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States of amino acid residues in proteins
ARCHIVES
OF
BIOCHEMISTRY
States IV. Bound
Tyrosine
AND
of Amino and by
YUJI
INADA,
Institute
(1964)
Acid
Residues
Tryptophan
Residues
Difference
AYAKO AND
From the Tokugawa
106, 326-332
BIOPHYSICS
for Biological
in Proteins in Pepsin
Spectrophotometry
MATSUSHIMA, MASAKO KAZUO SHIBATA Research,
Received
as Observed
Tokyo,’
October
KAMATA
and Tokyo Institute
of Technology,
Tokyo
17, 1963
The alkali denaturation of pepsin was studied between pH 1 and 13 by difference spectroscopy and by activity measurements at pH 1.8, and the tryptophan and tyrosine residues were differentiated into two and three groups, respectively that differed in state. As the pH value of a pepsin solution is raised progressively from 1.8, the denaturation proceeds in several steps. The proteolytic activity drops first with pK = 3.1, and the band shift of two presumed tryptophan residues follows the inactivation (pK = 4.0). The band shift of the remaining four tryptophan residues proceeds between pH 7 and 8 and in parallel with an irreversible inactivation. At higher pH values, the ionization of tyrosine residues in various states occurs successively; 11 rapidly ionizing residues with pK = 10.0, 5 rapidly ionizing residues with pK = 11.5, and 1 slowly ionizing residue with a higher pK value. These results are discussed with reference to data reported previously.
The spectral changes observable on acidifying protein solutions are smaller and of different origin. All the tyrosine, tryptophan, and phenylalanine bands in the 250-300 mu region shift toward shorter wavelengths. The difference spectra of neutral protein solutions referred to acidic solutions generally show three distinct positive peaks around 279, 286, and 295 rnp and several minor peaks at shorter wavelengths (3, 4), in which the peak at 295 rnp arises from the shift of the tryptophan band. Recent observations of this phenomenon with model derivatives of amino acids (4, 5) as well as with various proteins have shown that the shifts are caused by changes in the environment of the chromophores of these amino acid residues in proteins. As reviewed by Scheraga (6), the changes may be due to cleavage of hydrogen bonds or ionization of a residue in the close vicinity of the chromophores, or exposure of a residue in a hydrophobic portion of the protein to the hydrophilic medium. Thus, any conformation
The ultraviolet absorption bands of proteins change upon making their solutions acid or alkaline. The change observable with alkali is due to the ionization of the phenol group of tyrosine residues, and the difference spectrum of ionization shows a maximum at 295 rnp and a minimum around 275 mp. Spectrophotometric titrations based on this phenomenon were applied in previous studies to insulin, lysozyme, and catalase (1) and to chymotrypsinogen, chymotrypsin, and trypsin (2). The tyrosine residues in these proteins were differentiated into three types : an instantaneously ionizing residue with a pK value similar to 10.0-10.1 for free tyrosine; a slowly ionizing residue with a higher pK value; and a nonionizable residue, in which the latter two types of residues may be regarded as bound residues hydrogenbonded or embedded in the interior of the molecule. Earlier literature on other proteins is reviewed with these results in the previous papers (1, 2). 1 Mailing
address. 326
TYROSINE
AND TRYPTOPHAN
change or denaturation leading to these changes in state of tryptophan residues is reflected by the band shifts. In the present paper the states of tyrosine and tryptophan residues in pepsin as determined from these two types of spectra1 changes are described. The spectrophotometric titrations were carried out over the pH range between 1 and 13, and the titration curves obtained were compared with the pH dependence of the proteolytic activity, and with the reversibility of alkali denaturation as estimated in terms of activity. The spectral change of the enzyme on the acidic side of pH have already been studied by Blumenfeld and Perlmann (7) and by Edelhoch (8), but the data reported by these authors differ from each other. The results obtained in the present study are discussed with reference to these earlier investigations. Throughout this paper, AE represents the difference in absorbance (E) at a difference maximum, n the moles of free and/or bound residues of tryptophan or tyrosine per mole of protein, and m the order of a sigmoid titration curve, which is equal to the number of hydrogen ions involved in the unmasking or ionization of the residue titrated.
RESIDUES
327
IN PEPSIN
dues per mole of protein) with the As value, 2305, estimated previously (1). All of the spectroscopic measurements were made with a Cary recording spectrophotometer model 14M and 1.0.cm cells. The proteolytic activity of pepsin was measured as a function of pH by the hemoglobin method (10). Digestion took place at 30°C for 10 minutes. The extent of digestion was estimated from the absorbance value at 280 rnp of a trichloroacetic acid soluble fraction of the digest, and was taken as the relative measure of activity.
RESULTS
AND DISCUSSION
TRYPTOPHAN
RESIDUES
As a preliminary study the experiment reported by Donovan et al. (4) for free tryptophan was repeated. The difference spectrum obtained for a 6.66 X 1O-4 I!4 solution at pH 7.8 against the same eoncentration of tryptophan at pH 1.5 is shown by curve A in Fig. 1, and indicates a maximum at 293 ml.r. The pH dependence of the AE value at the maximum is shown by curve A in Fig. 2. The values of pK and m for the ehange on the acidic side were estimated to be 2.3 and 1.0, respectively; as concluded by Donovan et al. the change
to
EXPERIMENTAL A crude preparation of swine pepsin (Difco Laboratories Inc.) was recrystallized three times by adjustment of the pH of the solution (9); the crystals were used for the various measurements. The concentration of pepsin was determined photometrically, assuming its molar extinction coefficient (E) at 278 rnp to be 52.47 X 103 (7). Pepsin is stable and undergoes no denaturation or autolysis around pH 4. The titration curve on the acidic side was measured at 298 rnp for a 1.11 X 1OW M pepsin solution at a desired pH referred to the same concentration of the solution at pH 4.0. The sample solution was prepared by adding 1 ml of a mixture of 1.0 M HCl and 1.0 M KC1 solutions to 9 ml of a pepsin solution at pH 4.0. The ionic strength of sample solutions at various pH values was thus kept constant at 0.10. Similar measurements were made for free tryptophan. The titration curve on the alkaline side was measured at 295 rnp, with a pepsin solution at pH 8.5 as the reference. The concentration of pepsin was 2.41 X 1O-5 M, and the ionic strength was kept at 0.125 with mixtures of KOH and KCl. AE values were reduced to 12 (moles of ionized t,vrosine resi_I
to .6 to 2 +o / O,
-0. 2-
-A-c
26C1
0’ I
I
280
300
Wavelength
in
320 mp
: ,O
FIG. 1. Difference spectra for tryptophan and pepsin solutions. Curve A, 6.66 X IO-* M tryptophan between pH 1.5 and 7.8; curve B, 1.00 X low4 M pepsin between pH 1.8 and 8.1; curve C, 1.8 X 1F M pepsin between pH 8.5 and 13.2. The solutions at the higher pH values were measured against those at lower pH values as the reference.
328
INADA,
MATSUSHIMA,
KAMATA,
PH FIG. 2. Changes of spectrum
or activity
with
pH observed for tryptophan and pepsin. Curve A, tryptophan, AE at 293 rnp; curve B, 1.11 X 10-’ M pepsin, AE at 298 rnp; curve C, proteolytic activi-
ties of pepsin measured at various pH values; curve D, the degree of reversibility of alkali denaturation as measured in terms of activity.
is caused by the inductive effect of ionization of the carboxyl group. From the absorbance change between pH 0.8 and 7.7, the Ae value was estimated to be 365, which is greater than the value, 240, obtained by these authors. Above pH 7.7, the curve rises again and reflects the effect of proton dissociation from the amino group. The difference spectrum of 1.00 X 1O-4 M pepsin solution at pH 1.8 and 8.1 is shown by curve B in Fig. 1. The shape of the major positive peak is similar to that obtained for tryptophan but is located at the longer wavelength of 298 rnp. In addition to this peak, the spectrum shows a round maximum at 275 rnp, a small dip, and a hump at 287 and 289 rnp, respectively, and a sharp minimum at 292 rnp. In measurements of difference spectra of solutions of high absorbancy, maxima or minima are often
flattened
and deformed
when the slit
AND SHIBATA
then flattened the sharp minimum at 292 rnp. An increase of the voltage, however, showed no effect on the whole spectrum of curve B, and Beer’s law was obeyed at 298 rnp with a slit width of 0.2 mm, so that measurements of AE at 298 rnB in further experiments were conducted with this slit width. The difference spectrum of pepsin on the acidic side of pH has been observed by two different groups. Edelhoch (8) observed the difference spectrum of a pepsin solution of pH 7.4 referred to the solution at pH 5.8, and obtained maxima around 282, 290, and 300 rnp and minima at 279, 286, and 292 mF. He interpreted the 279 and 286 rnp minima to be due to the shift of the tyrosine band, and the 292 rnp minimum to the shift of the tryptophan band. On the other hand, the difference spectrum obtained by Blumenfeld and Perlmann (7) for a pepsin solution at pH 4.6 referred to the solution at pH 2.0 showed no negative reading of AE in the same spectral region, and three maxima appeared at 278, 285, and 294 mp. The result obtained by Edelhoch resembles ours at longer wavelengths; both results show a sharp minimum at 292 mp and a rather high maximum around 298 my. At shorter wavelengths, however, the sharp and deep minimum is much
at 286 rnp observed
by Edelhoch
less marked in our spectrum, and appears on the positive side of the spectrum. The spectrum of Blumenfeld and Perlmann is considerably different from either of these spectra, particularly in the positive readings of AE in the intermediate wavelength range near 290 mp. As illustrated below, these differences are due to the pH values selected for the sample and reference solutions. (i) The difference spectrum between pH 1.8 and 6.1 was similar to that obtained by Blumenfeld
and
Perlmann,
although
the
width is not narrow enough. The present 285 and 294 rnp maxima observed by them measurements were repeated for the same appeared at 287 and 298 rnp in our spectrum, sample and reference solutions under a respectively, and the 287 rnp maximum was variety of conditions during which the slit lower than the 298 rnp maximum. (ii) The width was varied arbitrarily by variation of difference spectrum obtained between pH the voltage applied to the photomultiplier. A progressive decrease of the voltage (an 6.1 and 8.1 was similar to that obtained by Edelhoch; the addition of these difference increase of the slit width) to less than that spectra gave the spectrum of curve B obused for the measurement of curve B first lowered the round maximum at 275 rnE.c and served between pH 1.8 and 8.1. (iii) AS
TYROSINE AND TRYPTOPHAN RESIDUES IN PEPSIN
demonstrated below, the spectrum changes in two steps on the acidic side of pH, and the change observed by Blumenfeld and Perlmann is in one of these two steps while
residue responsible for the first step and 375 for the residue in the second step. The value obtained for the residues in pepsin agrees approximately with these values for the
tryptophan residues in lysozyme. The calculations support the view that the total absorbance change in pepsin results from the band shift of the six tryptophan residues. Another noteworthy fact is that the absorbance change in the first step is very cIose to one third of the total change. This implies that two of the residues undergo the spectral change in the first step and the remaining four in the second step. The value of m for the first step is approximately 1.0. This indicates that the change is due to the ionization of a group in close vicinity to the tryptophan residues and, from the pK value of 4.0, the group is probably a carboxyl group. By contrast, the value of m for the second step is much greater and is discussed later with the activity data. Curve C in Fig. 2 shows the variation of dues starts above pH 8.6, and is illustrated the proteolytic activity with pH. The optilater. The pK values for the first and the mum pH was estimated from the curve to be second steps were estimated from the curve 1.8 & 0.2, and the pK value for the inactivation on the alkaline side of the curve was to be 4.0 and 7.2, respectively. The total absorbance change below pH 3.1. This value of pK is lower than the 8.2 in the first and the second steps is 0.250. value for the first step of the spectral change; When this value of AE is divided by the there seems to be no correlation between the product of pepsin concentration (1.11 X inactivation and the change in state of the 1O-4 n/r) and AC (365) determined for free tryptophan residues. The reversibility of the inactivation with tryptophan, we obtain n = 6.2 in agreement with the molar tryptophan content, 6, de- alkali was observed in the following manner. termined by Blumenfeld and Perlmann (12). Pepsin was first dissolved at pH 4.0, and If the calculation is reversed on the assump- the pH of this solution was changed to a tion that the total change is caused by the value between 1.8 and 9.4. The solution, band shift of six tryptophan residues, we after standing at 7°C for 2 minutes, was obtain Ae = 375 for each residue, a value readjusted to the optimum pH of 1.8 and the activity was measured. The activity close to that for free tryptophan. This agreement may be fortuitous, since the AC thus measured (curve I) in Fig. 2) is constant below 6.3 and drops at higher pH value for the band shift of free tryptophan due to the inductive effect of ionization of values. This implies that the inactivation below pH 6.3 is completely reversible while its carboxyl group should generally be different from the values for tryptophan residues that at higher pH values is irreversible. of different types in proteins. A noteworthy The pK value for the irreversible deactivafact concerned with this problem is the tion is 7.5, which is in approximate agreeresult obtained by Donovan et al. (4) for ment with the value of 7.2 for the second tryptophan residues in lysozyme. They ob- step of the spectral change. In addition, these tained two steps of absorbance change with activity and spectral changes occur in a pK values of 3.15 and 6.20, and the Ae value narrow pH range with an m value much calculated from their data is 438 for the greater than 1.0. It is inferred from these the change observed by Edelhoch is in the other. The spectra observed in the two pH ranges differ considerably below 290 rnp, and, as reviewed and discussed by Bigelow and Sonenberg (11) , these changes at shorter wavelengths are regarded to be due to the shifts of superposed tyrosine and tryptophan bands. At longer wavelengths, however, both spectra are identical in that they have a distinct maximum at 298 rnp, which is due to the shift of the tryptophan band alone. The AE value at 298 rn/* was measured as a function of pH, and the result is shown with reference to extremely acidic pH by curve B in Fig. 2. Two steps of absorbance change occur below pH 8.2, and the curve reaches a plateau between pH 8.2 and 8.6. The third step due to the ionization of tyrosine resi-
330
INADA,
MATSUSHIMA,
KAMATA,
results that a large conformation change or denaturation occurs between pH 7 and 8, and is reflected by the irreversible inactivation and the abrupt change of spectrum. Edelhoch (8) had demonstrated a close correlation in the same pH range between the inactivation and the liberation of protons with alkali. His potentiometric titrations also revealed the time-dependent nature of the liberation of protons and a discrepancy between the forward and the backward titration curves in this pH range. These results support the view of a large conformation change. TYROSINE RESIDUES Curve C in Fig. 1 shows the difference spectrum observed for 1.8 X 1O-5 M pepsin solution at pH 13.2 with reference to the same solution at pH 8.5. The spectral change due to the ionization of tyrosine residues is much greater than those obtained at acidic pH. The AE value at the maximum of 295 rnp was measured as a function of pH for pepsin solutions in 4.0 M guanidine, and the result is shown in curve A of Fig. 3. As the pH increases, AE increases with pK = 10.0 and m = 1.0, and reaches a level of
n -8
PH
FIG. 3. Ionization curves for the tyrosine residues in 2.41 X 10d6 M pepsin. Curve A, with 4 M guanidine; curve B, without guanidine, observed immediately after the preparation of the alkaline solutions; curve C, without guanidine, observed 3 hours after mixing; curve D, the theoretical curve for 11 tyrosine residues with pK = 10.0; curve F, the theoretical curve for 5 tyrosine residues with pK = 11.5. The AE values shown by open triangles on curve F were obtained by subtracting the values calculated for the 11 residues from the observed values on curve B.
AND SHIBATA
n = 17. This value of pK agrees with that for free tyrosine, and the value of n coincides with the molar tyrosine content determined by Li (13). It is therefore evident that all of the tyrosine residues are free to ionize in 4.0 M guanidine. The AE values for pepsin in the absence of denaturant measured immediately after making the solution alkaline are shown by solid circles on curve B in Fig. 3. The curve is flatter and is shifted toward a higher pH as compared with curve A. Moreover, the AE value at extremely alkaline pH is lower than the value on curve A, and corresponds approximately to n = 16. When strongly alkaline solutions of pepsin were left standing at room temperature, the absorbance values at 295 mp increased gradually. A constant reading of absorbance was obtained 1 to 3 hours after mixing, and the time was dependent on the pH value. Curve C in Fig. 3 shows the result obtained after 3 hours in alkali and deviates from curve B above pH 11.2. The value of AE at extremely alkaline pH agrees with that of curve A for guanidinedenatured pepsin. The difference in AE between curves A and B was calculated to correspond to An = 1.3. However, this value of An may include considerable errors because of the small difference between large readings of AE. The difference was directly measured with more concentrated pepsin solutions to estimate An more exactly, and the result was An = 1.0 f 0.1. It is evident that 16 of the 17 residues ionize instantaneously with alkali and the remaining residue ionizes slowly. The apparent pK value estimated from curve B is approximately 10.4, which is slightly higher than the value for free tyrosine, and the value of m is appreciably smaller than 1.0. Two different interpretations may be given for this deviation of m. (i) As reviewed comprehensively by Tanford (14), the titration curves of proteins are flattened by the electrostatic effect, so that we obtain titration curves of different values of m at different ionic strengths. (ii) Curve B may be a composite of ionization curves with m = 1.0 and with different pK values. An analysis based on this interpretation was successful when it was assumed that the 16
331
TYROSINE AND TRYPTOPHAN RESIDUES IN PEPSIN
TABLE I residues represent 11 residues with pK = VARIOUS TYPES OF TRYPTOPHAN AND TYROSINE 10.0 and 5 residues with pK = 11.5. Curve RESIDUES IN PEPSIN D in Fig. 3 is the ionization curveobtained
by calculationfor the 11freeresidues.Open triangles on curve F indicate the values obtained by subtracting the values for the 11 free residues from the observed values on curve B, and these differences agree with the AE values calculated for 5 residues with pK = 11.5 (curve F) . An interesting observation was made by Li (13)) who observed the reaction at pH 5.7 of iodine with native and urea-denatured pepsin. According to his kinetic analysis, 12 residues are free to be iodinated, and 3 of the remaining 5 residues are liberated with 7.1 M urea. This result offers support for our second interpretation, since it differs by only one residue from our estimate of 11 free and 6 bound residues. The effect of ionic strength on the ionization curve will clarify which interpretation is correct and awaits further experiment. On the other hand the difference in the number of strongly bound residues, 2 in the result of Li and 1 in ours, may be significant. It indicates that one of the two residues is of the rapidly ionizing type, but not reactive with iodine, and that one of these residues is transformed by the denaturation between pH 7 and 8 into the rapidly ionizing type. A similar result was obtained previously for tyrosine residues in insulin (1, 15). Three of the 4 residues ionized rapidly and the remaining residue slowly. However, only two tyrosine residues were reactive with cyanuric fluoride near neutral pH, which indicated that one of two nonreactive residues was transformed with alkali into the reactive type. The alkali denaturation of pepsin may be described as follows in terms of spectral and activity changes. When alkali is added to a pepsin solution at pH 1.8, inactivation is associated with a pK of 2.3, and the band shift of two tryptophan residues follows the inactivation. These activity and spectral chauges are completely reversible; the conformation change of the molecule may be limited. The band shift of the remaining 4 tryptophan residues proceeds between pH 7 and 8 in parallel with an irreversible in-
Residuetype’” Tryptophan (nt = G) Tyrosine (nt = 17)
n
PK
2 4 11 5 1
4.0 7.2 10.0 11.5 >11.7
Ionization rapid rapid slow
ant = total molar content of tryptophan tyrosine.
or
activation. These changes are considered to be due to a large conformation change. At higher pH values, the ionization of various types of tyrosine residues occurs successively; 11 free residues with pK = 10.0, 5 weakly bound residues with pK = 11.5, and 1 strongly bound residue with a higher pK value. It requires about 3 hours in strong alkali to unmask the most strongly bound residue. Different types of tryptophan and tyrosine residues distinguished in the present study are summarized in Table I with their pK values. REFERENCES 1. INADA, Y., J. Biochem. (Tokyo) 49, 218 (1961). Y., KAMATA, M., MATSLJSHIMA, A., AND SHIBATA, K., Biochim. Biophys. Acta,
2. INADA,
81, 323 (1964). 3. LASKOWSKI, M., JR., LEACH, S. J., AND OCHERAGA, H. A., J. Am. Chem. Sot. 82, 571 (1960). 4. DONOVAN, J. W., LASKOWSKI, M., JR., AND SCHERAGA, H. A., J. Am. Chem. Sot. 83, 2686 (1961). 5. WETLAUFER, D. B., EDS~LL, J. T., AND HOLLINGWORTH, B. R., J. Biol. Chem. 233, 1421 (1958). 6. SCHERAGA, H. A., “Molecular Biology” (N.O. Kaplan and H. A. Scheraga, eds.), Vol. 1 (“Protein Structure”), p. 234. Academic Press, New York, 1961. 7. BLUMENFELD, 0. O., ASD PERLMANN, G. E., J. Gen. Physiol. 42, 563 (1959). 8. EDELHOCH, H., J. Am. Chem. Sot. 80, 6640 (1958). 9. NORTHROP, J. H., J. Gen. Physiol. 30, 177 (1946). 10. ANSON, M. L., in “Crystalline Enzymes”
332
INADA,MATSUSHIMA,KAMATA, ANDSHIBATA
(J. H. Northrop, ed.), Columbia Biological Series, No. 12, p. 305. Columbia Univ. Press, New York, 1948. 11. BIGELOW, C. C., AND SONENBERG, M., Biochemistry 1, 197 (1962). 12. BLUMENFELD, 0. O., AND PERLMANN, G. E., J. Gen. Physiol. 42, 553 (1959).
13. LI, C. H., J. Am. Chem. Sot. 67, 1065 (1945). 14. TANFORD, C., in “Advances in Protein Chemistry” (M. L. Anson and J. T. Edsall, eds.), Vol. 17, p. 95. Academic Press, New York, 1962. 15. KURIHARA, K., HORINISHI, H., AND SHIBATA, K., Biochim. Biophys. Acta 74, 678 (1963).
OF
BIOCHEMISTRY
States IV. Bound
Tyrosine
AND
of Amino and by
YUJI
INADA,
Institute
(1964)
Acid
Residues
Tryptophan
Residues
Difference
AYAKO AND
From the Tokugawa
106, 326-332
BIOPHYSICS
for Biological
in Proteins in Pepsin
Spectrophotometry
MATSUSHIMA, MASAKO KAZUO SHIBATA Research,
Received
as Observed
Tokyo,’
October
KAMATA
and Tokyo Institute
of Technology,
Tokyo
17, 1963
The alkali denaturation of pepsin was studied between pH 1 and 13 by difference spectroscopy and by activity measurements at pH 1.8, and the tryptophan and tyrosine residues were differentiated into two and three groups, respectively that differed in state. As the pH value of a pepsin solution is raised progressively from 1.8, the denaturation proceeds in several steps. The proteolytic activity drops first with pK = 3.1, and the band shift of two presumed tryptophan residues follows the inactivation (pK = 4.0). The band shift of the remaining four tryptophan residues proceeds between pH 7 and 8 and in parallel with an irreversible inactivation. At higher pH values, the ionization of tyrosine residues in various states occurs successively; 11 rapidly ionizing residues with pK = 10.0, 5 rapidly ionizing residues with pK = 11.5, and 1 slowly ionizing residue with a higher pK value. These results are discussed with reference to data reported previously.
The spectral changes observable on acidifying protein solutions are smaller and of different origin. All the tyrosine, tryptophan, and phenylalanine bands in the 250-300 mu region shift toward shorter wavelengths. The difference spectra of neutral protein solutions referred to acidic solutions generally show three distinct positive peaks around 279, 286, and 295 rnp and several minor peaks at shorter wavelengths (3, 4), in which the peak at 295 rnp arises from the shift of the tryptophan band. Recent observations of this phenomenon with model derivatives of amino acids (4, 5) as well as with various proteins have shown that the shifts are caused by changes in the environment of the chromophores of these amino acid residues in proteins. As reviewed by Scheraga (6), the changes may be due to cleavage of hydrogen bonds or ionization of a residue in the close vicinity of the chromophores, or exposure of a residue in a hydrophobic portion of the protein to the hydrophilic medium. Thus, any conformation
The ultraviolet absorption bands of proteins change upon making their solutions acid or alkaline. The change observable with alkali is due to the ionization of the phenol group of tyrosine residues, and the difference spectrum of ionization shows a maximum at 295 rnp and a minimum around 275 mp. Spectrophotometric titrations based on this phenomenon were applied in previous studies to insulin, lysozyme, and catalase (1) and to chymotrypsinogen, chymotrypsin, and trypsin (2). The tyrosine residues in these proteins were differentiated into three types : an instantaneously ionizing residue with a pK value similar to 10.0-10.1 for free tyrosine; a slowly ionizing residue with a higher pK value; and a nonionizable residue, in which the latter two types of residues may be regarded as bound residues hydrogenbonded or embedded in the interior of the molecule. Earlier literature on other proteins is reviewed with these results in the previous papers (1, 2). 1 Mailing
address. 326
TYROSINE
AND TRYPTOPHAN
change or denaturation leading to these changes in state of tryptophan residues is reflected by the band shifts. In the present paper the states of tyrosine and tryptophan residues in pepsin as determined from these two types of spectra1 changes are described. The spectrophotometric titrations were carried out over the pH range between 1 and 13, and the titration curves obtained were compared with the pH dependence of the proteolytic activity, and with the reversibility of alkali denaturation as estimated in terms of activity. The spectral change of the enzyme on the acidic side of pH have already been studied by Blumenfeld and Perlmann (7) and by Edelhoch (8), but the data reported by these authors differ from each other. The results obtained in the present study are discussed with reference to these earlier investigations. Throughout this paper, AE represents the difference in absorbance (E) at a difference maximum, n the moles of free and/or bound residues of tryptophan or tyrosine per mole of protein, and m the order of a sigmoid titration curve, which is equal to the number of hydrogen ions involved in the unmasking or ionization of the residue titrated.
RESIDUES
327
IN PEPSIN
dues per mole of protein) with the As value, 2305, estimated previously (1). All of the spectroscopic measurements were made with a Cary recording spectrophotometer model 14M and 1.0.cm cells. The proteolytic activity of pepsin was measured as a function of pH by the hemoglobin method (10). Digestion took place at 30°C for 10 minutes. The extent of digestion was estimated from the absorbance value at 280 rnp of a trichloroacetic acid soluble fraction of the digest, and was taken as the relative measure of activity.
RESULTS
AND DISCUSSION
TRYPTOPHAN
RESIDUES
As a preliminary study the experiment reported by Donovan et al. (4) for free tryptophan was repeated. The difference spectrum obtained for a 6.66 X 1O-4 I!4 solution at pH 7.8 against the same eoncentration of tryptophan at pH 1.5 is shown by curve A in Fig. 1, and indicates a maximum at 293 ml.r. The pH dependence of the AE value at the maximum is shown by curve A in Fig. 2. The values of pK and m for the ehange on the acidic side were estimated to be 2.3 and 1.0, respectively; as concluded by Donovan et al. the change
to
EXPERIMENTAL A crude preparation of swine pepsin (Difco Laboratories Inc.) was recrystallized three times by adjustment of the pH of the solution (9); the crystals were used for the various measurements. The concentration of pepsin was determined photometrically, assuming its molar extinction coefficient (E) at 278 rnp to be 52.47 X 103 (7). Pepsin is stable and undergoes no denaturation or autolysis around pH 4. The titration curve on the acidic side was measured at 298 rnp for a 1.11 X 1OW M pepsin solution at a desired pH referred to the same concentration of the solution at pH 4.0. The sample solution was prepared by adding 1 ml of a mixture of 1.0 M HCl and 1.0 M KC1 solutions to 9 ml of a pepsin solution at pH 4.0. The ionic strength of sample solutions at various pH values was thus kept constant at 0.10. Similar measurements were made for free tryptophan. The titration curve on the alkaline side was measured at 295 rnp, with a pepsin solution at pH 8.5 as the reference. The concentration of pepsin was 2.41 X 1O-5 M, and the ionic strength was kept at 0.125 with mixtures of KOH and KCl. AE values were reduced to 12 (moles of ionized t,vrosine resi_I
to .6 to 2 +o / O,
-0. 2-
-A-c
26C1
0’ I
I
280
300
Wavelength
in
320 mp
: ,O
FIG. 1. Difference spectra for tryptophan and pepsin solutions. Curve A, 6.66 X IO-* M tryptophan between pH 1.5 and 7.8; curve B, 1.00 X low4 M pepsin between pH 1.8 and 8.1; curve C, 1.8 X 1F M pepsin between pH 8.5 and 13.2. The solutions at the higher pH values were measured against those at lower pH values as the reference.
328
INADA,
MATSUSHIMA,
KAMATA,
PH FIG. 2. Changes of spectrum
or activity
with
pH observed for tryptophan and pepsin. Curve A, tryptophan, AE at 293 rnp; curve B, 1.11 X 10-’ M pepsin, AE at 298 rnp; curve C, proteolytic activi-
ties of pepsin measured at various pH values; curve D, the degree of reversibility of alkali denaturation as measured in terms of activity.
is caused by the inductive effect of ionization of the carboxyl group. From the absorbance change between pH 0.8 and 7.7, the Ae value was estimated to be 365, which is greater than the value, 240, obtained by these authors. Above pH 7.7, the curve rises again and reflects the effect of proton dissociation from the amino group. The difference spectrum of 1.00 X 1O-4 M pepsin solution at pH 1.8 and 8.1 is shown by curve B in Fig. 1. The shape of the major positive peak is similar to that obtained for tryptophan but is located at the longer wavelength of 298 rnp. In addition to this peak, the spectrum shows a round maximum at 275 rnp, a small dip, and a hump at 287 and 289 rnp, respectively, and a sharp minimum at 292 rnp. In measurements of difference spectra of solutions of high absorbancy, maxima or minima are often
flattened
and deformed
when the slit
AND SHIBATA
then flattened the sharp minimum at 292 rnp. An increase of the voltage, however, showed no effect on the whole spectrum of curve B, and Beer’s law was obeyed at 298 rnp with a slit width of 0.2 mm, so that measurements of AE at 298 rnB in further experiments were conducted with this slit width. The difference spectrum of pepsin on the acidic side of pH has been observed by two different groups. Edelhoch (8) observed the difference spectrum of a pepsin solution of pH 7.4 referred to the solution at pH 5.8, and obtained maxima around 282, 290, and 300 rnp and minima at 279, 286, and 292 mF. He interpreted the 279 and 286 rnp minima to be due to the shift of the tyrosine band, and the 292 rnp minimum to the shift of the tryptophan band. On the other hand, the difference spectrum obtained by Blumenfeld and Perlmann (7) for a pepsin solution at pH 4.6 referred to the solution at pH 2.0 showed no negative reading of AE in the same spectral region, and three maxima appeared at 278, 285, and 294 mp. The result obtained by Edelhoch resembles ours at longer wavelengths; both results show a sharp minimum at 292 mp and a rather high maximum around 298 my. At shorter wavelengths, however, the sharp and deep minimum is much
at 286 rnp observed
by Edelhoch
less marked in our spectrum, and appears on the positive side of the spectrum. The spectrum of Blumenfeld and Perlmann is considerably different from either of these spectra, particularly in the positive readings of AE in the intermediate wavelength range near 290 mp. As illustrated below, these differences are due to the pH values selected for the sample and reference solutions. (i) The difference spectrum between pH 1.8 and 6.1 was similar to that obtained by Blumenfeld
and
Perlmann,
although
the
width is not narrow enough. The present 285 and 294 rnp maxima observed by them measurements were repeated for the same appeared at 287 and 298 rnp in our spectrum, sample and reference solutions under a respectively, and the 287 rnp maximum was variety of conditions during which the slit lower than the 298 rnp maximum. (ii) The width was varied arbitrarily by variation of difference spectrum obtained between pH the voltage applied to the photomultiplier. A progressive decrease of the voltage (an 6.1 and 8.1 was similar to that obtained by Edelhoch; the addition of these difference increase of the slit width) to less than that spectra gave the spectrum of curve B obused for the measurement of curve B first lowered the round maximum at 275 rnE.c and served between pH 1.8 and 8.1. (iii) AS
TYROSINE AND TRYPTOPHAN RESIDUES IN PEPSIN
demonstrated below, the spectrum changes in two steps on the acidic side of pH, and the change observed by Blumenfeld and Perlmann is in one of these two steps while
residue responsible for the first step and 375 for the residue in the second step. The value obtained for the residues in pepsin agrees approximately with these values for the
tryptophan residues in lysozyme. The calculations support the view that the total absorbance change in pepsin results from the band shift of the six tryptophan residues. Another noteworthy fact is that the absorbance change in the first step is very cIose to one third of the total change. This implies that two of the residues undergo the spectral change in the first step and the remaining four in the second step. The value of m for the first step is approximately 1.0. This indicates that the change is due to the ionization of a group in close vicinity to the tryptophan residues and, from the pK value of 4.0, the group is probably a carboxyl group. By contrast, the value of m for the second step is much greater and is discussed later with the activity data. Curve C in Fig. 2 shows the variation of dues starts above pH 8.6, and is illustrated the proteolytic activity with pH. The optilater. The pK values for the first and the mum pH was estimated from the curve to be second steps were estimated from the curve 1.8 & 0.2, and the pK value for the inactivation on the alkaline side of the curve was to be 4.0 and 7.2, respectively. The total absorbance change below pH 3.1. This value of pK is lower than the 8.2 in the first and the second steps is 0.250. value for the first step of the spectral change; When this value of AE is divided by the there seems to be no correlation between the product of pepsin concentration (1.11 X inactivation and the change in state of the 1O-4 n/r) and AC (365) determined for free tryptophan residues. The reversibility of the inactivation with tryptophan, we obtain n = 6.2 in agreement with the molar tryptophan content, 6, de- alkali was observed in the following manner. termined by Blumenfeld and Perlmann (12). Pepsin was first dissolved at pH 4.0, and If the calculation is reversed on the assump- the pH of this solution was changed to a tion that the total change is caused by the value between 1.8 and 9.4. The solution, band shift of six tryptophan residues, we after standing at 7°C for 2 minutes, was obtain Ae = 375 for each residue, a value readjusted to the optimum pH of 1.8 and the activity was measured. The activity close to that for free tryptophan. This agreement may be fortuitous, since the AC thus measured (curve I) in Fig. 2) is constant below 6.3 and drops at higher pH value for the band shift of free tryptophan due to the inductive effect of ionization of values. This implies that the inactivation below pH 6.3 is completely reversible while its carboxyl group should generally be different from the values for tryptophan residues that at higher pH values is irreversible. of different types in proteins. A noteworthy The pK value for the irreversible deactivafact concerned with this problem is the tion is 7.5, which is in approximate agreeresult obtained by Donovan et al. (4) for ment with the value of 7.2 for the second tryptophan residues in lysozyme. They ob- step of the spectral change. In addition, these tained two steps of absorbance change with activity and spectral changes occur in a pK values of 3.15 and 6.20, and the Ae value narrow pH range with an m value much calculated from their data is 438 for the greater than 1.0. It is inferred from these the change observed by Edelhoch is in the other. The spectra observed in the two pH ranges differ considerably below 290 rnp, and, as reviewed and discussed by Bigelow and Sonenberg (11) , these changes at shorter wavelengths are regarded to be due to the shifts of superposed tyrosine and tryptophan bands. At longer wavelengths, however, both spectra are identical in that they have a distinct maximum at 298 rnp, which is due to the shift of the tryptophan band alone. The AE value at 298 rn/* was measured as a function of pH, and the result is shown with reference to extremely acidic pH by curve B in Fig. 2. Two steps of absorbance change occur below pH 8.2, and the curve reaches a plateau between pH 8.2 and 8.6. The third step due to the ionization of tyrosine resi-
330
INADA,
MATSUSHIMA,
KAMATA,
results that a large conformation change or denaturation occurs between pH 7 and 8, and is reflected by the irreversible inactivation and the abrupt change of spectrum. Edelhoch (8) had demonstrated a close correlation in the same pH range between the inactivation and the liberation of protons with alkali. His potentiometric titrations also revealed the time-dependent nature of the liberation of protons and a discrepancy between the forward and the backward titration curves in this pH range. These results support the view of a large conformation change. TYROSINE RESIDUES Curve C in Fig. 1 shows the difference spectrum observed for 1.8 X 1O-5 M pepsin solution at pH 13.2 with reference to the same solution at pH 8.5. The spectral change due to the ionization of tyrosine residues is much greater than those obtained at acidic pH. The AE value at the maximum of 295 rnp was measured as a function of pH for pepsin solutions in 4.0 M guanidine, and the result is shown in curve A of Fig. 3. As the pH increases, AE increases with pK = 10.0 and m = 1.0, and reaches a level of
n -8
PH
FIG. 3. Ionization curves for the tyrosine residues in 2.41 X 10d6 M pepsin. Curve A, with 4 M guanidine; curve B, without guanidine, observed immediately after the preparation of the alkaline solutions; curve C, without guanidine, observed 3 hours after mixing; curve D, the theoretical curve for 11 tyrosine residues with pK = 10.0; curve F, the theoretical curve for 5 tyrosine residues with pK = 11.5. The AE values shown by open triangles on curve F were obtained by subtracting the values calculated for the 11 residues from the observed values on curve B.
AND SHIBATA
n = 17. This value of pK agrees with that for free tyrosine, and the value of n coincides with the molar tyrosine content determined by Li (13). It is therefore evident that all of the tyrosine residues are free to ionize in 4.0 M guanidine. The AE values for pepsin in the absence of denaturant measured immediately after making the solution alkaline are shown by solid circles on curve B in Fig. 3. The curve is flatter and is shifted toward a higher pH as compared with curve A. Moreover, the AE value at extremely alkaline pH is lower than the value on curve A, and corresponds approximately to n = 16. When strongly alkaline solutions of pepsin were left standing at room temperature, the absorbance values at 295 mp increased gradually. A constant reading of absorbance was obtained 1 to 3 hours after mixing, and the time was dependent on the pH value. Curve C in Fig. 3 shows the result obtained after 3 hours in alkali and deviates from curve B above pH 11.2. The value of AE at extremely alkaline pH agrees with that of curve A for guanidinedenatured pepsin. The difference in AE between curves A and B was calculated to correspond to An = 1.3. However, this value of An may include considerable errors because of the small difference between large readings of AE. The difference was directly measured with more concentrated pepsin solutions to estimate An more exactly, and the result was An = 1.0 f 0.1. It is evident that 16 of the 17 residues ionize instantaneously with alkali and the remaining residue ionizes slowly. The apparent pK value estimated from curve B is approximately 10.4, which is slightly higher than the value for free tyrosine, and the value of m is appreciably smaller than 1.0. Two different interpretations may be given for this deviation of m. (i) As reviewed comprehensively by Tanford (14), the titration curves of proteins are flattened by the electrostatic effect, so that we obtain titration curves of different values of m at different ionic strengths. (ii) Curve B may be a composite of ionization curves with m = 1.0 and with different pK values. An analysis based on this interpretation was successful when it was assumed that the 16
331
TYROSINE AND TRYPTOPHAN RESIDUES IN PEPSIN
TABLE I residues represent 11 residues with pK = VARIOUS TYPES OF TRYPTOPHAN AND TYROSINE 10.0 and 5 residues with pK = 11.5. Curve RESIDUES IN PEPSIN D in Fig. 3 is the ionization curveobtained
by calculationfor the 11freeresidues.Open triangles on curve F indicate the values obtained by subtracting the values for the 11 free residues from the observed values on curve B, and these differences agree with the AE values calculated for 5 residues with pK = 11.5 (curve F) . An interesting observation was made by Li (13)) who observed the reaction at pH 5.7 of iodine with native and urea-denatured pepsin. According to his kinetic analysis, 12 residues are free to be iodinated, and 3 of the remaining 5 residues are liberated with 7.1 M urea. This result offers support for our second interpretation, since it differs by only one residue from our estimate of 11 free and 6 bound residues. The effect of ionic strength on the ionization curve will clarify which interpretation is correct and awaits further experiment. On the other hand the difference in the number of strongly bound residues, 2 in the result of Li and 1 in ours, may be significant. It indicates that one of the two residues is of the rapidly ionizing type, but not reactive with iodine, and that one of these residues is transformed by the denaturation between pH 7 and 8 into the rapidly ionizing type. A similar result was obtained previously for tyrosine residues in insulin (1, 15). Three of the 4 residues ionized rapidly and the remaining residue slowly. However, only two tyrosine residues were reactive with cyanuric fluoride near neutral pH, which indicated that one of two nonreactive residues was transformed with alkali into the reactive type. The alkali denaturation of pepsin may be described as follows in terms of spectral and activity changes. When alkali is added to a pepsin solution at pH 1.8, inactivation is associated with a pK of 2.3, and the band shift of two tryptophan residues follows the inactivation. These activity and spectral chauges are completely reversible; the conformation change of the molecule may be limited. The band shift of the remaining 4 tryptophan residues proceeds between pH 7 and 8 in parallel with an irreversible in-
Residuetype’” Tryptophan (nt = G) Tyrosine (nt = 17)
n
PK
2 4 11 5 1
4.0 7.2 10.0 11.5 >11.7
Ionization rapid rapid slow
ant = total molar content of tryptophan tyrosine.
or
activation. These changes are considered to be due to a large conformation change. At higher pH values, the ionization of various types of tyrosine residues occurs successively; 11 free residues with pK = 10.0, 5 weakly bound residues with pK = 11.5, and 1 strongly bound residue with a higher pK value. It requires about 3 hours in strong alkali to unmask the most strongly bound residue. Different types of tryptophan and tyrosine residues distinguished in the present study are summarized in Table I with their pK values. REFERENCES 1. INADA, Y., J. Biochem. (Tokyo) 49, 218 (1961). Y., KAMATA, M., MATSLJSHIMA, A., AND SHIBATA, K., Biochim. Biophys. Acta,
2. INADA,
81, 323 (1964). 3. LASKOWSKI, M., JR., LEACH, S. J., AND OCHERAGA, H. A., J. Am. Chem. Sot. 82, 571 (1960). 4. DONOVAN, J. W., LASKOWSKI, M., JR., AND SCHERAGA, H. A., J. Am. Chem. Sot. 83, 2686 (1961). 5. WETLAUFER, D. B., EDS~LL, J. T., AND HOLLINGWORTH, B. R., J. Biol. Chem. 233, 1421 (1958). 6. SCHERAGA, H. A., “Molecular Biology” (N.O. Kaplan and H. A. Scheraga, eds.), Vol. 1 (“Protein Structure”), p. 234. Academic Press, New York, 1961. 7. BLUMENFELD, 0. O., ASD PERLMANN, G. E., J. Gen. Physiol. 42, 563 (1959). 8. EDELHOCH, H., J. Am. Chem. Sot. 80, 6640 (1958). 9. NORTHROP, J. H., J. Gen. Physiol. 30, 177 (1946). 10. ANSON, M. L., in “Crystalline Enzymes”
332
INADA,MATSUSHIMA,KAMATA, ANDSHIBATA
(J. H. Northrop, ed.), Columbia Biological Series, No. 12, p. 305. Columbia Univ. Press, New York, 1948. 11. BIGELOW, C. C., AND SONENBERG, M., Biochemistry 1, 197 (1962). 12. BLUMENFELD, 0. O., AND PERLMANN, G. E., J. Gen. Physiol. 42, 553 (1959).
13. LI, C. H., J. Am. Chem. Sot. 67, 1065 (1945). 14. TANFORD, C., in “Advances in Protein Chemistry” (M. L. Anson and J. T. Edsall, eds.), Vol. 17, p. 95. Academic Press, New York, 1962. 15. KURIHARA, K., HORINISHI, H., AND SHIBATA, K., Biochim. Biophys. Acta 74, 678 (1963).