3814
R. F. PASTERNACK, E. GIBBS, AND J. C. CASSATT
SA
=
( d(rSA) 7 r (dSA ) F) +SA
(4)
where SAis the differential entropy of the adsorbed molecules. The differential entropy curves obtained by treating the integral entropy curves according to eq 4 are given in Figure 6. The differential entropy of water physisorbed on both samples increases steeply to the entropy value of liquid water, and after the coverage reaches the T,i value, the entropy remains almost constant in the vicinity of the entropy level of liquid water. Observing the curves of the differential entropy in more detail, we can find a very weak hump just after the
attainment of the Vm value. This may probably indicate that the sudden increase in the adsorption amount at a moderate relative pressure region is accompanied by a sudden increase in entropy. The possibility of entropy increase in this region will probably arise from the rearrangement of physisorbed water molecules or from the onset of the second layer adsorption, which gives rise to a sudden increase in freedom of the adsorbed molecules. The detailed consideration of the mechanism of this phenomenon is difficult now, but will be developed in a subsequent paper. (22) T. L. Hill, P. H. Emmett, and L. G. Joyner, J . Amer. Chem. SOC.,73,5102 (1951).
The Complexation Kinetics of Leucine with Nickel(II), Cobalt(II), and Copper(11) by R. F. Pasternack,’ E. Gibbs, and J. C. Cassatt2 Department of Chemistry, Ithoca College, Ithaca, New York 14860 (Receited April 1, 1969)
The kinetics of leucine complexation with nickel(II), cobalt(I1) and copper(I1) have been determined with the temperature-jump technique. The reactions are of the type
ML,-*
+L
k*
ML, k -n
where M = Ni2+, Co2+, or CUP+,L refers to the attacking form of the ligand which is the anion in this case, and TZ = 1 or 2. At an ionic strength of 0.1 M and 2 5 O , the rate constants are: Ni2f kl = 1.7 X lo4 J4-l sec-l, IC-, 7.1 X 10-2sec-1, k z = 4.1 x 104M-lsec-l, k-2 = 1.5 sec-1; Co2+k1 = 1.3 X lo6-If-1 sec-1, kPl = 67 sec-l, k z = 1.7 x 106M-1sec-1, Lz= 600 sec-1; Cu2+kl = 1.6 x 109Jf1.1-1 sec-l, k-1 = 12 sec-l, k z = 8 x los 1M-l sec-1, L2= 15 sec-1. The rate constants for the reactions of Co2+and Cu2+ with the zwitterion form of the ligand were found to be zero within experimental error. These results are consistent with the dissociative mechanism suggested by Eigen. The nonpolar side chain of leucine appears to play no significant role in determining either the forward or reverse rates. Once again, as for other ligands, k P < k l C ~unlike the situation found for other metal ions.
Investigations of the kinetics of complexation reactions of labile metal ions with amino acids have entered a new phase. As a result of earlier it has become apparent that the zwitterion is a remarkably unreactive form and that, as a result, the reactions of the metal ions with the anionic form of the ligand can be studied at pH’s significantly below the pK of the ligand. Hence, although the pK of sarcosine
is about ten, the reaction The Journal of Physical Chemistry
+ JrCo(H20)4L+ + 2HzO
CO(HZO)~~+L-
could be studied at a pH of six to seven.e Many advantages accrue from working at these relatively low pH’s: (i) large excesses of metal ion can be employed to yield more precise values of the rate constants for the (1) Author to whom inquiries should be addressed. (2) Department of Chemistry, State University of New York a t Buffalo, Buffalo, New York 14214. (3) e.g., K. Kustin, R. F. Pasternack, and E. M. Weinstock, J . Amer. Chem. SOC.,88,4610 (1966). (4) $. C. Cassatt and R. G. Wilkins, ibid., 90,6045 (1968). (5) A. F. Pearlmutter and J. Stuehr, ibid., 90, 858 (1968). (6) R. F. Pasternack, K. Kustin, L. A. Hughes, and E. Gibbs, ibid., i n press.
KINETICSOF LEUCINECOMPLEXATION WITH Ni(II), Co(II), formation of the monosubstituted complexes and (ii) equilibria between the fully aquated metal ion and metal-hydroxy species need not be considered. As a result there is closer agreement between the rate constant for cobalt (11) complexation6-8 reactions and the rate constant for water exchange of this metal ion as obtained by Swift and Connick9 and, secondly, the complexation :kinetics of copper(I1) ion can now be studied routinely.6*6 This paper describes the reactions of several labile metal ions, Ni(II), Co(I1) and Cu(II), with the a-amino acid, leucine. Leucine has a nonpolar side chain (cf., Figure l), and it is of interest to determine whether this nonpolar portion of the molecule affects the kinetics of these complexation reactions through either hydrophobic interactions or through a decrease in surface of reaction as was suggested in an earlier paper. lo p8
Experimental Section The leucine used in these studies was obtained from Nutritional Biochemicals Corp. Baker reagent grade nitrate salts of potassium, cobalt, nickel and copper were used. Stock solutions of the transition metal ions were prepared and the concentrations of these solutions were determined using standard analytic techniques. The indicators used in this study were Allied Chemical methyl orange, methyl red, chlorphenol red and phenol red and Eastman Organic bromothymol blue. The temperature-jump apparatus has been described elsewhere.6 Solutions were freshly prepared from solid leucine and stock solutions of KNOs and the appropriate metal ion and indicator. The solutions were degassed and the p H was adjusted by the dropwise addition of to 0.01 pH unit. For dilute NaOH and/or "03 many of the solutions, the equilibration of the p H was relatively slow taking several minutes in some cases. The p H of solutions frequently were monitored during the course of the temperature-jump experiments. Each relaxation time represents an average of a t least three photographic determinations. The relative error for these measurements is 5 10%. Test solutions of either metal ion or ligand in the absence of the other showed no discernible relaxation effects. These "blank" experiments were carried out at concentration levels of the free metal ion and ligand characteristic of solutions containing a mixture of the two. The rate
CH \ CH, CH, /
Figure 1. The leucine anion.
AND
3815
Cu(I1)
constants obtained in this study are being reported to f25%. Results The reactions studied were of the type k.
MLn-l
+ L =NILn
(1)
k -n
where n equals 1 or 2: The symbol "L" refers to the form of the ligand attacking the metal ion and, therefore, eq 1 could in principle represent a series of reactions differing in the degree of protonation of the ligand. The analysis of the temperature-jump data requires, for such complicated systems, a prior knowledge of the appropriate equilibrium constants. The equilibrium constants used for the analysis of these leucine systems are given in Table I. Table I: Equilibrium Constants a t 25"
(p =
0.1 M )
" S. P. Datta, R. Leberman, and B. R. Rabin, Trans. Faraday Soc., 55,1982 (1959). Equilibrium constants have been corrected to an ionic strength of 0.1 M using the equation suggested by
D. D. Perrin and V. S. Sharma, J. Chem. SOC.,724(1967). Bretton, J. Chim. Phys., 54, 827, 837 (1957).
* P.
Whereas only two stability constants have been reported for each of the metal-leucine systems, experimental conditions for the kinetic runs were such that this limited data sufficed. The range of pH and concentrations of leucine and nickel(I1) available to us was (7) G. Davies, K. Kustin, and R. F. Pasternack, Inorg. Chem., in press. (8) R. L. Karpel, K. Kustin, and R. F. Pasternack, Biochim. Biophys. Acta, 177,434 (1969). (9) T. J. Swift and R. E. Connick, J . Chem. Phys., 37, 307 (1962). (10) A. Kowalak, K. Kustin, R. F. Pasternack, and 8. Petrucci, J . Amer. Chem. Soc., 89,3126 (1967).
Volume 73,Number 11 November 1060
R. F.PASTERNACK, E. GIBBS,AND J. C. CABSATT
3816 severely limited because of precipitation of what was presumed to be Ni(CsH1202N)~a t higher pH's and the relatively long relaxation times in the lower pH region. As a result of this experimental limitation it did not prove possible to analyze the nickel data in the same way that the cobalt and copper data were analyzed to determine the rate constant for the attack of the zwitterionic form of leucine. However, a stopped-flow study of this system showed that, within experimental error, the rate constant for this reaction is zero.I1 For both cobalt(I1) and copper(II), it proved possible to obtain relaxation spectra for solutions which contained a large excess of the metal ion. For such solutions it can be assumed that the only complexation reactions of importance are
+
&!I2+ CHJP--I)+ ML+ i
+ iH+
(2)
Only solutions containing [Go2+] 1 lO[CoL+] or [Cu2+] 14[CuL+] were used in this analysis. That the condition is less stringent for the copper case is a reflection of the fact that, for glycine,6 sarcosinea and serines klCU> k2Cu, unlike other metal ions. I n the p H range investigated, two forms of leucine needed to be considered; the anion L- and the zwitterion HL. Then
.+
If
Figure 2. Plot of (TB)-~ us. AB-' for cobalt(I1)-leucine system. See text for definitions of A and B. The slop0 of this line is kl and the intercept is h', both in units of M-l sec-'. /
(3)
By applying standard techniques for deriving relaxation time expressions, it can be shown that
.',1
=
Akl
8.4
I
/i
+ Bki'
where
LY=
P =
KI,
yl
[E+]-
Kza
+ PL-I
KID 3. [H+l [H+l [In-]
+
+
Plots were made of (TB)-~ vs. AB-' and are shown in Figure 2 for cobalt(I1) and Figure 3 for copper(I1). The slope of the lines are the respective kl's and the intercepts k1'. From the plots we find that for cobalt kl = 1.3 X lo6&!-' see-', kl' Si 0; for copper kl = 1.6 X lO9M-1 sec-1, kl' Si 0. The zwitterion is thereby once again shown to be a remarkably unreactive species toward metal ions. The Journal of Physical Chemistry
n/a
I
IO'
us. AB-' for Figure 3. Plot of (TB)-~ copper(I1)-leucine system.
For these solutions, the only complexation reaction which need be considered is (11) J. C. Cassatt, Ph.D. Dissertation, State University of New York a t Buffalo, 1969.
3817
KINETICSOF LEUCINECOMPLEXATION WITH Ni(II), Co(II), AND Cu(I1)
M2+
+ L-
kl
AIL+
k-1
(4)
It can be readily shown that a plot of 1 / os. ~ [M2+]/ (1 a) [L-.] will result in a straight line the slope of which is kl and the intercept lc-1.12 Figure 4 shows
+ +
such a plot for the cobalt(I1)-leucine system while Figure 5 shows the plot for the copper(I1)-leucine system. From the plots, a value of K1 = k l / k - 1 can be determined for each metal. For cobalt(II), the value so obtained is 2.2 X lo4M-I as compared to the literature value of 1.95 X lo4 M-l; for copper(II), we obtain 1.0 X lo8 J4-l and the literature value is 1.29 X lo8M-' (cf. Table I). For the remaining cobalt(I1) and copper(I1) solutions and for all of the nickel(I1) solutions, two complexation reactions need be considered M2+
+ L-
ML+
+ L-
kr
MI,+
Table I1 : Relaxation Spectra of Nickel(I1)-Leucine [Nin+]o x 108
2.56 7.96 2.56 2.56 2.56 2.56 10.3 5.14 7.72 2.56 5.14
x
[Lido 103
PH
4.00 12.0 2.50 4.00 6.00 12.0 2.52 2.55 2.40 4.00 2.50
6.71 6.71 7.01 7.01 7.01 6.51 6.52 6.57 6.46 7.41 7.01
kl
= 1 . 7 X l o 4 M-lsec-l
k-1
=
7.1
x
lowssec-1
Solutionsa
Tabs, ,380
Taalo, 88C
0.13 0.020 0.15 0.080 0.055 0.042 0.11 0.20 0.13 0.45 0.18
0.11 0.026 0.13 0.085 0.063 0.056 0.10 0.14 0.12 0.54 0.11
k z = 4 . 1 X l o 4 M-l sec-1 k-2 = 1 . 5 sec-1
5 All concentrations are molar. The subscript zero refers to the total stoichiometric concentration. p = 0.1 M , temp = 25'.
(5)
k -1 kz
Table I11 : Relaxation Spectra of Cobalt(I1)-Leucine Solutionsa
ML2 k -2
The standard treatment developed by Hammes and Steinfeld13 was used to determine the rate constants which resulted in the best agreement between calculated and experimenial relaxation times. The experimental conditions, experimental and calculated relaxation times and the determined rate constants for leucine with nickel(II), cobalt(I1) and copper(I1) are given as Tables 11, 111, and IV, respectively. The kl value we obtained for nickel(I1) and leucine is 1.7 X lo4 M-'sec-' as compared to a value of 1.5 X lo4M-lsec-l obtained using a stopped-flow technique. l1
2.68 2.68 5.35 2.14 2.14 5.35 8.03 8.03 8.03 8.03 kl
k-1
4.35 2.16 6.48 2.25 5.59 7.68 9.92 52.3 69.2 85.7
7.15 7.16 6.96 7.13 7.07 6.61 6.38 6.09 6.02 6.11
= 1.3 X 106M-lsec-* = 67 sec-"
1.0 1.4 0.65 2.6 1.1 0.65 0.80 0.36 0.30 0.28
0.90 1.4 0.49 2.3 0.87 0.51 0.61 0.45 0.49 0.36
kz = 1 . 7 X 106M-lsec-1 k-z = 600 sec-1
a All concentrations are molar. The subscript zero refers to the total stoichiometric concentration. p = 0.1 MI temp = 25'.
Discussion
e'---.
.l
$4
..
The isobutyl side chain in the leucine molecule might be expected to influence the kinetics of complexation in a number of ways. As has been suggested, the nonpolar group might decrease the surface of reaction of the ligand and thereby decrease the forward rate constants.10 However, hydrophobic interactions might also be of importance and these might arise from two sources. The first is an interaction in the complex itself. If the side chain of one leucine ligand interacted with the side chain of a neighboring complexed leucine, the K2M would be unusually high. Kinetically, it might be expected that this would be manifested in an .a
( r c o ~ * l / ( l + 4 4 ICl)
,.a t
(.a
103
+ +
Figure 4. Plot of 1 / us. ~ [Coz+]/(l a) [L-1. The slope of the line is kl in units of M-l sec-l and the intercept is k- 1 in sec-l.
(12) M.Eigen and L. DeMaeyer, Techniques in Organic Chemistry, Vol. VIII, Part 11,Znd ed, S. L. Fries, E. 8.Lewis, and A. Weissberger, Ed., Intersoienoe Publishers, Inc., New York, N. Y., 1963. (13) G.G.Hammes and J. I. Steinfeld, J. Amer. Chem. Soe., 84,4639 (1962).
Volume 79,Number 11
November 1969
3818
R. F. PASTERNACK, E. GIBBS, AND J. C. CASSATT
Table IV : Relaxation Spectra of Copper(I1)-Leucine Solutions0 [Cun+lo x 108
[Liglo 10'
x
PH
robs, m8ec
4.92 9.87 4.92 2.46 4.92 9.87 2.46 4.92 9.87 4.92
8.00 3.99 7.4 5.00 4.00 5.6 20.0 3.15 65 2.50 4.02 16 5.00 4.50 1.7 5.00 4.60 1.6 5.00 4.51 3.2 5.00 3.52 29 5.00 3.55 24 10.0 3.50 28 kl = 1 . 6 X 109 M-1 sec-1 kz = 8 x 108 M-1 k-I = 12 sec-l k-2 = 15 sec-1
roalo,
\bo.
msec
8.2 5.4 60 18 2.1 0.89 3.2 33 20 30 sec-1
I+O
.
110.
loo.
c
'z
a All concentrations are molar. The subscript zero refers to the total stoichiometric concentration. fi = 0.1 M , temp = 25'.
unusually small On the other hand it is possible that the side chains would sterically interfere with one another. This would result in an unusually low K z M with an attending large value for IG-2. An examination of models of metal leucine systems shows that the side
0
D
l
4
Figure 5. Plot of I/.
VS.
8
6
(tcul+l/(r r a t
t
(1'1)
IO
II
10'
[ c u ~ + c ]+ / ( ~a) + [L-I.
Table V : Forward Rate Constants f6r Metal Complexation Reactions" Serines
Glyoineh7
MZ' Nickel(I1) Cobalt(I1) Copper (11)
4.1 x 104 1 . 5 X lo6 4 . 0 x 109
a
All rate constants have units of
5.6 X lo4 2 . 0 x 106 4 x 108 8ec-I.
2.9 x 104 2.0 x 10' 2 . 5 x 109
1 . 3 x 104 9.2 X lo6 2.8 x 109
1 . 7 x 104 1 . 3 X 10' 1 . 6 X lo9
1 . 2 x 104 1 . 5 X 108 1 x 108
4 . 1 X lo4 1 . 7 x 106 8 x 108
+ L- 2 MLz 3.4 2.0 5
x x x
104 106 108
The temperature is 25" and the ionic strength is 0.1 M.
chains most likely do not interact and this type of inner interaction is unlikely. However it has been shown that the stability constants, KzM,for some leucine dipeptides were anomalously high.I4 It was suggested at that time that side-chain (hydrophobic) interactions were responsible for this unusual stability. A second possible effect of the nonpolar side chain might be in the ion-pair formation constant between the ligand and mono-complex. It seems possible that due to hydrophobic interactions between the side chain of the leucine attached to the metal and that in the ion-pair, the ion-pair might be unusually stable. This would result in a larger than normal k2. However, when the ligand is a simple a-amino acid, this interaction would result in an ion-pair in which the a-amino The Journal of Physical Chemistry
Leucine's
+ L- 5 ML+
ML+ Nickel(I1) Cobalt(I1) Copper (11)
Sarcosine8
acid portion would be pointed away from the metal. This configuration would probably be unreactive here but might be reactive in the oligopeptide case. The forward rate constants obtained for the metalleucine systems are compared with those obtained for similar systems in Table V.15 The table includes only those results which have been obtained since the advent of the low pH technique. For each metal ion, the kl values are in good agreement, with the nickel values showing the greatest variation. Quite clearly the large nonpolar group of leucine does not influence the forward (14) S.I?. Datta, R. Leberman, and B. R. Rabin, Trans. Faraday SOC., 55, 1982 (1959). (15) This work,
KINETICSOF LEUCINECOMPLEXATION WITH Ni(II), Co(II), rate constant for complexation which, therefore, indicates that the smaller than normal rate constants obtained for nickel(I1) and cobalt(I1) with a-aminobutyric acidlo cannot be explained in terms of a decreased surface of reaction. The lc, values are also in good agreement for each metal ion except for nickel(I1)- Bnd copper(I1)-sarcosine systems. Apparently the N-methyl group of sarcosine affects the complexation kinetics considerably more than does the isobutyl group of leucine which is attached to a nonbonding atom. There is no indication of steric hindrance nor hydrophobic interactions in the forward rates obtained for leucine. There is considerably more variation in reverserate constants than forward-rate constants as is shown in Table VI. This is to be expected since these values are in practice (and especially the k, values) obtained directly from the equilibrium constants, IC-, = kn/K,. Since, as has been shown, the for-ard rate constants are relatively invariant for these ligands, any differences in stability constants will manifest themselves in the reverse-rate constants. A comparison of the reverse-rate constants shows that the leucine values are not extreme except for copper(I1). That the k-2 rate constant is small would be consistent with a hydrophobic interaction between the nonpolar side chains. However, the k-I rate constant is comparatively even smaller. Therefore, it may well be that the variations are due more to experimental error than in any chemical interaction. We
AND
Cu(I1)
3819 ~
~~
Table VI: Reverse Rate Constants for Metal Complexation Reactions" Glycines37
Serine*
Sarcosine6
Leuoinels
MI,+ ? M % + + LNickel (11) Cobalt (11) Copper(I1)
0.057 34 34
0.11 93 32
ML% ~ ML+ Nickel(I1) Cobalt(I1) Copper (11)
0.93 330 50
0.041 57 32
0.071 67 12
+ L-
1.6 930 150
a All rate constants have units of sec-1. 25' and the ionic strength is 0.1 M .
0.50 570 22
1.5 600 15
The temperature is
conclude that the complexation kinetics of leucine are entirely normal and, therefore, that the large, nonpolar portion of the molecule has no influence on rates. Having now demonstrated that the complexation kinetics involving leucine itself are quite normal, this research is being extended to studies on the complexation kinetics of leucine dipeptides,
Acknowledgments. The authors gratefully acknowledge partial support from the Petroleum Research Fund for Grant 2982B and to the National Science Foundation for Grant GP-8099.
Volume 73, Number 11 November 1988
