INTERACTION OF POLYVINYLPYRROLIDONE WITH Azo DYES
Oct., 1957
1375
THE INTERACTION OF POLYVINYLPYRROLIDONE WITH SOME AZO DYES1 BY H. P. FRANK,^ S. BARKINAND F. R. EIRICH Contributionfrom the Institute of Polymer Research, Polytechnic Institute of Brooklyn, Brooklyn, New York Recaiued February 36,1067
Polyvinylpyrrolidone (PVP) dis lays strong binding affinities toward various organic anions such as the azo dyes orange
I1 (0-11) and benzo urpurin 4B &P), The binding equilibria were studied by a conductance method and by a dial sis technique. It was Pound thrtt one dyestuff ion is bound by chain segments of 7 or 10 monomer units for 0-11 and gP, respectively. It is believed that the dye ions align themselves with their long axis alongside the polymer chain and are
held in place by van der Waals forces. The effect of added potassium chloride on the binding equilibria was studied. The polyelectrolyte character of the PVP-dye com lex was demonstrated by its electroviscous effect. A viscosity reduction in the presence of salt was explained as a cross-h$ng effect due to aggregation of dye ions.
Introduction The interaction of PVP with dyestuff has recently attracted some a t t e n t i ~ n . ~PVP . ~ is a polar water-soluble polymer which will bind in varying degrees a great variety of chemically entirely different cosolutes, such as many dye anion^.^ One of the most striking features of these complexes is their polyelectrolyte character with the number of charges dependent on the degree of binding. I n the past we have studied complexes of PVP with triiodide ions.6 The behavior of the dye complexes is in many ways reminiscent of the PVPtriiodide system. At present we are working on complexes of PVP with long chain alkyl sulfates. I n the present study we have used the azodyes 0-11 and BP. The solution behavior of these dyes is described in the literature,6 and we have also made a thorough study of the dyestuff solutions as Experimental Reagents.-0-11 from water.’
(Eastman Kodak) twice recrystallized
23
-N=N-oOa-Na
+
B P (Allied Chemical and Dye Corp., National Aniline Division), twice recrystallized from water.’ NHz I
”2
I
I
SOa-Na+ Potassium chloride used was Mallinckrodt (A.R. grade). Polyvinylpyrrolidone: K 30, weight average molecular weight approximately 70,000; K 62, weight average molecular weight approximately 350,000. Both samples were obtained through the courtesy of Schenley Laboratories, Inc. Solutions of PVP samples were passed through ion(1) This work was supported b y the office of the Surgeon General, Dept. of the Army. (2) Chemistry Department, Illinois Institute of Technology, Chicago 16, Ill. (3) W. Scholtan, Makromol. Chem., 11, 131 (1953). (4) J. W. Breitenbacb and E. Wolf, ibid., 18/19, 217 (1956). (5) 8.Barkin, H. P. Frank and F. R. Eirich, “Ricerca Soi., Simposio Internazionale di Chimica Macromoleculare,” 1955. (6) E. Valko, “Kolloidchemische Grundlagen der Textilveredlung,” Vcrlag Julius Springer, Berlin, 1937. (7) H. P. Frank, J . Coll. Sci., in press.
exchange columns in order to remove ionic impurities. The conductance was reduced to a very small value which is probably caused by terminal carboxyl groups.* The PVP conductance was deducted from all reported conductance data. The water used for conductance measurements was twice distilled in the presence of potassium permanganate. The specific conductance is not particularly low, but sufficient for our purpose: approximately 2 X 10-6 Q-l cm.-‘ a t 25’. This value was deducted from all reported conductance data. Conductance.-Measurements were carried out at 25” by means of a bridge by Klett Mfg. Co. The cell .electrodes were black platinized. Frequencies of one and two thousand cycles per second were used. In no instance was there any frequency dependence of the conductance. All solutions used were unbuffered. The pH of all solutions was in the range between 6 and 7.5 (model G Beckman pH meter). This applies also to solutions used for dialysis and viscosity. Dialysis .-Dialysis experiments were carried out in sealed glass tubes at 30”.aJ,g Visking Gel Cellophane was used as membranes. The inside and outside compartments were 8 ml. in practically all cases. The samples were allowed to equilibrate for 5 days with occasional shaking. A spectrophotometric technique was used to determine the dyestuff concentration in the outside compartment. The molar extinction coefficient of 0-11 at 485 mp, E = 22,400 in the absence of salt and E = 21,800 in the presence of 0.1 M KCI. Beer’s Law was found to be valid in the concentration range used (0.6-3 X 10-6 M ) . 0.1 M KCl is a sufficiently large salt concentration to suppress any Donnan effect. A Donnan correction was applied for salt-free solutions. The gel cellophane itself has a certain capacity for dye binding. A blank curve of the shape of a Langmuir isotherm was obtained from which the appropriate corrections can be determined. Viscosity.-An Ubbelohde dilution viscometer was used. The efflux time for water was 158.0 seconds. The kinetic energy correction under these conditions is small and was neglected. All measurements were carried out a t 25”.
Results Conductance.-Three sets of measurements were carried out a t 0-11 concentrations of increasing normality: 0.001515,0.00379 and 0.00757 N . The results are plotted in Fig. 1. A quantitative evaluation of the results requires a number of assumptions. At zero polymer concentration the equivalent conductance is given by the sum of the ion conductances (dye ion plus SOdium counterion) : A = ADXN~+. A,, of 0-11 a t infinite dilution is 82 W 1c ~ . ~ . ~ J 0-11solutions in salt-free water do not show any indication of forming either micelles or ion pairs.’
+
(8) R. Woodhams, Doctoral Thesis, 1953, Polytechnic Institute of Brooklyn. (9) I. Klotz, F. Walker and R. Pivan, J . A m . Chem. SOC.,68, 1486 (1946).
H. P. FRANK, S. BARKIN AND F. R. EIRICH
1376
Vol. 61
XONst a t infinite dilution is 50.1. With increasing dyestuff concentration XNa+ is assumed to decrease 4 a t the same rate as A. Therefore AN&*. = XONa+A/ e; 80 0 70 Ao. We further have to assume that a t the maxi9 60 mum polymer concentration (2.3 X the mo0.00757 N 2 50 lar ratio between PVP and 0-11 is sufficiently large -0 to bind practically all 0-11 ions. The relative 80 70 charge density of the polymer complex will then be 2 60 00379 N given by the ratio between the molar concentration 4 50 of 0-11 and the base molar concentration of PVP. 2 80 The numerical value increases with increasing 0-11 70 concentration up to 0.036 (Table I). The increas60 0.001515 N ing charge densities will lead to a certain degree of 50 A & counterion retention (ion pair formation). Numer0 0.25 0.5 0.75 1.0 1.25 1.5 1.75 2.0 2.25 ical values for the degree of ion pair formation are Concn. PVP x 102 (g. cm.-3). taken from data on sodium polymethacrylate'0 and conductance A us. PVP K 62 concentraare given in Table I. We assume that only the Fig. 1.-Equivalent tion at three 0-11 concentrations. charge, and not the chemical nature of the ionized group, affects the degree of ion pair formation. In- XPD-. The height difference between the experiasmuch as ion pair formation occurs to a minor de- mental curve and line B was taken to be a measure gree only, any uncertainties thus introduced are of the concentration of free 0-11 ions. From these not likely to cause a major error. data and from the known value of XDJ a binding The conductance contribution of free sodium equilibrium constant KO-IIcan be calculated uncounterions is given by h a t = f h a + , where f is der the assumption that one 0-11 ion complexes the fraction of free sodium ions (Table I). We with a chain segment of definite length of about mdke the assumption that the degree of ion pair seven monomer units, which will be shown later formation (1 - f ) increases linearly with polymer KO-II= IpiD-I/[p7l[D-l (1) concentration (line A, Fig. 1) which is obviously incorrect. However, in view of the small values of [D-] is the molar free dyestuff concentration and (1 - f ) this procedure causes a negligible error only [P,] the molar concentration of chain segments with which can be demonstrated by calculating (1 - f ) seven units. An expression of the kind of equation at any polymer concentration using a numerical 1 implies that chain segments of seven monomer value for the charge density which has been ob- units are assumed to be kinetically independent tained by the above proposed approximation pro- units of equal reactivity regardless whether they are contained in one polymer molecule or in differTABLE I ent ones. The results of these calculations are PVP concn. at which given in Table 11. .lJ
Dye rroncn.,
N
Max. charge dens. (dye)/
(PVP)
0.001515 .00757
0.210 ,210
0-1I 0.0072 .036
0.00314 .00777
0.0315 .OG30
BP 0.100 .123
F
TABLE I1 (1
Dye' concn. (mole 1. -1)
0-11
0.0085 ,043
48.2 44 0
0.115
42.3
,141
40.0
0.1 6.4 24 19
cedure. At the maximum polymer concentration the difference A - XXNa+represents the conductance The contribution of the PVP-dye anion: XPD-. normality of PD- is identical with that of 0-11 if all 0-11 ions are bound. At still higher PVP concentrations the normality of PD- remains constant, but XPD- will be reduced due to the decreasing charge densities. Below the maximum polymer concentration the normality of PD- will decrease due to the dissociation of the complex. Accordingly, the conductance contribution of PD- will decrease, and we have approximated this decrease by the straight line B (Fig. 1). The height difference between lines A and B at any given polymer concentration represents the conductance of PD-, and a t the maximum polymer concentration it is (10) J. R. Huirenga, P. F. Grieger and F. T. Wall, J . Am. Chem. Soc.. 1 3 , 2636 (1950).
k
ky
8
complete dye bindmgis reached (base mole. 1. -1)
p
BP
PVP
concn. (bmole 1. -1)
0.001515 0.03 ,001515 .08 .001515 -12 .003785 .03 .003585 .08 ,00757 .03 ,00757 .08 .00757 .12 Av. value for KO-II= 325 f 33 0.001565 .001565 ,001565 .00389 .00380 ,00389
0.01 .02 ,025 .01 .02 .03 ,00389 .04 Av. value for KBP= 5400 f 1200
R 264 347 336 303 333 276 323 412
6100 4900 7400 4500 6600 4400 3700
The equivalent conductances XPD- a t the maximum PVP concentrations are given in the last column of Table I. The numerical values for X P D are of an order of magnitude which is consistent with previous data at comparable charge densities. lo Analogous conductance experiments were carried out with B P a t 0.00314 and 0.00777 N . The
i
.
Oct., 1957
INTERACTION OF POLYVINYLPYRROLIDONE WITH Azo DYES
I
.100h -?
i . -
1 1
nii c g
1
- = I - -
90
30
1
r
-
0 0.25 0.5 0.75 1.0 1.25 1.5 1.75 2.0 2.25 Concn. PVP x lo2(g. cm.-3). Fig. 2.-Equivalent conductance A us. PVP K 62 concn a t two BP concns. All conductance dat,a contain a conBtant contribution of ionic impurities in BP7 of the magnitude of six A-units, which makes the values of b,"t appear six units too high.
maximum concentration of PVP is approximately 2.4 X Results are shown in Fig. 2. The shape of the conductance curves of B P is different from those of 0-11 solutions. A conductance plateau is reached at much lower PVP concentrations indicating stronger binding of BP. The further reduction of the conductance a t increasing polymer concentration is due to decreasing charge densities of the polymeric anion. The evaluation procedure of the experimental data is essentially the same as in the previous case. From the shape of the curves we assume that practically complete binding had occurred upon reaching the plateau at 0.35 and 0.7 X PVP concentration. All the uncertainties of the 0-11 systems are magnified in this case, and additional complications such as ion pair formation of BP with itselfs.' must be considered. The equilibrium constant K B Pwas calculated assuming one B P ion to complex with a chain segment of ten units KBP = fp~oD-l/[P~olP-l (2) The results of the calculations are given in Table I and Table 11. The value of KBPis much more uncertain than the corresponding KO-11 since the linearity of line B in this system is physically unjustified. One can only hope to obtain the order of magnitude of the binding constant in this manner. If one replaces line B by curves of plausible shape (curve B', Fig. 2), one obtains values for the equilibrium constant which may be considerably greater. The values for XPD- in Table I are again of the expected magnitude considering the greater charge densities. Another possible method t o evaluate conductance data in terms of binding equilibria which is not basically different from the one used before, but which furnishes information on the number of monomer units interacting with one dye ion, is a procedure originally proposed for protein-dye binding by Kl0t5.~ This method has already been applied successfully t o PW-dye ~ y s t e m s . ~It, ~ assumes the presence of a certain number of equivalent binding sites, and it accordingly neglects any interaction between bound dye ions. The formulation is that of a Langmuir isotherm
1377
+
z
(3)
where r is the number of moles of dye bound per base mole PVP, n is the number of binding sites per base mole, CD is free dye concentration and K the binding constant. We can determine the amount of free and bound dye from conductance data, and we can obtain n and K by plotting l / r vs. l / C D (Fig. 3). The 0-11 plot contains two points which were determined by the dialysis method and which agree fairly well with the conductance data. The numerical value of KO-11is 333 which is in good agreement with the previous value of 325 33. The value for l/n is 7, Le., seven monomer units represent one binding site for 0-11. An analogous straight line is obtained for BP. The numerical value for the binding constant is 8000 and can only be taken as indication of its order of magnitude. The agreement between the two values for K B Pis rather poor which, however, is not surprising in view of the complications. The literature3 reports a numerical value for K B P which is several orders of magnitude greater than ours. This discrepancy is due to the use of a 0.1 M phosphate buffer which will cause formation of large B P micelles' which will profoundly affect the degree of complex formation and possibly also its nature. The value for l/n on the Klotz plot is 10 in this system. Dialysis.-In order to determine the degree of dye binding in the presence of salt, the results of dialysis equilibria of 0-11 in 0.1 M KCl using PVP K62 were evaluated. If one wants to avoid formation of 0-11 micelles, one must limit the experiments to a certain 0-11 concentration range and must not exceed moderate salt concentrations.' The results of the dialysis experiments corrected for dye binding by cellophane are given in Table 111.
*
TABLE I11 Base mole PVP x 104
Concn. PVP X 101 (bmole 1. -1)
Equil. concn. 0-11 X 105 (mole 1.
-1)
7.04 7.04 7.04 7.04 7.04 7.04 7.04 3.52 1.76 0.88
8.80 8.80 8.80 8.80 8.80 8.80 8.80 4.40 2.20 1.10
0.1 M KC1 179.8 77.1 31.15 9.82 3.09 1.48 0.81 64.9 122.0 163.3
6.98 6.98
8.72 8.72
No salt" 539.3 253.8
I/r
l/CD
7.98 12.58 26.7 152 677 1853 4045 13.7 9.6 8.6
560 1300 3210 10200 32300 67700 123000 1540 820 610
14.8 21.6
300 900
(11) The figurea in this section of the table are calculated from the Donnan equation: ( O X ) % = (0-1I)i X (O:II)i,t,,t where (0-1I)a and (0-II)i are the free 0-11 concentration in the outside and inside compartment, respectively, and (O-II)i, tot is the total concentration of 0-11 on the inside. Counterion retention on the inside will cause an uneven distribution of the free sodium ions. This effect however was neglected. There will also be an uncertainty in the correction for dye binding by the membrane because of the concentration difference of 0-11 in the inside and outiide compartment.
1378
H. P. FRANK, S. BARKINAND F. R. EIRICH
Unfortunately it is not possible to carry out analogous experiments on PVP-BP-KCl inasmuch as the formation of large BP micelles is caused even by small salt concentrations. The results listed on Table IV are plotted according to equation 3 on Fig. 3. Results obtained a t different PVP concentrations all fall on the same line. The value of the equilibrium constant KO-Il-Kcl is considerably greater than in the absence of salt: 1,000 as compared to 333, The intercept however is reduced t o a value of 5.5, Le., 5.5 monomer units forming one binding site. Viscosity.-The results of viscosity measurements on solutions of PVP K 62 and 0-11 and BP, respectively, are plotted on a conventional diagram (Fig. 4). The figure also shows viscosity curves for the system PVP-0-11 in the presence of KCI. The system PVP-BP could not be studied in the presence of salt because of micelle formation. Figure 4 also contains a reference curve R for PVP K 62 in water. Addition of moderate quantities of KC1 up to concentrations of 0.4 M causes almost no change of the reference curve. A great increase of the reduced viscosity is produced by both dyes and a sharp viscosity maximum is observed. The position of the maximum depends on the dye concentration. Addition of salt leads to a considerable reduction of the reduced viscosity as well as of [SI. Analogous, but less extreme, changes of the viscosity curves were observed on a comparatively low molecular weight PVP K 30. Discussion From conductance, dialysis and viscosity data, a fairly consistent picture of a polyelectrolyte complex of PVP with dye ions can be derived. No definite information concerning the nature of this interaction exists. It has been claimed that hydrogen bonds may at least partly be responsible for the binding,* but it seems difficult t o accept such a mechanism in an aqueous solution. We would like t o assume that primarily van der Waals forces (dispersion forces) between the aromatic system of the dye ions and the paraffinic chain of the PVP molecule are responsible for the binding. 0-11 as well as BP can assume a flat configuration and probably exercise maximum interaction by aligning themselves with the paraffinic chains. PVP is a fairly stiff polyvinyl chain with bulky substituents which do not allow much rotation around C-C bonds. A molecular mode1 of a tetramer shows this rather clearly and also demonstrates the accessibility of the paraffin backbone (Fig. 5 ) . The PVP-dye complex is closely related t o the complexes formed between proteins and organic ani o n ~ I.n these ~ ~ systems ~ ~ ~it has ~ ~been shown that binding is caused by electrostatic attraction between opposite charges plus intermolecular forces. The PVP molecule does not carry any ionizable groups so that binding has t o be accomplished by intermolecular forces alone. However, the lactame bond in the pyrrolidone ring represents a dipole which is likely t o undergo ion-dipole interaction with the dye ion and will aid the binding of (12) E. Fredericq. BulZ. 800. chim. Belo., 68, 158 (1954). E.Frederioq, ibid., 64,639 (1955).
(131
Vol. 61
100
75
r; 2
50 40 30
0
500
1000
1500 2000
2500 3000
l/CD.
Fig. 3.-Klotz plot: moles dye bound per basemole PVP us. reciprocal molar free dye concentration, l / r us. l/m. Absence of salt: (A) 0,0-11(conductance); a), 0-11 (dialysis); (C) 0 , B P (conductance). Presence of 0.1 M KCl (all data from dialysis): e,0-11, PVP concn. 8,0-11, PVP concn. 4.40 X 8 , 0-11, 8.80 X PVP concn.'2.20 x 10-2; e, 0-11PVP concn. 1.10 x 10 -2.
0
400
7 300 bb Io
i
/
v
4 g 200
100
R
0
0.25 0.5 0.75 1.0 Concn. PVP X 102 (g. cm.-8). Fig. 4.-Reduced viscosity vs. PVP K 62 concentration. R is the reference curve for PVP K 62-water. BP: ( B j 0 0.00139 M, (A) 0 , 0.00317 M ; 0-11: D)a),0.00152 M , (6)8,0.00380 M ; 0-11 0.4 M KCI; (E) 8, 0.00152 M , (F)Q, 0.00380 M . I
+
anions in such a way as to supply the necessary attraction force to bring the two components into
INTERACTION OF POLYVINYLPYRROLIDONE WITH Azo DYES
Oct., 1957
Fig. 5.-Molecular model of PVP chain segment with four monomer units.
A
C
B
Fig. 6.-Configuration of PVP-dye complex: A, in the absence of salt; B, in the presence of salt; C, after formation of dye double ions functioning as cross-links.
close contact. The short range dispersion forces will then stabilize the complex. The dispersion forces will be the greater, the more aromatic rings the dye anion contains. Accordingly, the binding of BP exceeds that of 0-11. We can estimate an energy balance if we can calculate the free energy of the binding reaction from the e uilibrium constants. I n order to do that our data ave to be converted into different units, expressing the binding in terms of moles of dye per mole PVP instead of per basemole (PVP K62: MW = 350,000, XN = 175,000). The results of these calculations are given in Table IV.
81,
TABLE IV l h z , mole
PVP/mole dye bound
0-11 BP
0.0044 0.0062
- A F ==
AHD (cal. mole-')
76000
6700
-3500
1300000
8500
-7000
RT In R P nxKX = K P (cal. mole-])
Klx is the binding constant for the first anion bound by a PVP molecule. AHD is the energy term for dispersion forces calculated on the basis of plausible estimateslo (1000 cal. for benzene and 2500 cal. for naphthalene). The difference between A F and AHD has to be attributed to additional AHterms (ion-Dipole) and to a AS-term which for such binding reactions is usually strongly positive3
1379
because of the replacement of hydrate water molecules on the PVP by dye anions. AF values for subsequently bound ions, after the first, will continually decline because of electrostatic repulsion, but this effect would be difficult to estimate. The number of monomer units which are contained in one binding site is seven for 0-11 and ten for BP. These numbers can be understood in terms of the dimensions of the dye ions: the length of an 0-11ion is approximately 13 8.which would correspond to a chain segment of six or seven units. The corresponding number for BP is approximately 20 8.for its length, corresponding to about ten or eleven monomer units.I4 The hypothesis of alignment of dye ions with their long axis alongside the polymer chain in order to achieve maximum interaction would thus appear sterically plausible. I n the presence of salt (0.1 M KCI) the electrostatic repulsion between dye anions is screened by a large excess of counterions and accordingly the binding affinity of the polymer is relatively increased as demonstrated by the increase of the equilibrium constant in the presence of salt. The accompanying reduction of the number of monomer units in one site (from 7 t o 5.5) may be taken as evidence for somewhat tighter packing of dye ions because of reduced electrostatic repulsion. The viscosity curves of Fig. 4 clearly show the polyelectrolyte character of the complexes. In the absence of salt the reduced viscosity increases with decreasing PVP concentration in a manner which is typical for polyelectrolytes because of the electrostatic repulsion of identical charges leading to an unfolding of the polymer molecules. The reduced viscosity passes through a maximum the position of which depends on the dye concentration. This effect is caused by the increasing concentration of free counterions with increasing dilution of PVP at constant dye concentration. The increased concentration of counterions will tend to suppress the electroviscous effect and thus at a definite polymer concentration will cause a viscosity maximum. In accordance with this concept the maximum is higher for BP than for 0-11because of the greater binding affinity of the former and it is shifted toward lower PVP concentrations by reducing the dye concentration. The addition of salt suppresses the electroviscous effect and should lead to EL viscosity curve which is essentially comparable to the reference curve R. However, a reduction of the viscosity is observed. [ q ] is reduced by a factor of about two. We have previously observed a similar, but more extreme, effect (reduction factor of ten) on PVP-triiodide c~mplexes.~ We must assume a mechanism which leads to a reduction of the hydrodynamic volume of the polymer complex below the volume of the uncharged polymer. One can only speculate on such a mecha(14) G . Scheibe, Koll. Ztschr., 188, 147 (19621, studied the fluoreacence quenching of the system PVP-eosine. He arrived at the conclusion that one eosine molecule interacts with 10 to 20 monomer units which is in good agreement with the order of magnitude of our results. In the original paper it is stated that one eosine molecule interacts with one to two monomer unib. This however is a numerical error (it should he 10-20) which Prof. Scheibe very kindly informed us about.
1380
LEROYG. ATJGENSTINE AND B. ROGER RAY
nism and the following hypothetical explanation can be offered: PVP molecules are practically saturated with dye ions and assume an extended shape in the absence of salt (Fig. 6A). Upon addition of salt the molecule folds up (Fig. 6B), and the local dye concentration may be sufficiently great so that some of the bound dye ions can aggregate in the presence of,salt and form double ions or small micelles which act as a cross-link, thus reducing the hydrodynamic volume of the system (Fig. 6C)’.A simple calculation will demonstrate the plausibility of this mechanism. We assume the dye bridge to act as a tetrafunctional cross-link, such as, divinylbenzene. We apply the theory for branched polymers of Zimm and Stockmayer.15 Let [710be the intrinsic viscosity of the unbranched PVP (in water) and [7] the corresponding parameter of the (15) B. H. Zimm and (1949).
W.H. Stockmayer, J . Chsm. Phys., 17,
1301
Vol. 61
“crosslinked” P W (0-11and KCl) [llIom(EZo*)*~:, Ill1 ..(B*)”3
(4)
where Rzoand Rz are the mean square radius of the unbranched and the cross-linked molecule, respectively. Assuming the solutions to be thermodynamically ideal, we can write g =
R“Po
(5)
and accordingly hl/[lllo (6) The numerical values for [7]0 and [7] are 80 and 40, respectively, so that gal/’= 0.5 and g = 0.63. From tables for tetrafunctional cross-links16we can derive a value of about three cross-links per molecule for g = 0.63. g’/r =
(16) C. D. Thurmond and B. H. Zimm, J . Polymer Sei., 8, 477 (1951).
TRYPSIN MONOLAYERS AT THE AIR-WATER INTERFACE. 11. E F F E C T O F X-RAY AND ULTRAVIOLET RADIATION UPON ENZYMATIC ACTIVITY‘ BYLEROYG. AUGENSTINEAND B. ROGERRAY Contribution from the Department of Chemistry and Chemical Engineering, Universitv of Illinois, Urbana, Illinois Received November 19, 1966
Compressed trypsin monolayers spread on ammonium sulfate support solution were irradiated, recovered, and the enzymatic activity determined. Contrary to the results reported by Maeia and Blumenthal, X-ray dosage of up to 1000 r. was found to produce no significant effects upon either film pressure or recoverable activity. For surface concentrations greater than 357/100 cm.2, the sensitivity (quantum yield) to ultraviolet radiation was found to be about one-third or one-half that reported for this enzyme in solution and was essentially independent of surface concentration, Le., film pressure, and inactivation appeared to be a one-hit event. For lower surface concentrations the data suggest that inactivation could be a multi-hit event.
Introduction The physical characteristics of proteins adsorbed at the air-water interface have been extensively investigated; however, there is relatively little information as to the retention of biological activity or whether the constraints imposed by the interface serve to modify the influence of factors such as radiation which are known to affect activity. Such information bears upon the general problem of protein structure and, as well, may have implications in cellular physiology. If the response t o radiation of an enzyme oriented in a monolayer is markedly different from its response in solution or as a solid, then, some further insight may be gained on the mechanisms involved in inactivation. One of us (LGA) has hypothesized regarding the steps involved in the heat denaturation of proteins.2 Extension of this “weak link” model suggests that the radiosensitivity of proteins should vary with pressure effects, in particular, such as are exerted within a monolayer. I n a sense, irradiation of enzyme monolayers allows study of the sensitivity as a function of physical state through a range of phases from ‘igaseous” to “solid.” (1) I n part from a theais by Leroy G. Augenstine submitted to the University of Illinois in partial fulfillment of the requirement for t h e Ph.D. degree in Physico-Chemical Biology, 1955. (2) L. Augenstine, “Information Theory in Biology.” ed. H. Quastler. 1953, Univ. of Ill. Press, Urbana, Ill., pp. 119-122.
I n an earlier paper3 we described the film pressure-concentration characteristics of compressed monolayers of trypsin and reported the amounts of enzymatic activity that could be recovered. It was found that the recoverable activity was dependent upon the manipulations given the film, the age of the film, and the methods used in preparing the support solut,ion. The data support the idea that the expanded film contains only unfolded trypsin molecules which have permanently lost all activity in the process of unfolding, whereas the film formed and kept under a compression greater than about 0.25 dyne/cm. consists of fractions of enzyme in globular and unfolded configurations. With few exceptions, irradiation of enzymes has been carried out in solution or in the solid state. Barron4 has reviewed much of the work involving X-irradiation of enzymes in dilute solution and McLaren6 has published an excellent review of the effects of ultraviolet irradiation on a number of enzymes both in dilute solution and dry state. The Yale Biophysics group6has irradiated enzymes and (3) R. Ray and L. Augenstine, THISJOURNAL,60, 1193 (1956). (4) G. Barron, “Radiation Biology.” Vol. I, Chap. 5, ed. A. Hollaender, McCraw-Hill Book Co., Inc., New York, N. y.,1954. (5) A. McLaren, Advances in Enzymology, 9, 75 (1949). (6) (a) W. P. McNulty, Jr., and F. Hutchinson, Arch. Biochem. Biophys., 60, 92 (1954); (b) C. L. Smith, ibid., 60, 322 (1954); (0)
E. Pollard, “Biochemical Aspects of Baaic Mechanisms in Radio-
*
.
