EQUILIBRIA OF THE BASIC AMINO ACIDS IN THE FORMOL TITRATION BY MILTON LEVY (From the Department of Chemistry, University and Bellevue Hospital Medical College, New York University, New York) (Received for publication, January 22, 1935) In a previous paper (5), the behavior of a number of amino acids in the form01 titration was systematized by the use of hypothetical equilibria and the appropriate constants. The present communi- cation deals with an extension of this system to the basic amino acids-arginine, lysine, and histidine. The titration curves of these amino acids may be treated as equivalent to the curves obtained by titrating mixtures of three monovalent acids* present in equivalent quantities and having the requisite titration constants (8, 9). Each of the single acids behaves in the mixture according to the Henderson-Hasselbalch equation and the amino acids can therefore be characterized by three constants, pK,, pK,, and pK3. Birch and Harris (1) have published titration curves, at one or two formaldehyde concentrations, for each of the basic amino acids. These curves are of the standard form predicted by the Henderson-Hasselbalch equation. This fact has been confirmed in the course of the present work. The problem of the behavior of the basic amino acids in the form01 titration is, therefore, to determine the laws governing the changesof the titration constants with formaldehyde concentration. The data of Birch and Harris are too few to give an adequate basis for the solution of the problem. EXPERIMENTAL The experiments were conducted at 30” in the manner previously described (5, 6), suitably modified to fit special peculiarities. To 1 The term acid is used in the Bronsted sense. 365 by guest on March 17, 2020 http://www.jbc.org/ Downloaded from
Transcript
EQUILIBRIA OF THE BASIC AMINO ACIDS IN THE FORMOL TITRATION
BY MILTON LEVY
(From the Department of Chemistry, University and Bellevue Hospital Medical College, New York University, New York)
(Received for publication, January 22, 1935)
In a previous paper (5), the behavior of a number of amino acids in the form01 titration was systematized by the use of hypothetical equilibria and the appropriate constants. The present communi- cation deals with an extension of this system to the basic amino acids-arginine, lysine, and histidine.
The titration curves of these amino acids may be treated as equivalent to the curves obtained by titrating mixtures of three monovalent acids* present in equivalent quantities and having the requisite titration constants (8, 9). Each of the single acids behaves in the mixture according to the Henderson-Hasselbalch equation and the amino acids can therefore be characterized by three constants, pK,, pK,, and pK3.
Birch and Harris (1) have published titration curves, at one or two formaldehyde concentrations, for each of the basic amino acids. These curves are of the standard form predicted by the Henderson-Hasselbalch equation. This fact has been confirmed in the course of the present work. The problem of the behavior of the basic amino acids in the form01 titration is, therefore, to determine the laws governing the changes of the titration constants with formaldehyde concentration. The data of Birch and Harris are too few to give an adequate basis for the solution of the problem.
EXPERIMENTAL
The experiments were conducted at 30” in the manner previously described (5, 6), suitably modified to fit special peculiarities. To
1 The term acid is used in the Bronsted sense. 365
investigate the variation of a particular group, a solution was prepared by dissolving the desired substance in water and adding the requisite quantity of NaOH or HCl to bring it to the mid-point (pH = pK) of the appropriate group. The resultant solution was diluted to volume and titrated with formaldehyde, the hydrogen electrode being used to measure the pH after each addition. The amino acid concentration was 0.01 M within 20 per cent, the varia- tion being due to dilution by the formaldehyde solution. Arginine did not come to equilibrium rapidly enough for this method. It was necessary to prepare a series of solutions at different formalde- hyde concentrations and to measure the pH after 24 hours. The pH did not then change in a further 48 hours. The course of the pH changes was investigated and found to follow smooth curves, which will be discussed in a later section.
The materials used and their amino acid contents, determined by the Van Slyke method, are given below.
In every case the titration values checked the expected within 1 or 2 per cent. No other data than the titration value are at hand on the imidazolelactic acid used because of the small quantity available.
Results
The experimental results are presented in the form of graphs showing the relations between the mid-point pH (pG,) values and the logarithms of the formaldehyde concentrations (see Figs. 1, 2, and 4). As records of the experimental results, the graphs seem self-explanatory. Detailed discussion will be found with the development of working equations and the calculations of con- stants for the postulated equilibria.
In order to unify the treatment a hypothetical amino acid con- forming to the type under discussion will be considered. At the isoelectric point it is symbolized by A*. Its behavior in an acid- base system, aside from the most acid group (which is not involved in the form01 titration) may be represented by Equations 1 and 2.
(1)
(2)
A-++ + A-f + H+ KS = bh/a a b h
A-+&A-+? b
Ka = ch/b c
The lower case letter below each symbol is used to represent both the concentration and the species in the equations and discussion. The K values are the hydrogen ion dissociation constants as deter- mined in water.
The entities represented by a, b, and c may be capable of react- ing with 1 or 2 molecules of formaldehyde as shown in the following reactions. The L values are the formaldehyde association con- stants and are identified by systematic subscripts.
(3)
(41
A-t++FeAF+ L11 = d/aF a d
$++F*AF-+ Liz = e/bF e
(5)
(6)
(7)
(8)
A-+ F&AR’-- & = g/cF c 0
A-‘+ + 2F FS AF,+ LI1 = i/aF* a i
$-+ + 2F F? A Fz-+ i
LB = j/bF’
A- + 2F F? AFz- Lza = m/cF2 # m
In the equations the lower case letters are used as before and the capital F represents the formaldehyde and its concentration.
The above equations were not, of course, written a priori, but are used to serve as a convenient framework to show the basic similarity of the varied experimental curves. Not all the equi- libria are necessary to describe the data in the specific case of each amino acid. Some of the particular reactions do not occur at all or they occur in inaccessible pH regions or formaldehyde concen-
trations. In Table I are listed the formulae of the compounds of arginine, lysine, and histidine corresponding to the generalized type of acid given above. A space without a formula indicates that for one of the reasons just stated equilibria involving the entity omitted need not be considered in deriving working equa- tions. Study of Table I will show that formaldehyde is considered as associating with the NH:, group only.
Besides the appropriate set of reactions among those above the analysis of the curves involves the principle that the sums of positive and negative ions shall be equal. In most cases the Hf and OH- ions do not contribute significantly to these sums, so that they are usually omitted from the equations for electroneu- trality.
The development of working equations and the determination of the constants are discussed separately for each amino acid.
The multiplicity of the constants might make it appear that they are of little practical value in the actual use of the form01 titration. For some of the constants this is certainly true. For practical purposes a single combination constant for each amino acid is more convenient. In the discussion of stoichiometry such constants are used. These with the other constants are collected in Table II under the heading pGF. This is defined as the appar- ent titration constant of the most alkaline group titrated in the form01 titration in 2.3 M formaldehyde. This is the constant which determines the end-point to be used with the conditions previously recommended (6).
Arginine
The region in which the acidity of the guanidine group of argi- nine becomes manifest is also a region in which the buffering power of formaldehyde is very great. The preponderance of the neces- sary corrections makes an accurate estimate of how much pK3 is affected by formaldehyde very uncertain from the data available.2
2 The implication in a recent note by Jukes and Branch (Jukes, T. H., and Branch, G. E. K., Science, 80, 228 (1935)) that the lack of a demon- strable titer for the guanidine group in the form01 titration proves that it does not react with formaldehyde is incorrect. The apparent strength of this group may have been increased by formol, without affecting the titer, to pH 9 if the constant of the group concerned still remained above 11.
When formaldehyde is added to arginine in a solution at pK, (pH 8.91), there is, as stated above, a change of pH with time. This change was investigated for a number of formaldehyde con- centrations and a family of curves obtained. The rate of the change increases with the concentration. It is probable that the electrode did not follow the changes as rapidly as they occurred. An attempt was made to derive kinetic equations which would fit, but the results were not consistent. It is enough to note that the major part of the change is soon over (5 minutes) even in M CHzO and that it is not dependent on the presence of the electrode or of Hz. The points in Fig. 1 were obtained after 24 hours stand- ing and did not change in a further 2 days. The lower formalde- hyde concentrations have been corrected for the amount required to react with arginine.
The points in Fig. 1 can be described in terms of Equations 1, 6, and 7 combined with the equation for electroneutrality a + i = b + j. The resultant equation is Equation 9.
(9) pG, = pKz - log (1 + L,, P) + log (1 + &I P)
When Lz2F2 is large compared to 1 and LZIP small
(10) pG, = pK& - 2 log F
Under these conditions the curve in Fig. 1 is a straight line with a slope of 2. The situation is realized over the lower part of the curve and the extrapolation of this part of the curve t.o log F = 0 as indicated by the dotted line gives pK2Lz2 = 3.50. pLz2 = -5.413 or Lz2 = 2.57 X 1Oj. This is a much larger association constant than any observed for the monoamino acids.
When F is large, Equation 9 reduces to a constant pG, = pK2L22IL21. The constant value which pGf approaches when F is large is 3.35. pLzl is therefore 0.15.
To show the agreement of the equations with the data the curve of Fig. 1 is plotted from the constants and the points are experi- mental.
It is interesting to note that pK2L2,/L21 is the hydrogen ion dissociation constant of the formaldehyde compound of arginine indicated by i in Table I. Its value, 3.35, is typical of a carboxyl
3 The symbol p is used as an operator with the usual significance as in pH, etc.
group in a compound carrying a distant positive charge and is therefore reasonable for this substance.
Stoichiometry-Serensen (10) found that arginine monohydro- chloride required 1 equivalent of alkali in the form01 titration and that this was practically independent of the formaldehyde concen- tration and the pH of the end-point. This conforms to the high value of L22. With the conditions recently recommended, arginine
FIQ. 1. The titration constants of arginine in formaldehyde. The ordinate represents pGf starting at pK2; abscissa, logarithms of the form- aldehyde concentrations in moles per liter.
should be titrated completely plus a small blank. If sufficient time is allowed to obtain equilibrium, quite low formaldehyde concentrations would allow complete titration to an end-point of 7 and with pure arginine these conditions would be much more favorable than the usual form01 titration. In mixtures, however, a titration of this kind would also include a considerable amount of histidine and a lesser amount of lysine. Monoamino acids would probably not interfere.
The titration constants associated with the two amino groups of lysine are changed by formaldehyde. Because these groups overlap somewhat, the stoichiometric mid pH values are not exactly equal to the pK values of the groups concerned. Formal- dehyde produces nearly parallel changes in the titration constants of the two groups and the discrepancy between mid pH and pK is
-2 -I log F 0 I
Fro. 2. The titration constants of lysine in formaldehyde. The co- ordinates are the same as in Fig. 1. The lower curve is started at pKa and the upper at pKn.
small in any case. The interpretation of the data is considerably simplified if the difference is ignored and the stoichiometric mid pH treated as equal to pK or pG, as the case may require.
In Fig. 2 pGf values are plotted against log F. The curves are of the same general form as those obtained with the monoamino acids (5). The same form of equation describes them.
When the curve starts with pK,, the equations are developed
from Equations 1, 4, and 7 and the statement of electroneutrality a = b + e + j. The manipulations described in (5) lead to
(11) Gf ( ) --1 f=dl+L+loOM K2
In Fig. 3 M is plotted against F. The slope of the straight line is 0.01 L,, and the intercept 0.01 LIZ. Another way of getting the constant L,, is to take advantage of the simplified equation
(12) pG, = pK&z - 2 log F
which indicates that the part of the upper curve in Fig. 2 having a slope of 2, when extrapolated to log F = 0, should have the intercept pK2Lzz. The values of the constants are given in Table II.
TABLE II
Constants Determining Behavior of Basic Amino Acids in Formaldehyde
In considering the variation of the second amino, pK,, group of lysine account must be taken of the reactions used in developing pG,,. Would the association of formaldehyde with b affect the tendency of the second amino group to dissociate hydrogen ion? The reaction of formaldehyde with‘b does not change the electrical charge produced by ionization. The charges seem to be the chief influences affecting the dissociation tendencies of other groups in the molecule. The answer to the question is therefore most probably negative. The test is to use the constant pKs deter- mined in the absence of formaldehyde in the equations developed. This was done and found to fit satisfactorily. This justifies the writing of 1 or 2 molecules of formaldehyde associated with the first amino group in c, g, and m as indicated by X in Table I. The hydrogen ion dissociation tendencies of b, e, and j are equal.
The equation for electroneutrality at the mid pH of the second amino group is b = c + g + m. Equations 13 and 14 are anal- ogous to Equations 11 and 12.
(13) Gl ( > --1 f=LIJ+L,,P= 1OOM KS
(14) pGn = pK& - 2 log F
The plot of Equation 13 on Fig. 3 gives a straight line with inter- cept L13 and slope Lz3. The extrapolation of the lower curve in
FIQ. 3. The formaldehyde association constants of lysine. The ordinate represents M of Equations 11 (lower curve) and 13 (upper curve); abscissa, F in moles per liter.
Fig. 2 in the proper manner gives pK3L23. The values of the constants may be found in Table II.
Stoichiometry-The amount of alkali used in the titration of lysine in the form01 titration depends on the initial pH as well as on the end-point (11). At the usual initial pH, 7, lysine is present as the monocation. Complete titration therefore requires that 2 equivalents of alkali be used. The pH of the end-point is deter-
mined by pGf8. With the conditions and equations developed in a previous paper (6) and pK,LZ3 = 8.1, the stoichiometric point is pHs/ = 9.7 + 0.5 log C where C is the lysine concentration at the end-volume. But in a mixture an end-point 0.1 unit more acid than this was recommended. By taking this into account the error in the titration of the last group would be 0.42/&. At 0.1 M lysine this would be 1.3 per cent. The other group would be completely titrated and the total error would be 0.65 per cent. In dealing with pure lysine the more favorable calculated end- point would give an error half as great.
S@-ensen investigated the stoichiometry of lysine and, with conditions under which other amino acids were nearly completely titrated, found that 92.5 per cent of the theoretical for one group was required. He started from the isoelectric point so that one group should have been titrated. Jodidi (3) explained this “abnor- mality” by assuming that the feeble basicity of one amino group affected adversely its ability to combine with formaldehyde. Actually the association constants of lysine are between 5 and 10 times as great as those for most monoamino acids. The greater these constants are the more favorable the acid is for accurate titration. Responsibility for the low values obtained would more justly be placed on the high value of pK3. S@rensen’s error is greater than that calculated above because he corrected for a blank which is not allowed for in the equations (6).
Hi&dine
Kossel and Edlbacher (4) adjusted an imidazole solution to pH 7, added formaldehyde, and titrated with alkali to a thymol- phthalein end-point. The quantity of alkali required was 84 to 85 per cent of the theory for one group. If the pK of imidazole is like that of its methyl derivative (7), this result would have been obtained whether the form01 had been added or not. The lack of a control without form01 led these workers to state that their experiment demonstrated a reaction between the imidazole and formaldehyde similar to that of amino groups. The experi- ment does not do this.
In order to determine whether or not the imidazole group reacts with formaldehyde, experiments were performed with 4-(or 5) methylimidazole and with imidazolelactic acid. The solutions
were adjusted to the mid-points of the imidazole groups by the addition of the requisite amount of acid or alkali and titrated with formaldehyde. The results are recorded in Curves I and II of Fig. 4. The effect is very much less than with an amino group. This may be taken to indicate that the imidazole group does not associate formaldehyde (the effect is then ascribed to solvent changes) or that both the acid and corresponding base react to only slightly different extents.
I
FIG. 4. The titration constants of imidazole derivatives in formalde- hyde. The coordinates are the same as in Fig. 1. Curve I, 4-(or 5-)methyl- imidazole; Curve II, imidazolelactic acid; Curve III, histamine starting from pK1; Curve IV, histidine, starting at pK2; Curve V, histamine starting at pK*; Curve VI, histidine starting at pK,.
In the case of histidine the major changes produced in pG by formaldehyde are interpretable on the basis that the imidazole group does not react and the formula? have been so written in Table I.
The data for Curve IV in Fig. 4 were obtained on the addition of formaldehyde to a histidine solution originally at pH 6, the mid- point of the imidazole group (7). The equilibria which must be
considered are represented in Equations 1, 3, and 4. The equa- tion for electroneutrality is a + d = b + e. At low formaldehyde concentrations d is small and may be neglected. Then Equation 15 may be derived.
(15) h = K2 + R*LzF
This equation is plotted in Fig. 5. From the slope of the line KzLl:, is 4.10 X low4 or pLIZ = -2.62.
I01 8
I
0; 0 .2 .4 F .6 .8
FIQ. 5. The formaldehyde association constants of histidine and his- tamine. Abscissa, Fin moles per liter; ordinates: upper curve, M of Equa- tion 16, lower curve, H X lo4 (Equation 15).
When F is large, pG, = pK2L12/L11. This is the constant value which Curve IV of Fig. 4 reaches and is equal to 3.15. pLll is therefore 0.23. The constant pK,LI,/LII is the pK of the acid d. Its value 3.15 is characteristic of a carboxyl group in the same molecule with an imidazole when the amino group is absent. This condition holds in imidazolelactic acid where pK1 is 2.99 (7).
The agreement is a qualitative confirmation of the absence of the charged amino group in d.
Curve VI of Fig. 4 was obtained on the addition of formaldehyde to a histidine solution originally at pH 9.17, the mid-point of the amino group. The equilibria that must be considered are Equa- tions 2, 4, and 5; and the equation for electroneutrality is b + e = c + g. The extensive changes occur at very low formaldehyde concentrations, in fact below stoichiometric quantity. This introduces a complication in that the total formaldehyde is far removed from the free formaldehyde. However, when F is large enough, it may be shown that pG/, = PK~L~~/L~~. This would indicate that Curve VI should reach a minimum constant value. Actually, it has a minimum followed by an increase. The mini- mum is at pG, = 7.3, which is consistent with the formulation of the acid e, when compared with imidazolelactic acid which it resembles. The increase of pG,, which follows, may be ascribed to solvent influences or to reaction of the imidazole group with form- aldehyde as mentioned above. If 7.3 is accepted as PK~L~JL~~ then pL13 = -4.5. Successive approximations applied to the low formaldehyde concentrations give pL13 = - 4.3 with considerable uncertainty. The agreement as to order of magnitude is sat- isfactory.
The behavior of histamine in formaldehyde was studied with the result that Curves III and V of Fig. 4 were obtained. If it is remembered that pK1 of histamine belongs to the imidazole group, and pK2 to the amino group (7), and that no carboxyl is present, it is easy to transfer the discussion of histidine to histamine. Here again the minimum pH reached by the amino group (7.2) in Curve V is characteristic of an imidazole uninfluenced by a charged amino group and is comparable to methylimidazole. In the study of Curve III it became evident that in addition to the reactions of Equations 1, 3, and 4, the association of histamine with 2 moles of formaldehyde corresponding to the reaction of Equation 6 had to be considered. The equation for electro- neutrality is a + h = b + d + e - h. When h is small Equation 16 holds.
This equation is plotted in Fig. 5. The intercept is L11 and the slope Lzl. pLll = -2.59 and pLzl = -2.86. When h becomes large, the flattening of the curve to an asymptote at pH 2.4 indi- cates that b and a are present in negligible amounts so that h = 0.5 (d + e). But d + e is the total histamine concentration in this case. The observed maximum h is 4 X lOA and that cal- culated is 4.17 X 10-3. The association constants for formalde- hyde are so great that it can displace H+ completely from histam- ine monohydrochloride.
Xtoichiometry-The stoichiometric behavior of histidine in the form01 titration was investigated by Sorensen (10) who found that theoretical values were approached as the end-point became more alkaline. The formaldehyde concentration at the end-point had very little effect. Even under the most favorable conditions histidine was not titrated completely. Henriques and Gjalbak (2) and Kossel and Edlbacher (4) also titrated histidine but obtained values greater than the theory for 1 equivalent. The difference between the results of these authors and those of Serensen is due to the difference in the initial pH. S@rensen started at the iso- electric point, whereas the others first adjusted the pH to 7. The recent paper of Van Slyke and Kirk (11) discusses the effect of the initial pH on the form01 titration. The conditions of the usual form01 titration cannot give the desired stoichiometric relation for histidine except through a balancing of errors.
The end-point conditions for histidine are quite different from those of the monoamino acids. The constant of the group that determines the end-point does not change greatly with the formal- dehyde concentration when it is large. The most favorable formaldehyde concentration for histidine is at about 0.1 M instead of 2.3 M as advocated for mixtures (6). Calculations based on the principles previously used show the stoichiometric end-point in 2.3 M formaldehyde to be 10 - 0.5 log C, where C is the histidine concentration. The error of the titration, when carried to the end-point for mixtures (9.6 - 0.5 log C), is 2.34/d/c or in a 0.1 M
histidine solution, 7.4 per cent. An error of greater magnitude is involved in the original adjustment to pH 7. Only by an acai- dental balancing of the errors would the theoretical 1 equivalent be found. If the titration concerns histidine in the absence of
other amino acids, much better results are theoretically possible by adjusting the initial pH to 4, where histidine is all in the form of the monocation, and using only small amounts of formaldehyde, less than 0.5 M. The end-point would then be at 9.5. The advantage in the initial step is that the pH suggested is in the most poorly buffered region of the histidine curve. The amino and imidazole groups are too close together to permit as accurate an adjustment. The use of dilute formaldehyde is to take ad- vantage of the minimum pG, shown. It has the further advan- tage that the amount of alkali used by formaldehyde is diminished so that the end-point is sharper.
DISCUSSION
It is a common conception that the titration constants shifted by formaldehyde are to be assigned to amino groups. Aside from the fact that imino groups as in proline also react, the interpreta- tion of formaldehyde shifts must be considered carefully on the basis of such possibilities as are illustrated by histidine and hista- mine. When a molecule contains other acidic and basic groups not far removed in strength from the amino group, the possibility of internal H ion shifts exists. Only a close study of such cases is likely to lead to correct conclusions about the assignment of constants. Conversely, in the presence of an amino group, a shift in a constant ordinarily assigned to another group does not necessarily imply that the second group reacts with formaldehyde. Internal shifts of H ions may occur and thereby produce changes in the apparent constants.
The constants determining the behaviors of the basic amino acids in the form01 titration, according to the unified system presented in the theoretical part, are collected in Table II. Per- haps the most striking thing about these constants is that they are usually much larger than the corresponding constants for the monoamino acids (5).
SUMMARY
A unified systematic treatment of the equilibria between dibasic amino acids and formaldehyde has been developed and applied to arginine, histidine, and lysine. Only the amino groups are
considered as reacting with formaldehyde, each of them reacting with 1 or 2 molecules of formaldehyde successively. The stoichi- ometry of each amino acid in the form01 titration is discussed with reference to its equilibria.
BIBLIOGRAPHY
1. Birch, T. W., and Harris, L. J., Biochem. J., 24, 1080 (1930). 2. Henriques, V., and Gjalbak, J. K., 2. physiol. Chem., 76, 379 (1911). 3. Jodidi, S. L., J. Am. Chem. Sot., 40, 1031 (1918). 4. Kossel, A., and Edlbacher, S., Z. physiol. Chem., 93, 396 (1915). 5. Levy, M., J. Biol. Chem., 99, 767 (1932-33). 6. Levy, M., J. Biol. Chem., 106, 157 (1934). 7. Levy, M., J. Biol. Chem., 109, 361 (1935). 8. von Muralt, A. L., J. Am. Chem. Sot., 62, 3518 (1930). 9. Simms, H. S., J. Am. Chem. Sot., 48, 1239 (1926).
10. Sorensen, S. P. L., Biochem. Z., 7, 45 (1907); Compt.-rend. trav. Lab. Curlsberg, 7, 1 (1907).
11. Van Slyke, D. D., and Kirk, E., J. Biol. Chem., 102, 651 (1933).
