064
G. L. Gardner
The Journal of Physical Chemistry, Vol. 82, No. 8, 1978
Effect of Pyrophosphate and Phosphonate Anions on the Crystal Growth Kinetics of Calcium Oxalate Hydrates G.
L. Gardner
Rensselaer Polytechnic Institute, Materials Engineering Department, Troy, New York 12 18 1 (Received December 12, 1977) Publication costs assisted by the National Institutes of Health
The effect of pyrophosphate and phosphonate anions on the spontaneous formation of calcium oxalate mono-, di-, and trihydrates has been studied at 25 and 37 "C in supersaturated solutions of low ionic strength and at high ionic strength in solutions simulating physiological urine conditions. The rate of crystal growth in the presence of these additives was found to be dependent on the square of the solution supersaturation. Calculated rate constants for calcium oxalate trihydrate were independent of additive concentration for both the pyrophosphate and phosphonate cases and only the induction period preceding growth was found to increase with additive concentration. Sodium pyrophosphate decreases the nucleation kinetics of calcium oxalate monohydrate at a concentration of 8.0 X loT7M and completely inhibits monohydrate growth at 5.0 X lo4 M. The kinetic growth parameters for calcium oxalate dihydrate were found to be unaffected by these additives.
Introduction Crystal growth of inorganic and organic crystals from solution is in general affected not only by the properties of the solvent medium but also by additional soluble impurities where the ionic charge density of the latter appears to be of principal importance at least for the case of crystals with some degree of ionic bonding. Surface adsorption of impurity molecules with a resulting influence on one or more of the energetic, steric, and flux barriers to crystal growth has been the central point in many models used to explain changes in kinetic rates, habit modification, and impurity incorporation in growing crysta1s.l Concentrations of impurities in the range 10+ to M were effective for systems of soluble crystals while for sparingly soluble salts trace concentrations of 10" to M are significant. The solubility of the crystallizing material and its supersaturation in solution appear to be the main factors influencing impurity effects. For example, in comparing growth kinetics on seed crystals with spontaneous crystal formation, which generally requires higher supersaturation, one finds that the concentration of inhibitor required for a similar growth reduction is considerably higher for the case of spontaneous nucleation and growth. The inhibitor properties of condensed phosphates of both the meta- and polyphosphate types have been extensively studied in relation to scale f o r m a t i ~ n ~and -~ calcification5* in biological systems. In the former case it has been shown that sodium pyrophosphate is an effective growth inhibitor when added to CaC03,2-3SrS04,7 and Bas048 (Na5P3010added) systems while in the case of the more soluble CaS04-2H209J0 this inhibitor appeared to have only a slight effect on the growth kinetics. The pyrophosphate anion has been found in many biological fluids12 and thus there has been considerable interest in establishing its role in the formation of crystalline materials associated with hard and soft tissues as well as its influence on pathological mineralization of kidney and bladder calculi. Pyrophosphate is effective in retarding both the seeded and spontaneous growth of CaHP04.2H20 (DCPD)ll and Ca5(P04)30H(HAP).13-15 In the case of HAP initial phase separation is not affected but only the final conversion of the precursor phase to crystalline HAP. Sodium pyrophosphate has been found to inhibit the growth of CaCz04.H20in both low and high ionic strength 0022-3654/78/2082-0864$01 .OO/O
s ~ l u t i o n s ' ~and ~ ' ~in addition the crystal aggregation rate is reducedls and also the aging of metastable calcium oxalate hydrates is modified.lg A surface adsorption mechanism involving a simple Langmuir isotherm model has been found useful in describing the effect of P2074-in many of these systems,9J1J6J7 and it appears that the bonding capacity of pyrophosphate for metal ions20 together with its flexible structurez1 can explain the adsorptive activity and resulting inhibitor control. With the rather limited hydrolytic stability of the pyrophosphate ion interest has developed in the structural related phosphonate compounds22where the P-0-P linkage is replaced by P-C-P yielding increased stability in biological systems. Phosphonate compounds are similar to pyrophosphate in regard to inhibitor activity and work in this area has been reviewed by Russell and Fleischa5v6 Calcium oxalate can form three different hydrated structures in aqueous solution: a stable monohydrate phase as well as metastable di- and trihydrates. This investigation attempts to study the effect of inhibitors on the spontaneous nucleation and growth of the metastable hydrates in order to establish possible differences in inhibitor activity between the di- and trihydrates and the stable monohydrate structure. A precise conductivity method was used to follow changes in the calcium and oxalate ion concentrations; radioactive tracers were used to monitor uptake of pyrophosphate ion, and x-ray and optical techniques were used for structural characterization.
Experimental Section Stock solutions of calcium chloride (CaCI2-2H20,reagent grade, Fisher Scientific Co.) or calcium nitrate (Ca(N03)2.4H20 reagent grade, Baker and Adamson) and sodium or potassium oxalate (Na2C2O4,primary standard grade, Fisher Scientific Co.; KZC2O4.H20, reagent grade, Fisher Scientific Co.) were prepared using doubly distilled water and were filtered through 0.2-pm filters (Type GSWP, Millipore Corp.). The filters were washed prior to use to remove residual wetting agents or surfactants, and all filtering steps were carried out using a glass filtration apparatus (Millipore Corp.). Stock solutions were analyzed as described p r e v i o ~ s 1 y . l ~Sodium ~ ~ ~ pyrophosphate (Na4P207.10Hz0)solutions were prepared from reagent grade materials (Fisher Scientific Co.) and the 0 1978 American Chemical Society
Crystal Growth Kinetics of Calcium Oxalate Hydrates
TABLE I: Composition of High Ionic Strength Medium Concn, M Sodium chloride (NaCl) Magnesium nitrate (Mg(NO,),) Sodium sulfate (Na,SO,) Sodium phosphate (Na,HPO,) Potassium citrate (K,C,H,O,) Urea (NH,CONH,) Hydrochloric acid (HCl) to given final (pH)
0.1027 2.632 X 1.559 X lo-' 1.982 X lo-' 2.896 X lo-' 0.2114 0.0206 (5.80)
The Journal of Physical Chemistry, Vol. 82,No. 8, 1978 865
iterative method involving successive approximations for the ionic strength, 1. The activity coefficient for the divalent ion, fii (Zi = 2), was calculated using the equation (298 K) proposed by Davied'
- log tii = 0.51152? The thermodynamic association constant for calcium and the solubility products (Ksp) used in oxalate, KCaCZo4, calculations were those found from s t ~ d i e s ' in ~ ?clean ~~ supersaturated solutions. Crystal growth of calcium oxalate was studied as a function of impurity concentration a t 25 and 37 "C and a t various levels of relative supersaturation, u, where the latter term at time t (ut)is defined by
phosphonic acids used in this study were prepared from samples that were kindly donated by Monsanto Chemical Co., St. Louis, Mo. Procedures used for studying spontaneous crystal growth of calcium oxalates and a description of the crystallization and conductivity cells, particle sampling, x-ray diffraction, ut(CaCzO,.xH,O) = { ( [ c a 2 ' 1 t [ c 2 0 4 2 - 1 t)1'2 and microscopy techniques can be found in earlier 2 1/2 (KSP, CaC,0,*xH,0/f22)1'2 1 /(KSP, CaC,O,.xH,O/f2 ) s t ~ d i e s . ~ ~The * ~ pyrophosphate ~p~~ or phosphonic acid solution was freshly prepared and added to the growth cell (2) following addition of calcium chloride before final temAll concentrations are in molar units. In supersaturated perature equilibration and addition of sodium oxalate. The solutions of high ionic strength with the composition pH of the supersaturated solution not containing additives indicated in Table I a number of protonated ligand and was 6.0-6.2; the pH of solutions containing Na4P207was metal-ligand complexes exist in solution. To calculate the 7.0-7.4; and solutions containing phosphonic acids had a free ion concentrations the method of Perrin and Sayce pH of 5.0-5.2. In the latter case small aliquots of a (Program COMICS)28i29 which uses iteration formulas instandard sodium hydroxide solution was added into the volving ratios of observed and calculated total concencell prior to adding sodium oxalate and the pH of the final trations of metals and ligands was used and modified to supersaturated solution was 5.8-6.0. take into account activity coefficients ieq 1) of the ionized Pyrophosphate uptake during crystal growth was folspecies. All calculations were carried out using an IBM lowed in several experiments using 32Pradioactive tracers 360167 computer. The acid association constants and the (Na4P2O732P,in NaCl solution, New England Nuclear) metal-ligand stability constants that were required are together with liquid scintillation counting (Intertechnique listed in Table 11. The important calcium complexes for SL-30 liquid scintillation spectrometer). Sufficient tracer the supersaturation range studied ( u3Hz0= 1.3-4.6) were levels were used to give initial sample count levels of CaHC6O7-(18% of total Ca a t low u to 30% a t high u ) , 10 000-30 000 cpm and changes in pyrophosphate conCaS04 (8.5-9.8%), CaH2P04+ (7.7-8.0%), CaHP04 centration were followed by frequent sampling of the (4.1-4.2%), CaH2c607 (3.4-2.0%), and CaC2O4(2.0-1-7%). growth solution. The 1:l calcium (16-38% of total oxalate) and magnesium The effect of additives on the crystal growth of calcium (24-17%) oxalate complexes were most important with the oxalate trihydrate was studied at low ionic strength (1.3-2.5 protonated species, HCzOd-, accounting for 1.0 and 0.7% X M) using the same procedure employed in studies a t low and high u, respectively. without additives, and a similar procedure was used in the P y r o p h ~ s p h a t eand ~ ~ p h ~ s p h o n a t e ~ions l - ~ ~are known case of calcium oxalate monohydrate except that lower to complex with various metals including calcium in soinitial supersaturations were required. Calcium oxalate lution and the computational method described above dihydrate formation could not be achieved under these (COMICS) was used to access the degree of complex forconditions and instead supersaturated solutions of high mation. At pH 7.2 and with the total pyrophosphate ionic strength (0.198-0.214 M) containing the electrolytes concentration 3600 16
2.4b 1.5b 1.6b d
7.2c 6.8c 5.F
f
a Ionic strength range = 1.18-1.60 X lo-' M. All experiments at 37 'C, stirring rate = 300 rpm. CaC,O,.H,O principal growth phase. CaC20,.3H,0 principal growth phase. Mixture of two hydrates, no rate constant calculated. e Pyrophosphate added 37 min following ti. f Calcium pyrophosphate crystals forming after 60 h.
TABLE VI: Effect of Pyrophosphate and Phosphonate Additives on the Crystal Growth of Calcium Oxalate at High Ionic Strengtha Exptno.
TcaX 103M
Tc,o, X lo4 M
551 552 694 7 04 705 706 707 278 282 283 28 9 289-A
3.812 3.741 5.498 5.398 5.349 5.349 5.448 10.59 10.30 9.670 10.20 10.20
4.775 4.68 5 4.685 4.600 4.558 4.558 4.642 4.792 4.659 4.376 4.616 4.616
Concn x Additive Na4P207
Na,P,07 Na4P207
Na4P207
Na,P,O, Na4P2 7 ' Na4P207
HEDP ENTMP
l o 5M
ti, min
Crystal type (morphology)
Crystal size, wm
0.0 3.5 0.0 3.5 8.8 8.8 1.8 0.0 3.7 11.7 4.4 1.9
42 95 17 20 20 21 17 4 2 2.5 4 4
CaC,04.3H,0 (prismatic) CaC,04*3H,0 (prismatic) CaC,O;3H,O (prismatic) CaC,O; 3H,O (prismatic) CaC,0,.3H,O (prismatic) CaC,04.3H,0 (prismatic) CaC, 0,.3H,O (prismatic) CaC,0,.2H20 (octahedrons) CaC,04*2H,0(octahedrons) CaC,04*2H,0 (octahedrons) CaC,04*2H,0(octahedrons) CaC,0;2H,O (octahedrons)
4-8 3-6 7-12 7-12 7-12 7-12 7-12 3-5 3-5 3-5 3-5 3-5
a Ionic strength range = 0.198-0.214 M. Experiments 278, 282, 283, 289, and 289-A at 25 "C; all others at 37 "C. Stirring rate for all experiments equal to 300 rpm. Relative supersaturations ( u ~ ~ ,Uo, H,, O , UH,O) = expt 694, 704-707 (1.7, 2.4, 4.0);expt 551-552 (1.3, 1.9, 3.2);expt 278, 282, 283, 289 (3.4-3.7, 4.3-4.6, 6.2-6.5).
TABLE VII: Effect of Sodium Pyrophosphate on the Crystal Growth Kinetics of Calcium Oxalatea DehydrationHydrate Process Nucleation Growth aging CaC,O,.H, 0 CaC,O,.H,O CaC,0,.3H,O
Seeded Spontaneous Spontaneous
CaC,O,. 2 H,O
Spontaneous
+ = effective inhibitor.
Reference 16.
(+ 1" (-), ( + ) low u and
(+ )b
(+IC (-
1
(+Id
Crystal morphology
(+I
(+, high u )
high [P20,4-] (- )
(- )
Effect reduced under static growth conditions.
supersaturations (uSHZ0> 2.6, > 3.2) the major solid phase is calcium oxalate dihydrate while for low u levels ( C T ~ H ~< O ,2.0, uZHzO< 2.5) calcium oxalate trihydrate is the principal growth form. Intermediate concentrations produce mixtures of the two crystal phases and at the lowest supersaturation level studied ( u = 0.5, ~ uZHz0 ~ =~
(-
1
Reference 19.
0.9, uHZo= 1.75) again the trihydrate structure formed with only a trace of the monohydrate phase. The effect of pyrophosphate and phosphonate additives on crystal growth in these supersaturated solutions is summarized in Table VI, and comparing results for dihydrate growth ~in clean supersaturated solutions (expt 278) with results
The Journa/ 3f Physical Chemistry, Vol. 82, No. 8, 1978 869
Crystal Growth Kinetics of Calcium Oxalate Hydrates
TABLE VIII: Phosphonate E f f e c t on Crystal Growth of C a l c i u m Oxalatea Hydrate
Process
CaC,O,.H,O CaC, 0,. H, 0 CaC,O,.3H,O CaC, 0,-3H, 0 CaC,O,.2H,O
Seeded Spontaneous Spontaneous Spontaneous Spontaneous
t = effective inhibitor. Reference 16. l o w e d by aging in s o l u t i o n of ENTMP.
Nucleation
Growth
Reference 19.
in the presence of additives (expt 282,283,289) it is seen there is no effect on the measured induction period, crystal morphology, or crystal size. Under conditions where CaC204-3H20growth is important results agree with those found in dilute solutions and here again the induction period increases a t low c levels with added Na4P207(expt 551 and 552). Increasing the relative supersaturation decreases the effect of the latter on ti (expt 694 and 704-707). Tables VI1 and VI11 summarize results found in this study on the effect of sodium pyrophosphate and phosphonate anions, respectively, on calcium oxalate crystal growth.
Discussion The kinetics of crystal growth of calcium oxalates from supersaturated solutions containing added impurities are described by a rate equation (eq 3) analogous to that used in clean solutions. A similar equation has been found valid for a number of sparingly soluble salts including several calcium salts (CaS04.2H20,CaC03, CaHP04.2H209s4*11). Impurity adsorption at specific sites of the growing crystal surface is probably occurring, and in the case of pyrophosphate inhibition of CaC204.H20seeded growth a model involving a simple Langmuir adsorption isotherm has been used to successfully explain the rate data.16 This analysis allows for a calculation of the free energy of adsorption (AGads)1ap35and for the case of calcium oxalate monohydrate, following the method outlined by Davey and Mullin, the value found (AGad, = -2.1 kcal/mol) indicates that ledge sites are more important than edge or kink sites for adsorption since in the later cases AG,h has been found to be larger (5-10 kcal/rn01).~~ In the case of ledge sites the flux of pyrophosphate molecules and the resulting Ca-P207 bonding are probably more easily accommodated. Pyrophosphate and phosphonate additives had no significant effect on the growth rate of either metastable form of calcium oxalate. Calculated rate constants for calcium oxalate trihydrate were within experimental error equal to those found in clear solutions and these additives similarly have no effect on HAP growth. Thus for both spontaneous and seeded growth of HAP the growth rates of the initial metastable phases ae unaffected while alternatively these additives have a strong retarding action on the final growth rate of the thermodynamically stable Tracer studies indicate that crystalline HAP phase.13-15@v37 adsorption and possible incorporation of P207molecules does occur during CaC204.3H20phase separation and thus it is possible, as has been suggested by Sears38 and Cabrera,39that for certain crystalline materials (metastable structures) the surface growth fronts are still able to move around or flow over adsorbed impurity molecules, thus maintaining a constant growth rate. Nucleation and growth rates as reflected in the measured induction periods for CaC204.3H20decreased with increasing inhibitor concentration, and in contrast to the calculated rate constants they appear to be dependent on supersaturation, showing a greater decrease in the nu-
Crystal morphology
(+Ib
(+I (- 1
(+ 1 (- 1
(-
Dehydrationaging
1
(CaC,O,.3H,O
1
(+IC (-Id
(-
1
(--I
g r o w n in clean supersaturated s o l u t i o n fol-
cleation rate as the relative supersaturation is lowered. Phosphate impurities did not change the induction period for CaC204.2H20growth, and crystal size and morphology were similar to clean solutions runs, suggesting that the growth rate was similarly unaffected. These results agree with those of Miller,4O who found sodium pyrophosphate to have a negligible affect on the crystal size distribution and growth rates of the dihydrate phase as determined from continuous crystallizer experiments. to lo4 M) found At pyrophosphate concentrations in urine12 it would appear that spontaneous or epitaxial formation of CaOx-H20would be decreased or eliminated (under suitable flow conditions) while growth of the diand trihydrate phases would not be retarded. Aging processes involving dehydration to the stable monohydrate structure are modified by pyrophosphate as was found in earlier s t ~ d i e s and ’ ~ ~aggregation ~~ of these metastable crystal phases, which has been indicated1* as a possible critical step for calculi growth, has also been found to be reduced by pyrophosphate and phosphonate additives. Acknowledgment. This study was supported by the National Institutes of Health.
References and Notes (1) (a) R. J. Davey, J . CrystalGrowth,34, 109 (1976); (b) J. R. Bourne and R. J. Davey, ibu., 36, 278 (1976); (c) M. Ohara and R. C. Reid, “Modeling Crystal Growth Rates from Solution”, Prentice-Hall, Englewood Cliffs, N.J., 1973; (d) R. Boistelle in “Industrhl Ctystalliration”, J. W. Mullin, Ed., Plenum Press, New York, N.Y., 1976, p 203. (2) B. Raistrick, Discuss. Faraday Soc., 5, 234 (1949). (3) B. Raistrick, Chem. Ind., 40, (1952). (4) G. H. Nancolhs and M. M. Reddy, Soc. Pet. fng. J., 14, 117 (1974). (5) H. Fleisch and R. G. G. Russell, J . Dent. Res., 51, 324 (1972). (6) (a) R. G. 0. Russell and H. Fleisch in “The Biochemistry and Physiology of Bone”, Vol. 4, G. H. Boume, Ed., Academic Press, New York, N.Y., 1976, Chapter 2; (b) H. Fleisch et al. in “Phosphate Metabolism”, S. G. Massry and E. Riz, Ed., Plenum Press, New York, N.Y., 1977, p 279. (7) J. R. Campbell and G. H. Nancolhs, J. Phys. Chem., 73, 1735 (1969). (8) G. H. Nancollas and S. T. Llu, Soc. Pet. f n g . J . , 509 (1975). (9) S. T. Liu, Ph.D. Thesis, State University of New Ywk at Buffalo, Buffalo, N.Y., 1972. (10) S. T. Liu, unpublished data. (1 1) R. W. Marshall and G. H. Nancolhs, J. Phys. Chem., 73,3838 (1969). (12) H. Fleisch and S. Bisaz, Am. J . Physiol., 203, 671 (1962). (13) H. Fleisch et al., Calc. Tiss. Res., 2, 49 (1968). (14) J. D. Termine and A. S. Posner, Arch. Biochem. Biophys., 140,307 (1970). (15) W. G. Robertson, Caic. T i s . Res., 11, 311 (1973). (16) G. H. Nancolhs and G. L. Gardner, J. Cryst. Growth, 21, 267 (1974). (17) J. L. Meyer and L. H. Smith, Invest. Urol., 13, 36 (1975). (18) W. G. Robertson, M. Peacock, and B. E. C. Nordin, Clin. Chem. Acta, 43, 31 (1973). (19) G. L. Gardner, J . ColloidInterface Sci., 54, 298 (1976). (20) L. G. Sillen and A. E. Martell, Chem. SOC.Spec. hrbl., No. 17 (1964). (21) N. S. Mandel, Acta Crystalbgr., Sect. B , 31, 1730 (1975). (22) R. Engel, Chem. Rev., 77, 349 (1977). (23) G. L. Gardner, J . Cryst. Growth, 30, 158 (1975). (24) R. H. Doremus, G. L. Gardner, and W. McKay in “Colloquium on Renal Lithiasis (Proceeding of an International Colloquium on Renal Lithiasis, Galnesvllle, Fla, 1975, U.S.A.)”, B. Finlayson and W. C. Thomas, Jr., Ed., The University Presses of Florida, Gainesville, Fla., 1976, p 18. (25) G. L. Gardner, J . Cryst. Growth, submitted for publication. (26) G. L. Gardner and R. H. Doremus, J . Invest. Urol., in press. (27) C. W. Davies, “Ion Association”, Butterworths, London, 1962. (28) D. D. Perrin and I.0. Sayce, Talanta, 14, 833 (1967). (29) G. Ginzburg, Talanta, 23, 149 (1976).
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The Journal of Physical Chemistty, Vol. 82, No. 8, 1978
(30) R. R. Irani and C. F. Callis, J. fhys. Chem., 64, 1398 (1960). (31) S. Westerback, K. S. Rajan, and A. E. Martell, J. Am. Chem. Soc., 87, 2567 (1965). (32) R. P. Carter, 8. L. Carroll, and R. R. Iranl, I m g . Chem., 6, 939 (1967). (33) R. L. Carroll and R. R. Irani, J. Inow. Nucl. Chem., 30, 2971 (1968). (34) H. Wada and Q.Fernando, Anal. Chem., 44, 1640 (1972). (35) R. J. Davey and J. W. Mullin, J . Cryst. Growth, 26, 45 (1974).
Robert P. Liburdy (36) (37) (38) (39)
J. L. Meyer and G. H. Nancollas, Calc. Tiss. Res., 13, 295 (1973). M. D. Francis, Calc. Tiss. Res., 3 , 163 (1969). G. W. Sears, J. Chem. Phys., 29, 1045 (1958). N. Cabrera and D. A. Vermilyea in “Growth and Perfection of Crystals”, R. H. Doremus, 8. W. Roberts, and D. Turnbull, Ed., Wiley, New York, N.Y., 1958. (40) J. Miller, Ph.D. Thesis, The University of Arizona, 1978.
N-(3-Pyrene)succinimidothioethanol. A Fluorescent Probe Sensitive to pH and Redox Potential Robert P. Liburdy Radiation Sciences Division, USAF School of Aerospace Medicine, Aerospace Medical Division, USAF Systems Command, Brooks Air Force Base, Texas 78235 (Received July 7, 1977; Revised Manuscript Received October 31, 1977) Publication costs assisted by the Department of the Air Force, USAF School of Aerospace Medicine (AFSC)
The fluorescent P-mercaptoethanol adduct of N-(3-pyrene)maleimide, PM-BME, shows sensitivity to pH and to redox potential. PM-BME fluorescence at 370 and 390 nm increases reversibly as pH is increased from values of 3.0 to 8.0. Above pH values of 8.0 an irreversible red shift to wavelengths of 385 and 400 nm occurs with a marked enhancement of fluorescence. The observed rate constant for the base-induced red shift is pH dependent and correlaks closely with the rate of simple base hydrolysis of the succinimidothio ring on PM-BME. PM adducts of L-cysteine, N-acetyl-L-cysteine,and gluthathione also undergo a base-catalyzed red shift, with PM-L-cysteine exhibiting markedly quenched fluorescence. Following treatment with base, PM-L-cysteine is ninhydrin negative, suggesting that quenching of its red-shifted fluorescence is due to loss of its free amino group to form a thiazine adduct. Treatment of PM-BME with redox agents at constant pH results in a reversible increase in fluorescence emission associated with increased electron mobility. As with strong base (pH >12), strong oxidizing agents induce a new, broad, nonexcimer-associated emission band at 440 nm. This new peak is generated at the expense of emission at 385 and 400 nm and is reversed by acid, reducing agents, or by UV radiation. In one application, PM-BME sensitivity to redox potential is enhanced by the electron-transporting protein cytochrome c, indicating that PM adducts may prove useful in the future study of electron mobility and protein conformation.
Introduction Fluorescent probes may be used to study protein or synthetic biological and chemical structures since changes in the microenvironment around a fluorophore may significantly alter the probe’s fluorescence-emission propertie~.’-~It is necessary that a probe have sensitivity to well-defined chemical and physical influences; in this way similar influences occurring a t or near the site of fluorophore residence may be elucidated. N-(3-Pyrene)maleimide (PM)4is a recently synthesized nonfluorescent dye that forms highly fluorescent covalent adducts with reactive sulfhydryl and amino groups (Figure 1). Subsequently, the contractile and regulatory proteins of striated m ~ s c l e , ~50 - ~ S ribosomal proteins,8 and immunoglobulinsg have been conjugated with P M in an effort to study molecular structure during active functional states of these proteins. Recently,’O studies of the fluorescence properties of a simple short chain, aliphatic, thiol adduct of PM, N-(3-pyrene)succinimidothioethanol (PM-BME), were initiated in an effort to quantitate P M fluorescence using this simple model compound. Presented here are the results of experiments elucidating PM-BME fluorescence sensitivity to hydrogen ion concentration, electron mobility, and UV radiation. In addition, the results of fluorescence experiments are presented that employ PM-BME as a potentially useful probe of cyto-
chrome c-associated electron mobility in the methylene blue/H202 redox system.
Experimental Section Materials. N-(3-Pyrene)maleimide was synthesized as described previously.4 The N-(3-pyrene)maleimide adduct of P-mercaptoethanol was synthesized as described earliera7 All other low-molecular-weight thiols were purchased from Sigma Chemical Corp. Organics were reagent grade or better. Glass-distilled deionized water was used throughout. Cytochrome c was purchased from Calbiochem. N-(3-Pyrene)maleimide Adducts of Low-MolecularWeight Thiols. L-Cysteine, N-acetyl-L-cysteine, and glutathione (1M in water, pH 6.0) were each titrated with P M (8 M in acetone) until no further increase in fluorescence was observed. The calculated final concentration of P M in the reaction mixtures was 1M. The reaction mixtures were kept in darkness at 25 “C for 12 h and subsequently lyophilized to dryness. The residues were solubilized in acetic acid and recrystallized twice from acetone. Acid-Base Titration. Continuous titrations were performed using a Beckman Research pH meter and a Corning semimicro combination glass electrode. A water-jacketed glass titration well (50 mL) was maintained
This article not subject to U S . Copyright. Published 1978 by the American Chemical Society
