some of the solutes are shown in Fig. 1. The plots are linear but do not have the same intercept as G ( - R F ) is dose dependent at lower pHs and we have plotted the actual G ( - R F ) values at the dose employed. From the slope and intercept of these plots one can calculate the rate constant for the unknown solute, knowing the rate constant for the standard according to

1 k x + o n . 1 Slope= GOH s*on ann Intercept = Gon'

where kx.on and ks+on are the rate constants for reaction of OH with the unknown solute (X) and the standard (S), respectively. Knowing ks+on, kx+on can be calculated.

The OH rate constant for riboflavin itself was found by this method using ethanol as the start-

Riboflavin as a new versatile solute for kinetic technique 243

TABLE I . G(H202) AND G(-RF) IN AIR SATURATED 'y- IRRADIATED RIBOFLAVIN SOLUTIONS AT VARIOUS pH's

G(-mr) Q(R2o2)

0 1.35 2.65

1.5 1.23 2.40

3.0 1.26 2.30

4.3 1.20 2.28

6.8 1.22 2.27

9.0 1.25 2.30

10.0 1.27 2.20

11.0 1.30 2.10

C~val~es given here are initial @-values extrapolated to

• ~ 0 dO8@*

dard and following the bleaching of riboflavin at 445nm. Ethanol was chosen for this purpose because the value of koH+E,OH (=1.85× 109din 3 tool- ' s - ' ) is well established, a'~2'm3) From the slope and intercept of the plot in the riboflavin- ethanol system, kOH+RF was calculated to be 1.2_+ 0.05 x 10 m dm 3 mol -~ s - ' . This value does not change with pH over the range 1-11 (see Table 2). Thus this standard can be used for determining rate constants over a much wider pH range than was hitherto possible with other standards.

Using the above value of kO..RF, OH rate con- stants have been determined for a number of compounds and compared with literature values in Table 3. The agreement is good in many cases. In the case of a number of B-group vitamins OH rate constants have been determined earlier by Moor- thy and Hayon "''~6) by following the growth of one of the transients in pulse radiolysis experiments. These values however cannot be considered

B r - ( p H Z }

THIAMINE (hH6 '8 )

ISOPROPANOL (pH 6'8)

Br" (pH g)

I I I I 5 10 15 2 0

ix] [RF]

FIG. I. Competition kinetic plots for the reaction of OH radicals with various solutes.

244 KAMAL KISHORE et al.

TABLE 2. O H RATE CONSTANT FOB RIBOFLAVIN AT VARIOUS pH's

pH 8taada_~ k0H+Staadard k0H+Rib°flavin

(dm3mol-ls -1 ) (dm3mol-ls -1 ) (a) (b)

1

2

3

4.S

6.8

8.0

9.0

11.0

Thymine

Ethanol

Carbonate

4.7 x 109 1.17 x 1010

1.85x109 1.25 x 1010

" 1.18 x 1010

" 1.18 x 1010

" 1.20 x 1010

" 1.20 x 1010

" 1.20 x 1010

4.1 x 108 1.23 x 1010

(a) Prom Ref. 17 (b) Present data

TABLE 3. O H RATE CONSTANTS FOR VARIOUS COMPOUNDS kOII+RIBOFLAVIN = 1.2 X 1O l0 dm 3 tool-' S -t

Compound pH Rate constaat as determined by using Literature Value riboflavin as standard

(~3mol-ls "1 ) (a~3=of'la-1)

Metl~d on which Literature value is based (a)

Thymine 6.8 4.2 x 10 9 4.7 x 10 9

Isopropanol 6.8 2.2 x 1~ 2.0 x 10 9

Dioxsae 6.8 2.2 x 10 9 2,4 x 10 9

CNS- 6.8 9.6 x 109 1.1 x 1010

I" 6.8 2.0 x 1010 1,0, 3.4 x 1010

Bz- 2.0 6.7 x 10 9 5x10 9, lx1010

Br- 6.8 1.4 x 109 1.2 x 109

Br- 9.0 1.3 x 109 1.1 x 109

CI" 2.0 4.0 x 10 7 1.5 x 1010

TI + 6.0 7.0 x 10 9 7.6 x 10 9

Thiaxcine 6,S 5.0 x 10 9 3.0 x 109

( ~ - 4 . 7 )

Niootinsmside 6,8 1,3 x 109

Pyzidoxia 6.8 8.6 x 10 9 6.3 x 10 9

Folio acid 6.8 1.05x 1010 9.6 x 10 9

( ~ " 5.1) Pemthothemio acid 6.8 3.6 x 109 4.5 x 109

10 1010 Tryptophan %0 1.35x 10 1,2 x

RRjC Ump. with FezTocysnido

tt

i~22~Comp, with f~m~ation

PR Prod. form

PR P=od. ,form

PR,Prod. form, S

PR Prod. form

S (m~A)

PE Prod, form

PE Prod. form PR Prod. forum (16)

re, pred. form (14) ~ Proa. r ~ (15)

PR Prod. form (16)

PR Prod. form

Notes: (a) Ref 17 umloss otherwise stated. PR - P~Ise Radiolyede; Prod. forum . Product tzansient formation! S - steady state competition kinetics (cOmpetition with nol-te i-dioated)

Riboflavin as a new versatile solute for kinetic technique 245

representative of the compound as a whole because of the presence of many sites for OH attack in these complex molecules. The pulse radiolysis values obtained by following the rate of build up of OH product transient in such complex molecules can be expected to be lower than the true value and this appears to be the case for thiamine, pyridoxin and folic acid, although the difference between the present value and that reported by Moorthy and Hayon is only marginal in the case of the last compound. In the case of pantothenic acid the deviation is in the opposite direction. In view of the difficulty involved in following weak absorbance changes of the product transient from this compound, the present value may be taken as more authentic.

The k O H + C l - value at (pH2) is much lower than the accepted literature value. It would appear that the C12- formed by reaction of Cl- with OH reacts with riboflavin. This was verified in N20 saturated solutions containing 0 .1moldm -3 Cl- where riboflavin decomposition was still observed. No decomposit ion is expected if Cl2- formed by reaction of OH with Cl- did not react with riboflavin.

Thus riboflavin appears to be a useful standard for determining OH radical rate constants by the competit ion kinetic method except in those cases where the radicals formed by reaction of OH with the compound of interest themselves react with the standard. In any given case this aspect must be verified experimentally before hand.

R E F E R E N C E S

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3. G. E. ADAMS, J. M. BOAG and B. D. MICHAEL, Trans. Faraday. Soc. 1965, 61, 1417.

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5. J. F. WARD and L. S. MYERS JR., Radiat. Res. 1965, 26, 483.

6. D. G. MARKETOS, Z. fur Physt~ Chem. Neue Folge, 1969, 65, 306.

7. E. J. LAND and A. J. SWALLOW, Biochemistry, 1969, 8, 2117.

1. B. L. GUPTA, Microchem. J. 1973, 18, 363. 9. G. E. TREADWELL, W. L. CAIRNS and D. E. METZLER,

J. Chromatogr. 1968, 35, 376. 10. A. J. SWALLOW, Nature, 1955, 176, 793. l 1. M. J. GmioN and J. A. RYND, Biochem. Biophys. Res.

Comm. 1969, 34, 594. 12. J. H. BAXENDALE, E. M. FIELDEN and J. P. KEENE

in Pulse Radiolysis, (Edited by M. Ebert), p. 217, Academic Press, New York, 1965.

13. R. L. WILLSON, C. L. GREENSTOCK, G. E. ADAMS, R. WAGEMAN and L. M. DORFMAN, Int. J. Radiat. Phys. Chem. 1971, 3, 211.

14. P. N. MOORTHY and E. HAYON, J. Am. Chem. Soc. 1975, 97, 2048.

15. P. N. MOORTHY and E. HAYON, J. Org. Chem. 1976, 41, 1607.

16. P. N. MOORTHY and E. HAYON, J. Org. Chem. 1977, 42, 879.

17. L. M. DORFMAN and G. E. ADAMS, 1973, NSRDS- NBS 46.