J. Phys. Chem. 1986.90:6345-6349. Downloaded from pubs.acs.org by UNIV OF NEW SOUTH WALES on 08/26/15. For personal use only.
J. Phys. Chem. 1986,90,6345-6349 function itself. Here experimental studies are essential. One point of considerable importance that could be investigated by measurement of the equilibrium distribution of the conformers of N-methylalanylacetamide in aqueous solution is the origin of the hydrogen bonding interaction. Two types of models are in current use, and the integral equation method of this paper has been applied to both of them. The first, considered here, has an important contribution to the hydrogen bond energy (approximately 50%) that is nonelectrostatic (the so-called 10-12 term in potential function). The second, considered in a previous report,44dispenses with a special hydrogen bond term and obtains corresponding energies by the use of larger charges; i.e., the dominant contribution to the hydrogen bond is electrostatic. It is clear from the theory developed here, as can be seen by comparing Figure 3 of ref 44 with Figure 15 of the present paper, that the solvent effect is much more important in the second model than in the first; i.e., because the dominant effect of the aqueous solvent is to produce dielectric shielding, the electrostatic interaction is strongly decreased, while the explicit hydrogen bonding potential is unaffected. A measurement of the relative populations of hydrogen-bonded and non-hydrogen-bonded species would make it possible to determine which representation of the hydrogen bond is more accurate. The interplay of structure and dynamics plays an essential role in the function of biological m a c r o m ~ l e c u l e s . ~Much ~ ~ of the (46) Alber, T.;Gilber, William A.; Ponzi, Dagmar R.; Petsko, G.A. CZBA
Found.Symp. 1982, 93, 4. Karplus, M.; Swaminathan, S.; Ichiye, T.; van Gunsteren, W. F. Ibid., p 271 and references therein.
6345
theoretical progress in this area has come from the analysis of dynamical simulations of proteins and nucleic acids based on potential functions that are constructed from classical site-site models similar to those used here. However, most of the simulations have been restricted to treating the system in vacuo, although explicit solvent has been included in a few cases. The present approach to the potential of mean force when coupled with a reduced description of the dynamics, such as a Langevin algorithm, makes possible a fuller examination of the low-frequency dynamical behavior of polyatomic solutes in condensed-phase environments. It should be possible, for example, to perform simulations for systems like oligopeptides in an implicit solution environment that extend over nanoseconds or even microseconds. These could be used to obtain a detailed picture of conformation transitions and rate constants and would make possible a quantitative comparison with N M R relaxation data and other measurements of the intramolecular dynamics. Acknowledgment. B.M.P. would like to thank the Robert A. Welch Foundation for partial support of this work. P.J,R. acknowledges the support of the National Institutes of Health, and M.K.acknowledges support from the National Science Foundation. P.J.R. is an Alfred P. Sloan Foundation fellow and the recipient of an NSF Presidential Young Investigator Award and an NIH Research Career Development Award from the National Cancer Institute, DHHS. Registry No. N-Methylalanylacetamide, 19701-83-8.
Microspeciation of Polypeptides
Department of Inorganic and Analytical Chemistry, L. Edtvos University, Budapest, H- 1443, Pf.123, Hungary (Received: March 13, 1986)
A new principle to estimate the concentrations of differently protonated polypeptide species (microspecies) has been elaborated. This method uses a recent type of thermodynamic parameter: group constant, which is a submolecular measure of basicity. The applicability of the principle is discussed, and the relevant relations are deduced. To study the main characteristics of this type of microspeciation, a comparative analysis has been made on glutathione, using either its group constants or its previously published microconstants. The full efficiency of the method is exemplified by the 32 amino acid containing microspecies concentrations and their distributions.
Microspecies are multidentate (bi0)ligands of distinctive number and site(s) of the bound protons. This d e f i t e stage of protonation (and the inherent fine structure) is a sine qua non of their specific biochemical interactions. Speciation is a recent tendency of environmental and clinical analytical chemistry. It provides not only the analytical concentration of a component in a system but also its distribution among the different species. Microspeciation in addition distinguishes between the different isomeric products of protonation, complexation, and other associations. Due to its principal significance, this paper focuses on the basic questions of protonation microspeciation. To date the only way for microspeciation (determination of concentrations of microscopic protonation isomers) was their calculation in the knowledge of the pH, the total concentration, and the equilibrium macro- and microconstants. Two species, the zero and the maximum proton containing ones, do not require the knowledge of microconstants. All others (where isomeric protonation occurs) do. For example, in the solution of a dibasic unsymmetrical acid (the simplest type of compound for microequilibria), four microspecies exist: H2A, HA-, AH-, and A2-. H2A is the acid, A20022-3654/86/2090-6345$01.50/0
is its anion, and HA- and AH- are nonidentical protonation isomers with bound protons at different sites. H2A can be malic acid, amino acids, etc. In this system the relations 1-7 are valid kA = [HA-]([H+] [A2-])-' kB = [AH-]([H+][A2-])-'
(1)
kBA= [H2A]([H+][AH-])-'
(3)
kAB= [H,A]([H+] [HA-])-' + [HA-] + [AH-] + [H2S]
(4)
C, = [A2-]
= kA
+ ke
(5)
(6) (7)
where superscripts of k microconstants indicate the group (site) protonating in the process under consideration, subscripts (if at all) denote the other group@)bound to proton during the process, CAis the total concentration of the ligand, and 8, and O2 are the complex products. From eq 1-7 any microspecies concentration can be expressed, e.g., [AH-]. If CA= 1, eq 8 gives the proportion 0 1986 American Chemical Society
6346 The Journal of Physical Chemistry, Vol. 90, No. 23, 1986
(relative concentration or occurrence probability) of AH- in the system. [AH-] = kBCAIH+](l+ &[H+]
+ /32[H+]2)-1
(8)
J. Phys. Chem. 1986.90:6345-6349. Downloaded from pubs.acs.org by UNIV OF NEW SOUTH WALES on 08/26/15. For personal use only.
In a similar way microspecies concentrations of more complicated molecules can also be calculated from an analogous set of data. Total concentrations, pH, and macroconstants are usually available or determinable, but microconstants are scarce in the literature, and the feasibility of their determination is rather the exception than the rule. In fact, microconstant (and so microspecies) determination was only possible for relatively simple molecules (catecholamines,'** some amino acids,3 small peptides? etc., for example), provided that protonation of a t least all but one basic site could selectively be monitored by UV, NMR, or other solution spectroscopies. This is one of the difficulties why the efficiency of even the best evaluation method is restricted to two groups of overlapping equilibria and to four corresponding microconstants. Moreover, as the number of groups in the molecule linearly increases, there is an exponential increase in the number of microspecies and microconstants to be determined and also in the equations to be handled. This causes inevitable proliferation of the errors originated from two or more parallel used experimental techniques (pH-metry plus spectroscopy). The few attempts to determine microconstants in molecules of three proton binding sites have always used thermodynamic or spectroscopic data of auxiliary molecules (simplified derivative^).^-^^ For lack of a generally applicable microconstant determination method (for more details, see also ref 1l ) , the number of systems where reliable microequilibrium studies could be published is a few dozens. Due to the same reason no microspeciation data have appeared on multidentate bioligands such as polypeptides or nucleic acids, although there are some macromolecules where the protonation behavior of one or two groups could selectively be monitored.'* Concerning these large molecules, group constants can serve as thermodynamic parameters to estimate the equilibrium microconcentrations as described below.
Theory As it was expounded for peptides in the group constant paper," the electron attracting effect of protonation at one site cannot reach even the nearest other site, due to the quenching effect of the intervening bonds of the molecular backbone. Consequently, these sites can influence each other's protonation electrostatically or by mutual H-bond formation only, where the sites act as bridgehead groups. On this basis the groups can be sorted into two classes: (1) These groups are independent of the other protonating groups. They protonate singly and exist in two possible stages of protonation: the zero and the proton holding ones (A and AH, respectively, where the charges are not considered). It should be (1) Martin, R. B. J . Phys. Chem 1971, 75, 2657.
(2) Kiss, T.; Tath, B. Talanra 1982, 29, 1982. (3) Rabenstein, D. L.; Sayer, T. L. Anal. Chem. 1976, 48, 1141. (4) Rabenstein, D. L.; Greenber, M. S.; Evans, C. A. Biochemistry 1977, 16(5\. 977. (5) Neuberger, A. Biochem. J. 1936, 30, 2085. (6) Martin, R. B.; Edsall, J. T.; Wetlaufer, D. B.; Hollingworth, B. R. J . Biol. Chem. 1958, 233, 1429. (7) Ishimitsu, T.; Hirose, S.; Sakurai, H. Chem. Pharm. Bull. 1976, 24(12), 3195. (8) Ishimitsu, T.; Hirose, S.;Sakurai, H. Chem. Pharm. Bull. 1978,26(1),
.
. ..
74
(9) Ishimitsu, T.; Hirose, S.; Sakurai, H. Talanta 1977, 24, 555. (10) Rigler, N. E.; Bag, S. P.; Leyden, D. E.; Sudmeier, J. L.; Reilley, C. N . Anal. Chem. 1965. 37. 872. (11) Noszll, B. J. Phys. Chem., in press. (12) Rabenstein, D. L. J . A m . Chem. SOC.1973, 95, 2797. (13) NoszB1, B.; Scheller-Krattiger,V.; Martin, R. B. J. Am. Chem. Soc. 1982. 104. 1078. (14) Martin, R. B.; Edsall, J. T. Bull. SOC.Chim. Biol. 1958, 40, 1763. (15) Noszlll, B.; Burger, K. Znorg. Chim. Acta 1979, 3, L387. (16) NoszAl, B.; Burger, K. Magy. W m .Foly. 1980, 87, 175. (17) Burger, K.; Farsang, Gy.; Ladlnyi, L.; NoszSl, B.; PWi, M.; Takacsi Nagy, G. Bioelectrochem. Bioenerg. 1975, 2, 329. (18) Tanokura, M. Bioc. Biop. Acra 1983, 742(3), 586.
Noszzl noted, however, that nonprotonating parts of the molecules may be attached to these groups. The relevant protonation equilibrium is characterized by one group constant (eq 9). kA = ([AI [HI )-I (9) The probability of being protonated at such a group can be expressed by the ratio
PAH= [AHI([Al + [AHI)-' Similarly the probability of being unprotonated is
(10)
= [AI([Al + [AH])-' Substituting (9) into (10) and (11) gives
(1 1)
PAH
= kA[HI(1 + kA[HI)-'
PA= (1
(12)
+ k~[H])-l
(13) (2) These groups are divided into pairs with three stages of protonation: (a) the zero proton containing uncoupled stage, B,C; (b) the one proton containing coupled stage, holding the proton mutually B-H-C; (c) the two proton containing also the uncoupled stage, holding one proton each: BH,CH. Equilibrium between these positions can be depicted by the formation and the decomposition protonation constants of the H-bond: kw,c = [B-H-CI([B,Cl kdB+C [BH,CH]([B-H-C] [HI)-'
(14) (15)
The probability of these successive stages is expressed by the group pair concentrations: PB,C = [B,C]([B,C] [B-H-C] + [BH,CH])-' (16)
+
+ [B-H-C] + [BH,CH])-' PBH,CH = [BH,CH]([B,C] + [B-H-C] + [BH,CH])-' PB.H-c
= [B-H-C]([B,C]
(17)
(18) By making use of equilibrium equations (14) and (15), (16)-( 18) may be rewritten as (19)-(21).
+ kfB,CIHl + kfB,CkdB,C[H12)-1 (19) (20) PB-H-C = kfB,C[Hl (1 + kfB.CIH1 + kfB,CkdB,C[H12)-1 PBH,CH = kfB,CkdB,C[HI2(1 + kfB,CIHl + kfB,CkdB,C[H12)-l PB,C
= (1
(21) Equations 12, 13, and 19-21 show that the probability of any stage of individual or coupled groups can be expressed by the appropriate group constants and hydrogen ion concentrations. The above equations illustrate that an uncoupling group occurs at two levels of protonation and a coupling pair of groups exists in three stages. Accordingly, a molecule of h group pairs and n - 2h uncoupling groups may occur in 2"-Zh3hdifferent microscopic forms. The occurrence probability of the molecule in any protonation stage a t a given pH is the product of probabilities belonging to (independent or coupled) groups in the appropriate protonation stage at that particular pH. For example, if a molecule contains two independent groups (A and D) and one group pair (B,C), it may occur in 2e231 = 12 microscopic forms, which are combinations of the elementary forms of the groups. The occurrence probability of the molecule when group A is bare, D is protonated, and the B,C pair binds one proton is PA,BH-C,DH = PAPB-H-CPDH This can be exuressed in terms of Ywouur constants
(22)
= ( l + kAIHI)-'kf&CIHl ( l + kfB,CIHl + kfB,CkdB,C[H12)-'kD[HI(1 + kD[HI)-l (23) A more general formulation of (22)-type relations for the occurrence probability at a molecular level is PA,B-H-C.DH
N
Px =
np,
i=I
(24)
The Journal of Physical Chemistry, Vol. 90, No. 23, 1986 6341
Microspeciation of Polypeptides TABLE I: Equilibrium Macroconstants, Microconstants, and Group Constants of Glutathione cumulative macroconst microconstants group mnst log k" 2.07 2.09 0.05 3.12 3.36 2.33 8.93 9.13 9.28 9.08
*f 0.05 f 0.10 i 0.01 f 0.04 f 0.04 f 0.10 0.02
*
log
ky
3.38
log ks
8.82
log kN
9.39
The actual concentration of the molecule in any particular microscopic form can be calculated by multiplying the given probability by the total (analytical) concentration (CL) of the substance (eq 25). J. Phys. Chem. 1986.90:6345-6349. Downloaded from pubs.acs.org by UNIV OF NEW SOUTH WALES on 08/26/15. For personal use only.
[A,B-H-CPHl = P A , B - H - C , D H ~ L
(25)
In this paper the above theory is applied to two vitally important natural molecules. The first one (y-glutamylcysteinylglycine) is the largest and most significant peptide, which has been profoundly studied at the level of microscopic equilibria. Thus, for this molecule microspecies concentrations could be calculated in the traditional way too, as a basis for comparison. The other polypeptide, corticotropin, is already a macromolecule, where (for lack of method) no attempt for microspeciation has been made. Applications Full Microspeciation of Reduced Glutathion (GSH). Glutathione is considered a molecule of twice two protonating groups of overlapping equilibria. In the basic pH region (8-9), the glutamyl amino and the cysteinyl thiolate protonate, while around pH 2-3 the glycyl carboxylate and the glutamyl carboxylate protonate predominantly. Since these groups are well-separated by several intervening covalent bonds, their degree of protonation can be selectively monitored by N M R spectroscopy. Accordingly, the protonation processes of the molecule could be depicted in terms of twice four microconstants, taking into account four species in the basic and also four species in the acidic region (altogether seven, because the predominant one in the neutral region is common). There is no doubt that these seven species are the most abundant (major) microspecies in a glutathione solution. However, a molecule of 4 groups exists in 24 = 16 microscopic forms, and the major species are not necessarily the reactive ones, especially in biological processes, as proven by kinetic studies.I3 Using the literature microconstant values, we have calculated the relative concentrations of those seven major species. Besides, calculations based on group constants have also been made, either to compare the results gained by the traditional and the new types of constants and methods or to estimate the concentrations of the remaining nine (minor) microspecies, which is not possible by means of the strict microconstant-based treatment. Constants used in these calculations are summarized in Table I. The source of all these constants is Rabenstein's article.I2 Microconstants are in the original form; macroconstants are protonation ones, rearranged for group constant calculations. Microconstants and the relevant group constants are in the same row. The general relations between macroconstants and group constants for a molecule of four groups in the absence of group-linking intramolecular H-bonds are1'
+ ks + k y + (ku)* & = kNks + kNky + kNkU + ksky + (ksku + kyku)* /33 = kNksky + kNkskU + kNkykU + (kskykU)* 81 = kN
04
= kNkSkYkU
(26) (27)
(28)
TABLE 11: Numerical Values of Those MicroconcentrationsThat Can Be Calculated with either Microconstants or Group Constantsa log re1 concn of microspecies at pH 7 calcd by NS Y U microconst mouu const HHHH -8.56 -8.56 HHHO -3.65 -3.63 HHOH -4.68 -4.94 HOHH -10.38 OHHH* -8.98 HHOO -0.008 33 -0.008 40 -5.45 HOHO OHHO -6.02 -6.76 HOOH OHOH* -5.36 -10.80 OOHH* HOOO -1.94 -1.83 OH00 -2.14 -2.40 OOHO -7.84 000H* -7.18 0000 -4.22 -4.22 ON, S, Y , and U are group legends as in the text. H and 0 signals below them (regardless of the charge) indicate whether the given group holds proton (H) or not (0).A set of four such signals in a horizontal row defines a particular stage of a glutathione molecules. pH 7 has been chosen arbitrarily.
the whole system of eq 26-29 can be expressed by a polynomial of the fourth degree" k4 - &k3 + P2k2- &k
+ p4 = 0
(30)
where k group constants are the squares of eq 30. When we refer to glutathione, N, S, Y, and U subscripts denote the amino, thiolate, glycyl carboxylate, and glutamyl carboxylate sites of the molecule, respectively. The most important chemical precondition of using group constants is that the number of group-separating bonds should exceed three. In the case of glutathione, this is valid for the N-Y, N-S, S-Y, S-U, and Y-U distances, but it is not true for N-U. For this reason, the ks and ky values as squares of eq 30 are real group constants, representing the thiolate and glycyl carboxylate basicities, regardless of the protonation state of any other group. However, the glutamyl amino and a-carboxylate moieties of the molecule are remote enough to be independent of the other two sites, but they are in close vicinity and in strong interaction with each other. The amino basicity is several orders of magnitude greater than that of the adjacent carboxylate. Consequently, the kN "restricted group constant" refers to the amino basicity, whatever the protonation states of the S and Y groups are when U is anionic. Similarly, kU is also practically independent of S and Y, but its value is characteristic of the U carboxylate basicity only, when the neighboring amino is cationic. This is why those terms in (26)-(28), which represent a form where the glutamyl carboxylate holds the proton but the more basic nearby amino does not, are in parentheses with an asterisk. Each of the 15 terms on the right-hand side of eq 26-29 represents a microspecies holding 1-4 proton(s). The concentrations of 1 1 of them without an asterisk can be calculated purely by means of group constants as described in the theoretical part by eq 23. Since glutathione is not a typical peptide, owing to its amino acid like residue, to calculate the concentration of the remaining four microspecies designated with an asterisk needs further consideration. Each species with an asterisk has its protonation isomeric counterpart, where the glutamyl amino group holds the proton instead of the U carboxylate. In monomeric amino acids the concentration ratio between the amino and carboxylate protonated isomers is about 2.2 X lo5. To get the concentration of the asterisk holding species we have divided its N-protonated counterpart by 223 000, which is the value for g1y~ine.I~The
(29)
Due to its symmetry, starting at any of the four group constants,
(19) Cohn, E. J.; Edsall, J. T. Proteins, Amino Acids ond Peptides; Reinhold: New York, 1943; Chapter 4.
6348
The Journal of Physical Chemistry, Vol. 90, No. 23, 1986
TABLE III: Abundance Probabilitv (Relative Concentration) of Some Microspecies at pH 7“
NWY
-4 -6 -8 -10 -12 -14 -16 -18
\\I
J. Phys. Chem. 1986.90:6345-6349. Downloaded from pubs.acs.org by UNIV OF NEW SOUTH WALES on 08/26/15. For personal use only.
Nosziil
o i 2 3 4 5 6 i 8 9~ ~ I ~ B K - P H Figure 1. Relative concentration of 16 glutathione microspecies as a function of pH, calculated by means of group constants. The identifying legends are as in Table 11. In this representation microconstant-based points fall on the group-constant-based lines. substitution of the glycine’s second methylene proton by the rest of the glutat hione molecule obviously influences the basicities of the N amino and U carboxylate groups but not their ratio. When the data in Table I1 are interpreted, the following observations are remarkable. (a) Concentrations of the fully protonated and nonprotonated species ( H H H H and 0 0 0 0)can be calculated from macroconstants only. Thus, their parallel values are necessarily equal. (b) Species H H 0 0 is overwhelmingly dominant among those, which contain two protons, so the striking agreement between its corresponding values up to the third digit (at any pH) is also a matter of course. (c) Standard deviation of microconstants involves greater uncertainty a t the most abundant three and one proton containing species H H H 0 and H 0 0 0 than the difference between their microconstant- and group-constant-based concentrations. (d) The inferior protonation isomers ( H H 0 H and 0 H 0 0) are much more sensitive of the same differences between the corresponding microconstants and group constants than their superior counterparts (H H H 0 and H 0 0 0). As it can be seen from Table I, these differences are 0.02 and 0.11 log units. These are essentially the sources of the concentration differences. When the significance of these deviant concentration values is judged, it must be kept in mind that the minor concentrations as well as the belonging minor microconstants contain much more ambiguity than the major ones, although it is rarely manifested in their standard deviations. For example, the same microconstants of glutathione, determined under practically identical circumstances published by the most recognized schools of the field,I49l2differ by up to 0.16 log unit, without the uncertainty represented by the standard deviation, which may additionally be up to fO.10 log unit. In other words, the effect of different types of calculations from the same source data is greater for the minor isomers than for the other microspecies, but it is still markedly less than the “effect” (ambiguity) of different (in their kind definitely the best) microconstant determinations. (e) The microconstant-type treatment cannot handle the least occurring nine species. Thus, the only way to estimate their concentration is based on the group constant, without comparison possibility. A more visual presentation of the microspecies distribution as a function of pH is in Figure 1. Analogous comparisons with similar results can be made for a few other di- and tripeptide-containing systems, where microconstants are a ~ a i l a b l e . ~ Theoretical considerations and the above comparative analysis make it possible to summarize the essential characteristics of the group-constant-based microspeciation: (1) At normal peptides, concentrations of every possible species can be evaluated, including the least occurring minor ones. (2) The H-bond-forming protonation can be perceived, and its effect can be taken into account. Microconstant-type protonation
state of group ABCDEFGHI JKLM OOOOOOHHHHHHH OOOOOOOHHHHHH OOOOOHHHHHHHH OOOOOHOHHHHHH
re1 concn 0.578 0 196 0.155 0 052
Remaining 6 137 Microspecies HHHHHOOOOOOOO 6.05 x HHHHHHHOOOOOO 4.81 x HHHHHHOOOOOOO 1.63 x
10-37 10-37 10-37
It can be seen that the most occurring species has the favorable state of protonation at every site of the molecule.
schemes normally do not consider the two-sites-aided proton bindings. (3) For group-constant-based microspeciation, it is enough to use one single experimental technique: pH-metry. (4)This method cannot be used with satisfactory results in the case of molecules, where the intervening atoms between the protonation sites are not able to diminish the effect of protonation at the neighboring group to a negligible level. (5) Agreement between the differently calculated microspecies concentrations is excellent or fairly good. (6) Last but not least, there is not any other way to evaluate microspecies concentrations of multidentate ligands. Since the main features of the method were studied, their use was possible for the multifunctional polypeptide, corticotropin. Concentrations of ACTH Microspecies. In our earlier works several aspects of corticotropin (ACTH) protonation and complexation equilibria were but this is the first paper devoted to its microspeciation. The 32 amino acid containing N-terminal fragment of corticotropin includes-among others-one aspartic acid, three glutamic acid, one histidine, four lysine, and two tyrosine constituents. Their free functional groups plus the N-terminal and C-terminal moieties are responsible for the molecule’s proton mobilizing processes. Some other polar sites (arginine guanidinc, alcoholic hydroxyl, tryptophane indole side chains, and the peptide bonds) do not play a role within the investigated pH range. As expected, when we start the alkalimetric titration from a substance, which is twice lypohilized perprotonated material, altogether 13 protons dissociate up to pH 12, in good agreement with the number of functional groups. Their values and interpretations as well as experimental parts have been discussed elsewhere.” Here are the pure log k values, according to magnitude: log k , = 3.54; log kB = 3.59!; log kc = 4.10; log kD = 4.25; log kE = 5.03!; log kF = 6.43; log kc = 7.47; log kH = 9.70!; log kl = 10.13; log kj = 10.45; log kK = 10.82; log kL = 10.94; log kM = 10.98. The first five group constants obviously belong to carboxylate, the sixth to imidazole, the seventh to terminal amino, and the remaining six to t-amino and phenolate groups. An entry with an exclamation mark means that the group to which that particular value belongs takes part in H-bond forming or another type of group coupling. Namely, groups with constants 3.59 and 5.03 are glutamic carboxylates, in strong interaction with each other.” The value 9.70 is unusually low for any lysine t-amino or tyrosine phenolate group. It is obviously also linked with another group, but the proton connecting these groups leaves the bond at extremely high OH- concentration, beyond our studied pH limit. These above statements show that in the ACTH,-,, molecule one must count with 13 groups. Two of them (designated B and E, glutamyl carboxylates at positions 28 and 30) are connected with one another. The third (H), which is also part of a coupled unit, can be handled as an individual one, since its counterpart is out of our reach. This means that ACTH,_32within the normal pH region may occur in 2n-2h3h= 2113 = 641 1 microscopic forms. In the knowledge of the group constants, when the principles introduced in the theoretical section are used, the concentration of any arbitrarily chosen microspecies can be estimated. Relative
-
J . Phys. Chem. 1986, 90, 6349-6353 concentrations of a few most and least abundant microspecies at pH 7 are listed in Table 111. Resume. Microspeciation can be carried out by the microconstant-based or group-constant-based procedure. Microconstants provide more detailed information, and their use is exclusive for small molecules, when the number of bonds between the protonation sites is also small. However, microconstant determination is not feasible for macromolecules. Fortunately, in several well-defined cases microconstants can be substituted by group constants, which are their simplified derivatives. The group constants provide a tool to calculate concentrations for every state of individual groups or
6349
group pairs, as well as to estimate any protonation stage of whole multifunctional molecules. In macromolecular interactions, when both parties are in rapid protonation interconversion, the knowledge of microscopic concentrations for these stages of protonation is particularly important. Presently these concentrations can be approached by no other means than group-constant-based microspeciation. Acknowledgment. I am indebted to Prof. G. Farsang for helpful discussions. Registry No. GSH, 70-18-8;
9002-60-2.
J. Phys. Chem. 1986.90:6345-6349. Downloaded from pubs.acs.org by UNIV OF NEW SOUTH WALES on 08/26/15. For personal use only.
Correlation of Chemical Structure to Photoconductivity: Octacyano- and Octamethoxy-Substituted Zinc Phthalocyanine H. Meier,* W. Albrecht, Staatliches Forschungsinstitut fur Geochemie, 8600 Bamberg, FRG
D. Wohrle, and A. Jahn Organische und Makromolekulare Chemie, Uniuersitat Bremen, 2800 Bremen 33, FRG (Received: March 18, 1986)
The results of photoelectric measurements in octacyano- and octamethoxy-substituted zinc phthalocyanine are described. Photoelectric measurements in surface-type photocells show that ZnPc(CN), is about 2 orders of magnitude more photosensitive than ZnPc(OCH,)*. On the basis of the Onsager mechanism of free carrier generation, a hypotheses about the influence of electron-withdrawing and electron-releasing substituents on the photoconductivity of p-type ZnPc is given.
Introduction Photoelectric properties of phthalocyanines have been intensively studied because of their potential use, e.g., in electrophotographic systems,'v2 diode laser printer^,^ photovoltaic cell^,'^ photoelectrochemical devices,lO-" and vidicon television pickup t u b e ~ . ' ~ J ~ These studies indicate that dark and photoconductivity of phthalocyanines may be affected by substitutions of the central metal atom.I4*l5 Moreover, photoconductivity has been reported to be influenced by the formation of bridged structuresI6 and polymer^.'^-'^ In addition, photoconduction in phthalocyanines (1) Weigl, J. W.; Mammino, J.; Whittacker, G. L.; Radler, R. W.; Byrne, J. F. Curr. Probl. Electrophotogr. Eur. Colloq., 3rd 1912, 287. (2) Grammatica, S.; Mort, J. Appl. Phys. Lett. 1981, 38(6), 445. (3) Kakuta, A.; Mori, Y.; Takano, S.; Sawada, M.; Shibuya, I. J. Imag. Technol. 1985, 11, 7. (4) Meier, H.; Albrecht, W. Ber. Bunsen-Ges. Phys. Chem. 1964,68,64. ( 5 ) Ghosh, A. K.; Morel, D. L.; Feng, T.; Shaw, R. F.; Rowe, C. A. Jr. J. Appl. Phys. 1974, 45, 230. (6) Hall, K. J.; Bonham, J. S.; Lyons, L. E. Aust. J. Chem. 19l8,31, 1661. (7) Dodelet, J. P.; Pommier, H. P.; Ringuet, M. J. Appl. Phys. 1982, 53, 4270. (8) Shimura, M.; Toyoda, A. Jpn. J. Appl. Phys. 1984, 23, 1462. (9) NeSphrek, S. Czech. J. Phys. 1984, 34, 222. (10) Meier, H.; Albecht, W.; Tschirwitz, U.; Geheeb, N.; Zimmerhackl, E.Ber. Bunsen-Ges. Phys. Chem. 1911,81, 592; Chem.-1ng.-Tech.1919, 51, 653. (11) Loutfy, R. 0.; McIntyre, L. F. Sol. Energy Marer. 1982, 6, 467. (12) Meier, H.; Albrecht, W. Eer. Bunsen-Ges. Phys. Chem. 1969, 73, 86. (13) Meier, H. Top. Curr. Chem. 1916, 61, 85. (14) Meier, H. Organic Semiconductor: Dark- and Photoconductivity of Organic Solids; Verlag Chemie: Weinheim, 1974; pp 149, 130, 319. (15) Loutfy, R. 0. J. Phys. Chem. 1982, 86, 3302. (16) Loutfy, R. 0.; Hsiao, C. K. Polym. Prepr. (Am. Chem. SOC.,Diu. Polym. Chem.) 1982, 23, 237; Chem. Abstr. 1984, 100, 171. (17) Meier, H.; Albrecht, W.; Zimmerhackl, E. Polym. Bull. (Berlin) 1985, 13, 43. (18) Meier, H.; Albrecht, W.; Zimmerhackl, E.; Hanack, M.; Fischer, K. J. Mol. Electronics 1985, 1 , 47.
depends also on the type of crystal m o d i f i ~ a t i o n and ' ~ ~ on ~ ~special ~ doping agent^.^'-^^ However, no experimental results known to us show an influence of substituents at the ligands on the dark and photoelectric properties of phthalocyanines. Information on the effect of substituents is restricted to triphenylmethane dyes,24p y r a ~ i n e s , ' ~ . ~ ~ indandiones,26n i t r o f l ~ o r e n e sand , ~ ~ other dyes not belonging to phthalocyanine Therefore, studies seem necessary to complete data on the effect of substituents in organic photoconductors by including phthalocyanines. These investigations may give an insight into influences of substituents on elementary processes of conductivity concerning carrier generation, recombination, or mobility. As a consequence it should be expected that by a clear understanding of the effect of substituents rules for optimizing phthalocyanine-based photoconductors by synthesis can be outlined. In this report the first results of studies on dark and photoconductivity of zinc 2,3,9,10,16,17,23,24-octacyanopkthalocyanine (la) and zinc 2,3,9,10,16,17,23,24-octamethoxyphthalocyanine (lb), abbreviated ZnPC(CN), and ZnPC(OCH&, are given.
Experimental Section ZnPc(CN)*. This compound was prepared as described.28 (19) Meier, H.; Albrecht, W.; Zimmerhackl, E.; Hanack, M.; Metz, J. Synth. Met. 1985, 1 1 , 333. (20)Twarowski, A. J . J . Chem. Phys. 1982, 77, 4698. (21) Kearns, D. R.; Tollin, G.; Calvin, M. J. Chem. Phys. 1960, 32, 1020. (22) Meier, H.; Albrecht, W. Z . Phys. Chem. (Frankfurt am Main) 1963, 39, 249. (23) Meier, H.; Albecht, W.; Tschirwitz, U. Ber. Bunsen-Ges. Phys. Chem. 1969, 73, 795. (24) Meier, H. Die Photochemie der organischen Farbstoffe; Springer Verlag: Berlin-GBttingen-Heidelberg, 1964; pp 164, 251, 173, 196. (25) Golubovic, A. J . Phys. Chem. 1969, 73, 1352. (26) Dimond, N. A.; Mukherjee, T. K.Discuss. Faraday SOC.1971, 51, 1. (27) Mukherjee, T. K. J . Phys. Chem. 1966, 70, 3848.
0022-3654/86/2090-6349$01.50/00 1986 American Chemical Society
