INTERACTION OF ACRIDINE ORANGE WITH POLY-a-~-GLUTAMIC ACID
1615
The Interaction of Acridine Orange with Poly-a-L-glutamic Acid’
by Gordon G. Hammes and Coli D. Hubbard Department of Chemistry, Cornell University, Ithaca, New York, and the Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts (Received December 13, 1966)
A kinetic study has been made of the monomer-dimer reaction of acridine orange (AO) and of the reaction between poly-a-L-glutamic acid (PGA) and A 0 with the temperaturejump method. Related spectral measurements have also been made. The formation of the A 0 dimer is essentially diffusion controlled, which indicates the dye “stacking” occurs in lo-’” sec or less. The observed relaxation effect in the PGA-A0 system can be described quantitatively by assuming two relaxation processes occur. Measurement of the relaxation times was made at pH 4.7, where PGA is predominantly in a helical conformation, and at pH 7.5 where PGA is in the form of a random coil. At both pH values, both relaxation times are independent of polyglutamic acid concentration at high concentrations of polymer, but show a marked dependence on the polyglutamic acid concentration when the polymer concentration is lowered. Only at low concentrations of PGA do the relaxation data show a significant dependence upon the acridine orange concentration. A possible mechanism of the PGA-A0 interaction is one in which the rate-controlling steps in the over-all complex formation are intramolecular and probably involve the displacement of solvent molecules and/or counterions from the vicinity of the polymer. However, other plausible mechanisms involving dye stacking and polymer aggregation cannot be excluded. The same general mechanism appears to be operative for both the helical and random-coil forms of PGA, although the relaxation times are considerably longer for the former.
Introduction Several investigators have examined the equilibrium properties of aqueous solutions of dyestuffs and polyanions. Acridine orange (AO), in particular, has been used for this purpose; this has resulted in an accumulation of data concerned with the spectra of A 0 bound to such polyanions as polyglutamic acid, polyadenylic acid, polyuridylic acid, RNA, DNA, etc.2 From changes in the visible absorption spectrum as the concentration of acridine orange is varied, it has been established that A 0 aggregates in aqueous solution. Parallel changes in the spectrum of A 0 upon addition of polyelectrolyes have led to the hypothesis that dye molecules bound to the polymer may be sufficiently closely located on the polymer molecule to interact with one another to form aggregates analogous to those found in free dye solution.28 The phenomenon of A 0 molecules aggregating while bound to a polymer is generally termed “stacking.” Bradley and Wolf28
have introduced a parameter called the “stacking tendency” as an arbitrary measure of the strength of interaction between such dye molecules. Other workers have suggested that the color changes occurring in A 0 when the amount of added polymer is varied can be attributed to interaction of the dye with different types of binding sites on the polymer.2b-d This latter idea, however, is inconsistent with the results obtained with electrolytes having only one type of monomer unit. By determining the kinetic parameters characteristic (1) This work was supported by grants from the National Institutes of Health (GM 07803 and GM 13292). (2) For example, (a) D. F. Bradley and M . K. Wolf, Proc. Natl. Acad. Sci. U . S., 45, 944 (1959); (b) R. F. Steiner and R. F. Beers, Sczence, 127, 335 (1958); (c) R. F. Steiner and R. F. Beers, Arch. Biochem. Biophys., 81, 75 (1959); (d) R. F. Beers, D. D. Hendley, and R. F. Steiner, Nature, 182, 242 (1958); (e) A. L. Stone and D. F. Bradley, J . Am. Chem. SOC.,83,3627 (1961); (f) A. M. Michelson, Ann. Rev. Biochem., 30, 133 (1961). (3) V. Zanker, Z . Physik. Chem., 199, 15 (1952).
Volume 70, Number 6 May 1966
1616
of the reaction between poly-a-L-glutamic acid (PGA) and A 0 in aqueous solution and of the association reaction between two single dye molecules to form a dimeric dye molecule, we hoped to gain some insight into the nature of the complex species present at equilibrium and to obtain information about the mechanistic processes which lead to such species. Of particular interest are the rates and mechanism of stacking because of the importance of stacking in nucleic acid structures. The association of A 0 with itself and with PGA are both very rapid processes, so that the temperature-jump method was used for all kinetic measurements. Since at room temperatures and high salt concentration (approximately 0.1 M ) PGA exists predominantly as an (Y helix in aqueous solution below about pH 5, and as a random coil above pH 7,4the role of the polyenion structure in the dye-PGA interaction can be explored. However, the kinetic and spectral data obtained permit only qualitative features of the over-all mechanism of the AO-PGA interaction to be assessed.
Experimental Section Materials. Acridine orange from the National Aniline Division was recrystallized twice from methanol. Poly-a-L-glutamic acid (Lot No. G-54, molecular weight given as 65,000) was obtained from Pilot Chemicals. All other materials were standard reagent grade chemicals. Deionized distilled water was used for the preparation of all solutions. Spectra. The absorbancy of solutions was measured in the range 430-520 mp at 25.0" with a Beckman hlodel DU spectrophotometer fitted with a thermostated cell housing. In practice, solutions were made simply by adding the appropriate amounts of stock solutions of polyglutamic acid and acridine orange to a buffer solution, which upon bringing to a standard volume wa:$ either 0.1 M sodium acetateacetic acid (pH 4.7), or 0.1 M sodium acetatetris(hydroxymethy1)aminomethane (pH 7.5). All pH measurements were made with a Radiometer pH meter. The over-all association constant for reactions between other polyariions and A 0 has been reported to be markedly decreased as the high ionic strength is increased :2a therefore, the identification of complex species has been possible only at buffer concentrations less than or equal to 10-3 M . On the other hand, temperature-jump experiments require an ionic strength of about 0.1 M . Consequently, spectra of PGA-A0 solutions were measured over a range of buffer concentration from to 10-' M . Kinetic Experiments. Details of the temperaturejump apparatus have been amply doc~mented.~-' The Journal of Physical Chemistry
GORDON G. HAMMES AND
COLIND. HUBBARD
Temperature-jump experiments to determine the rate constants for association and dissociation of dimeric A 0 were performed over a range of total A 0 concento 5 X M in 0.1 M tration from 1.75 X sodium acetate-acetic acid buffer(pH 4.7). The relaxation effect (observed a t 446 mp) has a relaxation time approaching the time resolution of the apparatus. To minimize any cavitation effects, solutions were made from freshly boiled distilled water. However, within experimental error, the relaxation times were identical with those determined when this precaution was omitted. Before a series of kinetic runs on the PGA-A0 system was performed, solutions of the background electrolyte, with and without A 0 and PGA, were introduced into the cell of the temperature-jump apparatus and tested to verify that no relaxation effects other than those relating to PGA-A0 interactions were being observed. (Although a very fast relaxation process due to A 0 aggregation was observed with A 0 in buffer solution, the amount of free A 0 in solution when polymer is present is sufficiently small so that only a negligible amount of A 0 dimers is present and, consequently, this relaxation effect is not observed.) At both pH values investigated, 4.7 (ahelix) and 7.5 (random coil), the relaxation spectrum was obtained over a range of PGA concentrations from about 5 x l W 4 to 2 x loT2 M (the molarity of PGA is expressed in terms of the monomer) and at A 0 concentrations of 5.0 X and 2.5 X M . All kinetic runs were performed at 25.0" in 0.1 M buffer. The wavelengths used for observation of the chemical relaxation were 495 mp at pH 4.7 and 506 mp at pH 7.5. Because of the known sensitivity of acridine dyes to light, solutions containing A 0 were exposed to light for the absolute minimum of time; in the temperature-jump experiments the incident light was blocked from the cell during the period between successive temperature jumps. Results and Treatment of Data. The large differences in spectra of other polyanion-A0 complexes at low buffer concentration which occur upon varying the mole ratio of reactants have already been mentioned.28 The peak observed at 440-450 mp when the polymer/ A 0 ratio ([P]/[AO]) is near 1, gives way with increase in [P]/[AO] to one a t 460-470 mp. Further increase in [PJ/[AO] yields a band in the region of 500 (4) P. A. Doty, A. Wada, J. T. Yang, and E. R . Blout, J. Polymer Sei., 23, 851 (1957). (5) G.G. Hammes and J. I. Steinfeld, J. Am. Chem. Soc., 84, 4639 (1962). (6) G. G. Hammes and P. Fasella, ibid., 84, 4644 (1962). (7) R.E.Cathou and G . G. Hammes, ibid., 86, 3240 (1964).
INTERACTION OF ACRIDINE ORANGE WITH POLY-WL-GLUTAMIC ACID
1617
s 6
e
f
?' o
4b
4;O
4;O
4k
'
I
I
I
4& 490 500 510 520 X (mpl
Figure 2. Spectra of solutions a t pH 4.7, 25.0°, and M : - [PGA] = 0, 0.1 M buffer; [AO] = 2.5 X , [PGA] = 3.34 X M ,0.1 M buffer; [PGA] = 3.34 X lO-'M, 0.001 M buffer; a -,[PGA] = 2.0 X 10-2 M , 0.1 M buffer; and . ., [PGA] = 2.0 X 10-2M, 0.001 M buffer.
-_--.-- ..
X (mpb Figure 1. Spectra of solutions a t pH 7.5, 25.0°, and [AO] = 5.0 X 10-6M: , [PGA] = 0, 0.1 M buffer; -, [PGA] = 1.33 X 10-8 M , 0.1 M buffer; *l [PGA] = 1.33 X 10-8 M , 0.001 M buffer; [PGA] = 3.37 X 10-2 M , 0.1 M buffer; and . . ., [PGA] = 3.37 X 10-2M, 0.001 M buffer.
- -
-- . .
e ,
--
--]
mp, with a corresponding decline in the one a t 460470 mp. The value of the mole ratio required to produce these changes was found to be strongly dependent upon the particular polymer. The shortwavelength peak was considered to be characteristic of a highly stacked complex, while the longer-wavelength peaks were thought to represent the cases of two dye molecules interacting (dimer stacks) and no stacking, respectively. Observations on the PGA-A0 system at' pH 7.5 a t buffer concentrations of M are, in general, consistent with those for other systems described, although there was no evidence of a predominating peak near 500 mp in our measurements. Some typical spectra are shown in Figure 1. It may be that a higher ratio of [PGA]/[AO] than was used is required to produce the absorption band a t 500 mp, or that only small amounts of unstacked complexes are formed when the polyanion is polyglutamic acid. Interpretation of kinetic data would be greatly facilitated by an exact knowledge of the species present at equilibrium, but unfortunately a characterization of
the solutions used for the kinetic experiments in terms of the stacking model cannot be definitive owing to the dissociation of complexes and concomitant masking of the spectra which occurs at high buffer concentrations. It is difficult to ascertain whether the appearance of the band a t 495 mp should be attributed to free dye in monomeric form or to dye in monomeric form bound to polyglutamic acid or to a combination of both of these species. Most probably, solutions used for kinetic measurements contained a mixture of a small amount of dimer dye-polymer complex, single dyepolymer complex, and unbound dye. The spectra afford little assistance in assessment of the composition of equilibrium solutions when the PGA is in a helical form since only negligible changes of peak intensity and peak position occur when the total PGA concentration is varied over two orders of magnitude, the total A 0 concentration is varied over a factor of 2, and the buffer concentration is changed from lo-' to M (see Figure 2). However, the parallel in kinetic behavior suggests that the complexes formed are similar to those formed at pH 7.5. We will first consider the kinetic results obtained for the monomer-dimer system in acridine orange solutions. A plot of the logarithm of the amplitude of the light intensity change vs. time was linear for all Volume 70,Number 6 May 1966
1618
GORDON G. HAMMES AND COLIN D. HUBBARD
solutions, indicating a single relaxation process. For an association reaction of the type kiz
2A
Az ksi
the reciprocal relaxation time is 1/7 = 4h2 [AOM1 -k
(2)
k21
where [AOM]is the equilibrium concentration of the monomeric species. From previous data2 the equilibrium constant for the dimerization reaction can be estimated t o be 1.5 X lo4 M-' at 25.0", so that the concentrations of monomer and dimer species can be calculated for any value of the total acridine orange concentration. Figure 3 shows a plot of 7-1 us. 4[AOM]frorn which the following rate constants were determined: k12 = 2.7 X lo8M-' sec-' and k21 = 1.8 X 1 0 4 sec-1. The ratio of rate constants gives an equilibrium constant of 1.5 X lo4 M-I, in good agreement with the value cited above. The relaxation spectrum of the PGA-A0 system is considerably more complex than that of the A 0 system. For all solutions, a plot of the logarithm of the amplitude of the light intensity change us. time was curved, but the assumption of two relaxation times was sufficient to represent the data quantitatively. A typical plot of the data is shown in Figure 4, together with the calculated theoretical curve assuming two relaxation times, that is, assuming the signal amplitude can be A*e-t'r'. represented as Ale-t'" The relaxation times were obtained by first determining the longer relaxation time from the linear portion of the curve at long times; the linear portion of the curve was then extended to shorter times and was subtracted from the experimental curve. The value of the shorter time can be determined from the resultant straight line. The faster relaxation time at pH 7.5 could not be obtained very precisely since, except a t very low concentrations of PGA, the relaxation time was of the order of 20 psec, which is close to the limit of time
+
' 00
104, ~ O [ A O M ](M)
Figure 3. Plot of 7-l us. ~ [ A O Mfor ] the dimerization of acridine orange.
The Journal of Physical Chemistry
U
t hwc)
Figure 4. Plot of the logarithm of amplitude of light intensity change us. time; [PGA] = 2.7 X M, [AO] = 5.0 X 10-6M and pH 7.5. Circles are experimental points. The solid line is the theoretical curve obtained as described in the text. The equation for the theoretical curve is S A (millimeters) = 23.2e-*'o.o6 7.6e-1'0J7, where t is in milliseconds.
+
10'tPl (MI 1 a t pH 7.5 and 25.0": Figure 5. Plot of ~ ~ us.- [PGA] for [AO] = 5.0 X 10-6 M , circles are experimental points, solid lines are theoretical curves; for [AO] = 2.5 X M, squares are experimental points, dashed lines are theoretical curves. The theoretical curves are calculated with eq 6 and the values of n given in the figure.
resolution of the equipment. The estimated error in the other relaxation times is about &20%. I n Figure 5, the values of 1 / are ~ plotted ~ against [PGA] for both concentrations of A 0 employed. The over-all profiles are of similar form for both A 0 concentrations, but the relaxation times are quantitatively different a t low concentrations of A 0 and PGA. Unfortunately, in the critical region of very low PGA concentration, relaxation data cannot be reliably obtained because the interaction between PGA and A 0 is very weak. At both A 0 concentrations T~ reaches a constant value of approximately 200-250 psec at high concentrations of PGA. Relaxation times for dye interacting with the CYhelical form of polyglutamic acid were an order of magnitude slower, which allowed both of the relaxation times to be quantitatively measured. Both 71
INTERACTION OF ACRIDINE ORANQEWITH POLY-CY-L-GLUTAMIC ACID
and r2depend markedly upon the concentration of PGA until the ratio [PGA]/[AO] is about 200, whereupon T~ remains constant at 400-500 psec, while 7 2 reaches a concentration-independent value of about 3 msec. These concentration-independent limits are both also virtually independent of the acridine orange concentration. Again, as at pH 7 . 5 , the relaxation times differ at low PGA and A 0 concentrations, but the 1/r[PGA]profiles are of the same general form.
Discussion The results obtained for the monomer-dimer A 0 system indicate that the aggregation process is quite rapid. The maximum possible value for the secondorder rate constant is about 109 M-1 sec-', s so that dimer formation essentially is a diff usion-controlled process. The over-all mechanism of complex formation cqn be depicted as ks
kD
A+AJ-A.*ASAz
(3)
k- D
where A. -A represents a complex which forms and dissociates at rates controlled by diffusion and ks is the rate constant for "stacking." I n order for the over-all association rate to be diffusion controlled, ks must be much greater than k - ~ . The magnitude of k - ~can be estimated as lo1" sec-lv 9 so that the rate constant associated with "stacking" must be greater than 1Olo sec-'. The mechanistic explanation for the relaxation spectra of the PGA-A0 system is not so simple. If we assume that the actual relaxation processes being observed do not change AS the PGA concentration is changed and that the rehxation times reach a constant limiting value at high PGA concentrations, the processes being observed are almost certainly intramolecular. In general terms, a plausible mechanism is one in which a dye molecule binds very rapidly to a site on the polymer; this process is followedby a slower intramolecular rearrangement. The question as to whether dimeric, monomeric, or both forms of A 0 bind directly to the polymer will be considered later, along with possible explanations for the occurrence of two relaxation times. In terms of this mechanism, a lower bound can be estimated for the second-order rate constant characterizing the initial combination of dye and polymer by assuming the relaxation time for this process is less than 10 psec; in this case
I--"
-
108 M - 1
1619
tion in the A 0 system. (The rate constant for the PGA-A0 interaction would be expected to be larger, since it involves a reaction between positively and negatively charged molecules.) An alternative mechanism is one in which the measured relaxation times are considered to be primarily associated with the combination of dye and polymer, but at high PGA concentrations other processes (for example, the transfer of a dye molecule between polymers) become coupled to this simple one-step mechanism causing a plot of 7-l vs. [PGA] to level off rather than continuing as a straight line as predicted by the simple mechanism. For this mechanism, the characteristic second-order rate constant would be given approximately by the initial slopes of Figures 5-7. The maximum rate constant estimated by such a procedure is approximately 3 X lo6M-l sec-', which is considerably less than the value found in the A 0 system. If direct dye transfer were involved, the rate would presumably be slower for the random coil because of unfavorable electrostatic interactions. Therefore, this type of mechanism seems unlikely. If we assume the relaxation times characterize an intramolecular process, a simple mechanism which is consistent with the dependence of the relaxation times upon the polymer concentration, but excludes the various possible aggregation states of the dye, can be formally written as kza
kiz
nP
+ D J_ P,D
1_ P,D' ksz
kzi
(4)
where P and D designate polymer and dye, respectively. Equation 4 means that n polymer molecules are associated with each dye molecule. Assuming the first step is equilibrated rapidly, the slow relaxation time for eq 4 can be written as (see Appendix for details) 1/T
=
k32
+
k23
(5)
1.
Since in the present experiments n2(D)(P)n-lis usually much smaller than (p)n, eq can be approximated as 1/7 =
k32
+
k32
I+---
(6)
kzi kl2
[PI"
Theoretical curves for equal to 1, 2, and 3 were fit to the data by a trial and error procedure. Some of these curves are shown in Figures 5-7. In all cases the best fit is obtained for n equals to 2 or 3, although the fit is
sec-1
which is similar to the value found for dimer forma-
(8)
P. Debye, Trans. Electrochem. Soc., 8 2 , 265 (1942).
(9) M. Eigen,
Z. Physik. Chem. (Frankfurt), I ,
176 (1954).
Volume 70,Number 6 May 1966
GORDON G. HAMMES AND COLIND. HUBBARD
1620
Table I : Kinetic Parameters for the Polyglutamic Acid-Acridine Orange System 10~[AOI. M
PH
process
2.5 2.5 5.0 5.0 2.5 2.5 5.0 5.0 2.5 2.5 5.0 5.0
4.7 4.7 4.7 4.7 4.7 4.7 4.7 4.7 7.5 7.5 7.5 7.5
1 1 1 1 2 2 2 2 2 2 2 2
Relaxation
kidkn, M -n
5x 3x 3x 1.3 x 1x 4.5 x 7 x 2.5 X 3x 1.6 X 4x 2x
krr,
kn.
8ec -1
800 -1
2.3 X 2.2 x 2.8 X 2.4 X 3.1 X 2.9 X 3.7 x 3.6 X 4.2 X 4.1 X 4.7 x 5.1 X
102 107 105 107 108 106 10% lo6 10s
lo6 10s 107
x x
108 lo*
3 9
lo8
50 6X 20 1x 20 5 2x 1.6 X 50 50
108 lo2 lo2 102
lo2
10' lo8 10' lo*
n
102 102
lo2 102
102 108
1 3 1 3 1 2 1 2 1 2 1 3
-._I
i
d 4 r,:
1 0 3 ~ (M) ~1
Figure 6. Plot of z2-1 08. [PGA] at pH 4.7 and 25.0': for [AO] = 5.0 X 10-6 M, circles are experimental points, solid lines are theoretical curves; for [AO] = 2.5 X 10-5 M, squares are experimental points, dashed lines are theoretical curves. The theoretical curves are calculated with eq 6 and the values of n given in the figure.
data. The values of k12/k21, k ~ and , k 3 2 which describe the data best are summarized in Table I. If n is equal to 1, the mechanism is easily understood: it simply implies that the rate-controlling step in complex formation is an intramolecular process, presumably desolvation and/or displacement of counterions. If n is equal to 2 or 3, the mechanism does not appear plausible since this implies two or three polymer molecules are binding a single dye molecule. The above mechanism is obviously a gross simplification since it does not take into account the fact that two relaxation times are observed and that the relaxaThe Journal of Phgsical Chemistry
IbI:
, a l I , I b A
103CPl (Mi
Figure 7. Plot of ~ 1 - l us. [PGA] a t pH a t 4.7 and 25.0': M , circles are experimental points, for [AO] = 5.0 X solid lines are theoretical curves; for [AO] = 2.5 X M, squares are experimental points, dashed lines are theoretical curves. The theoretical curves are calculated with eq 6 and the values of n given in the figure.
PnD'
+ mP + D 1-Pn+mD2
Pn+mD2' (7)
Calculation of the relaxation spectrum of this mechanism with several assumptions about the relative equilibration rates of the different steps yields quite cumbersome expressions, which depend upon the dye concentration in a complex manner, but reduce to a form similar to that of eq 5 in certain limits. The data are not precise enough to merit further refinement along these lines except to say that enough parameters are available so that the data can be fit about as well as the simple mechanism (eq 4) with n equal to 1.
INTERACTION OF ACRIDINE ORANGE WITH POLY-LI-L-GLUTAMIC ACID
Many other types of mechanism have been investigated in trying to correlate the experimental data. For example, a “stacking mechanism” such as
P
+ 2D JJ P D + D J_ PDz JJ PD2’
(8)
cannot be reconciled with the data. A rather improbable mechanism which yields a fit to the data similar to the above case where n is equal 1, is one in which two polymer chains, each with a bound dye molecule, interact to form a complex in which the two dyes are shared by the two polymers, i.e.
P
+ D zP D + P D JJ PzDz
PzDs’
(9)
It does not appear likely that two random-coil polymer chains with or without bound dye molecules will combine, because of the unfavorable electrostatic conditions for such an interaction, although spectral data concerned with the interaction of polyadenylic acid and acridine orange led to the speculation that in the presence of excess of polyadenylic acid, bound dye is shared by the macromolecules. 2b Another possible mechanism is one in which after a very rapid initial polymer-dye interaction, two consecutive intramolecular processes occur which represent the faster and slower relaxation processes, respectively. This may be written P
+ D J_ PD J_ PD’ ZPD”
By making the assumptions that the initial step is equilibrated very rapidly compared with the last two, and that the final step is very slow compared with the previous one, expressions for both relaxation times can be derived which are similar to eq 6, when n equals 1. Finally, we have considered the effect of including the monomer-dimer equilibrium of acridine orange in the mechanisms: in fact, this refinement produces only minor changes in the shapes of the theoretical curves regardless of what assumptions are made concerning the nature of the species being bound. To conclude, an exhaustive examination of possible mechanisms of the PGA-A0 interaction has not led to a quantitative fit of the data. This may be due to the fact that discrete relaxation times are not being measured. The results do seem to indicate that the initial PGA-A0 interaction is quite rapid and is followed by relatively slow intramolecular processes. However, this simple mechanism is clearly not sufficient to explain all of the results quantitatively. If the aggregation or “stacking” of acridine orange upon the polymer chain is responsible for the observed relaxation processes, then this occurs considerably more
1621
slowly than in free solution. Conceivably “stacking” along the polymer chain could involve displacement of solvent and counterions and therefore might be slower than in the A 0 dimer. However, another possibility is that the rate-controlling steps being observed are involved in the actual mechanism of complex formation between A 0 and PGA, which would also involve displacement of solvent molecules and counterions. The actual “stacking” interaction probably occurs very rapidly as in the A 0 dimer (k > 1O’O sec-l). Alternatively, polymer aggregation might be occurring, but this seems unlikely for PGA at high pH values. Unfortunately, our results yield no criterion upon which a distinction between the various mechanisms can be made. The geometrical form of the polyglutamic acid appears to have a negligible part in influencing the overall mechanism or stoichiometry of the interaction with acridine orange, but does affect the magnitude of the kinetic parameters. A smaller electrostatic attraction of the cationic dye for the polymer when more of the carboxyl groups of the latter are protonated presumably accounts for the slower reaction of acridine orange with the helical form of the polymer. The observation that the spectrum of a solution of acridine orange at pH 12.8 is only negligibly changed by the presence of polyglutamic acid at a concentration of M indicates that when acridine approximately 2 x orange is essentially unprotonated, its interaction with polyglutamic acid is very weak. Furthermore, this suggests that the binding between PGA and A 0 is primarily electrostatic in nature. Little conclusive information about the structure of PGA-A0 complexes is available and reports about the form of the DNA-A0 complex indicate the variance of opinion in this field2a~eJo-1zThe results obtained here indicate that the structure of the complexes should be qualitatively similar for both geometrical forms of PGA. A preliminary investigation of the interaction between polynucleotides and acridine orange by the temperature-jump method has shown that in these systems the relaxation effects are even more complex. However, hopefully, further studies of these and analogous systems will lead to clarification of the mechanism of interaction of the polyglutamic acid-acridine orange system.
(10) L. S. Lerman. J . Mol. Biol., 3, 18 (1961); PTOC. Natl. Acad. Sci. U.S., 49, 94 (1963). (11) S. F.Mason and A. J. McCaffery, Nature, 204,468 (1964). (12) L. Stryer and E. R. Blout, J . Am. Chem. Soc., 8 3 , 1411 (1961).
Volume 70, Number 6 May 1966
1622
PETERG. BOWERS AND GERALD B. PORTER
Appendix Derivation of Equation 5 From eq 4, the rate law near equilibrium can be written as
Mass conservation requires that
+ 6[P,D] + 6[P,D'] = 0 B[P] + nb[P,D] + n6[P,D'] = 0 6[D]
(A21 L
--1
(A3) =
and differentiation of the equilibrium constant
1 -6[P,D] 7
(A8)
and use of eq A6 gives the expression for the relaxation time in eq 5.
Kinetics of Excited Molecules.
V.
Photochemistry of Hexafluoroacetone
by Peter G. Bowers1 and Gerald B. Porter Department of Chemistry, University of British Columbia, Vancouver, Canada
(Received December IS, 1966)
The photochemical decomposition of hexafluoroacetone has been examined over a wide pressure range at several different exciting wavelengths. A mechanism for the primary photochemical process is proposed to interpret these results and those of the emission spectra. All of the rate constants for the elementary reactions are evaluated. The natural lifetime of the triplet state is estimated to be about 0.03 sec. The RRK theory of unimolecular reactions is applied to the rate constants for dissociation of singlet excited molecules.
Introduction Various aspects of the photolysis of hexafluoroacetone (HFA) have been studied to elucidate the primary pr0cess.2-~ HFA is a particularly good substance for this type of investigation because its photochemistry is simple for low conversions, except at elevated temperatures.6 Carbon monoxide and hexafluoroethane, formed in equal amounts, are the only products, and the quantum yield of either can be assumed with certainty to represent the quantum yield of primary dissociation, The Journal of Physical Chemistry
#. The chemical mechanism of the reaction is adequately represented by the following two steps. (1) Holder of a University of British Columbia Fellowship, 19621963,and a National Research Council Bursary, 1963-1964. (2) P. B.Ayscough and E. W. R. Steacie. Proc. Roy. SOC.(London), A234, 476 (1956). (3) G. Giacometti, H.Okabe, and E. W. R. Steacie, Proc. Roy. SOC. (London), A250, 287 (1959). (4) H. Okabe and E. W. R. Steacie, Can. J. Chem., 36, 137 (1958). (5) A. S. Gordon, J. Chem. Phys., 36, 1330 (1962).
