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Hydrogen-deuterium exchange study of amino acids and proteins by 200- to 230-nm spectroscopy
ANALYTICAL
BIOCHEMISTRY
Hydrogen-Deuterium
110,
242-249 (1981)
Exchange Study of Amino Acids and Proteins by 200- to 230~nm Spectroscopy
TETSUO TAKAHASHI,MAMORU Faculty
of Pharmaceutical
Sciences,
NAKANISHI, AND MASAMICHI TSUBOI
University
of Tokyo,
Hongo,
Bunkyo-ku,
Tokyo
113, Japan
Received June 9, 1980 Deuteration of asparagine, glutamine, arginine, tryptophan, and tyrosine has been found to cause a small but appreciable spectral change in the 200- to 230-nm region. By the use of a stopped-flow ultraviolet absorption method, the rates of deuteration of these amino acids have been determined. On the basis of these results, and on the basis of our previous study on the hydrogen-deuterium exchange of the peptide groups, a few comments are given as for how to apply the method to protein study.
In a recent issue of this journal, Englander et al. (1) described that hydrogen-deuterium exchange behavior of peptide bond-containing molecules can be measured in an extremely simple manner by ultraviolet spectrophotometry. This is just what we had shown in our earlier paper (2). There, we showed that a time-dependent decrease in absorbance at 227 nm was observed when N-methylacetamide or poly-D,L-alanine was suddenly brought into a D,O medium by a stopped-flow device. The work described in this paper forms a part of our effort to bring this stopped-flow 200- to 230~nm spectrophotometry method into a form directly applicable to an actual protein study. Some of the sidechains of proteins have absorptions in the 200- to 230nm region, and their intensities may change on deuteration; these may interfere with the kinetic measurement of the mainchain deuteration. Therefore, we should first examine in this respect all the sidechains which have labile hydrogen atoms and have ultraviolet absorptions in the 200- to 230-nm region. They are asparagine , glutamine, arginine, histidine, lysine, serine, threonine, cysteine, tryptophan, and tyrosine. We can then fix a proper wavelength to use for the 0003-2697/81/010242-08$02.00/O Copyright 0 1981 by Academic Press, Inc. AU rights of reproduction in any form reserved.
242
mainchain deuteration kinetic measurement that gives minimum interference and error. It is also expected that, in such examinations, a method might be newly established to trace some of the sidechain deuteration reactions without interference with the mainchain kinetics. MATERIALS
AND METHODS
Imidazole was purchased from Nakarai Company, Ltd.; phenol, L-tryptophan, and L-tyrosine were from Wako Pure Chemical Industries, Ltd.; L-lysine, L-serine, Lthreonine, L-cysteine, L-glutamine, Lasparagine, and L-arginine, from Kyowa Hakko Company, Ltd. ; and deuterium oxide (99.75%) from CEA-CEN-SACLAY. The hydrogen-deuterium exchange reactions were traced with a Union Giken stoppedflow spectrophotometer RA-401. This is equipped with a rapid mixing device with a dead time of 0.5 ms; the ultraviolet spectrophotometer has a focal length of 25 cm, a sensitivity of 0.0001 O.D. units (absorbance), and a response time of 0.1 ms. The optical path length of the cell is 10 or 2 mm. This equipment was connected to a UnionGiken data processor RA-450, a monitor
HYDROGEN-DEUTERIUM
Wavelength
1 “In
EXCHANGE
)
FIG. 1. Absorption spectra of L-asparagine (-), L-glutamine (- - -), and L-arginine (- - -). The amount of he (difference in the molar extinction coefficient) which is considered to be solely caused by the deuteration of asparagine (0), glutamine (O), or arginine (A) is also given as a function of the wavelength.
scope, and an XY plotter. Ultraviolet absorptions and difference spectra were observed by use of Hitachi EPS-3 spectrophotometer. The hydrogen ion concentration of the solution was measured with a HitachiHoriba MSS pH meter. In this paper, we use the notation pH even for the deuterium ion concentration of a deuterium oxide solution, and pH meter readings are always given without any corrections.
BY ULTRAVIOLET
243
SPECTROSCOPY
sorption coefficient is observed at any of the wavelengths in the 200- to 220-nm region. An example of the recorded curve in such an experiment is shown in Fig. 2. A replot of such a curve with a logarithmic scale along ordinate gives a straight line; the deuteration proceeds as a single first-order reaction at every temperature in the IO-40°C range, and at every pH in the 3.5-6.5 range. Extrapolation of such a straight line to zero time gives a total amount of Ae (where E is the molar extinction coefficient) which is attributable to deuteration (note that this is 50% deuteration because the ultimate solvent is 1: 1 H,O + D,O). This be value is plotted against the wavelength of the ultraviolet light used for the kinetic measurement as shown in Fig. 1 with white and black circles (for L-asparagine and L-glutamine, respectively), In Fig. 3, the deuteration rate constants (k,) are plotted along a logarithmic scale against pH. As is seen here, asparagine shows a minimum at pH 4.4, while glutamine at pH 4.8. In both acidic and alkaline sides of these minima, the relations are given nearly by straight lines with slope l/l. Thus, the k,-pH relation is given by k, = k,+[H+]
+ k&OH-]
+ k,,,
[1]
where k. is a constant independent of [H+] and [OH-]. The catalytic constants kH+ and kOH-are estimated to be 2200 M-’ s-l and 2.2 Y
f A =r
RESULTS Asparagine and Glutamine
Both of these amino acids show a sharp rise of their absorption coefficients on changing the wavelength from 210 to 200 nm (see Fig. 1). When a H,O solution of asparagine or glutamine is rapidly mixed with DzO, a time-dependent decrease of the ab-
TIME
(SEC
1
FIG. 2. A recorded curve in a stopped-flow ultraviolet absorption study of the H -+ D exchange reaction of L-asparagine. At 18.5”C, L-asparagine in H,O (1 mg/ml in HpO) was rapidly mixed with D,O (1: 1 in volume), so that the final concentration of L-asparagine was 0.5 mg/ml. A time-dependent decrease in absorbance at 202 nm was observed and the data were stored. A 2-mm cell was used.
244
TAKAHASHI,
NAKANISHI,
AND TSUBOI TABLE
HYDROGEN-EXCHANGE AT 20°C) AND
koH-,
MOLAR CAUSED
AMOUNT
1
RATE CONSTANTS OF CHANGE
EXTINCTION COEFFICIENT BY DEUTERATION
(E)
h+ Sample
o.tk , PH
. 4
,
, 4
5
6
FIG. 3. Hydrogen-deuterium exchange rate constant for r-asparagine (0) and for L-glutamine (0) at 39.W plotted against pH. The continuous lines show calculated relations on an assumption thatk,, = 2200 and k,,a- = 2.2 X 108 M-’ s-r for L-asparagine, and k,, = 5500 and kc,,- = 1 x lo8 M-’ s-* for L-glutamine.
lo8 M-’ s-l, respectively, for asparagine, and 5500 M-’ s-l and 1 X lo8 M-’ s-l, respectively, for glutamine, both at 395°C. The catalytic constant values for 20°C are given in Table 1. In Fig. 4, a few Arrhenius plots for the deuteration rate constants k, are illustrated. Here, a straight line is obtained in every case, and from its slope activation energy (AH+) of each deuteration reaction is estimated. It is 13.5 kcal/mol for asparagine at pH 2.7 and 19 kcal/mol at pH 6.5. For asparagine and glutamine sidechains, Molday et al. (3) examined tritium (T-H)exchange rates at 0°C. The T-H rate constant for poly-o,L-asparagine, for example, is estimated, from their Fig. 5, to be about 6 min-’ at pH 6.5 and at 0°C. This is nearly equal to what is expected from our Arrhenius plot (Fig. 4A); at 0°C (and at pH 6.5) the hydrogen deuterium exchange rate of asparagine comes out to be about 0.1 s-l (=6 min-‘). The values of the activation energy given above are also in agreement with what was obtained by Blomberg et al. (4) (17.7 kcal/mol at pH 4.8) with the 15Nmagnetic resonance method. x
Arginine
The absorption spectrum of L-arginine is shown in Fig. 1. When L-arginine in H,O is rapidly mixed with D,O (at pH 2.95 and
(M-’
L-Asparagine L-Glutamine L-Arginine -NH-NH*
AT
nm
206
km-
S-‘)
s-‘)
(M-l
530
1300 2 220
POly-DL-akWIiIK.
Poly-rglutamic Poly-L-lysine
(kH+ AND IN THE
(de)
acid
AC
8.0 x 10’ 3.6 x 10’
30 25
3.9 x 108 1.9 x 100 9.3 x 10’ 2.0 x lo8 4.3 x 108
20 20
33 28
40
a 50% deuteration (see text). I
I
I
I
I
(A) 1
I
\
3k---. 32
I
40
I
3.3
35 3’0 Temperature
l/T
I
3-4 x lo-’
d5 2’0 (“C 1
FIG. 4. (A) The rate constant k, of the hydrogendeuterium exchange reaction of L-asparagine at pH 6.5 plotted on a logarithmic scale against reciprocal absolute temperature (Arrhenius plot). (B) A similar Arrhenius plot for L-asparagine at pH 2.8. (C) A similar Arrhenius plot for L-arginine (terminal C Y-Y NH,) at pH 2.1.
HYDROGEN-DEUTERIUM
EXCHANGE
s< 0
10
20 TIME
30 ( SEC
40
!iO
1
FIG. 5. The time dependence of the decrease in absorbance at 202 nm observed when L-arginine dissolved in H,O (1 mg/ml) is mixed with DIO (1: 1 volume; final concentration of L-arginine is 0.5 m&ml at pH 2.95 and 395°C. This is a photographic reproduction of the curve recorded on the plotter connected with a Union Giken stopped-flow spectrophotometer RA-401 and a data processor RA-450.
39.5”C, the final concentration is 0.5 mg/ml), a time-dependent decrease of the absorbance at 202 nm is observed as shown in Fig. 5. A replot of such data, as illustrated also in Fig. 5, shows that the absorbance decrease takes place as two first-order processes, fast and slow ones. By extrapolating the straight line corresponding to the faster reaction to zero time, we are able to determine the total change Act in the molar extinction coefficient associated to the deuteration. The Act value measured in this way is plotted against wavelength in Fig. 1. When the straight line corresponding to the slower reaction is extrapolated to zero time, the change (he,) in the extinction coefficient corresponding to the slower reaction is obtained. It was found that Ar, = 0.33 Act for every wavelength examined. The rate constants of the fast and slow reactions are plotted on a logarithmic scale against pH of the solution at 39.5”C in Fig. 6. The rate constant for the slow reaction shows a minimum at pH 2.2, while that of the fast reaction seems to come at about 3.5. In the pH region of 3.5 to 4.5, the estimation of the two reaction rates could not
BY ULTRAVIOLET
245
SPECTROSCOPY
straightforwardly be made. Here, the first guess was made by drawing a straightline in the log k,-pH space (Fig. 6) from the point corresponding to the slower rate constant at pH 3.0 with the slope of l/l, and by assuming that one of the two rate constants is on this straight line at every pH in the pH 3.5-4.5 region. After such a first guess a small adjustment of the rate constants caused a fit of the experimental kinetic data. Such an estimation shows that, on going from lower pH region to higher, the order of the rate constant values seems to be inverted at pH 3.5. Therefore, it is not proper to call the two reactions “fast” and “slow” ones. Instead, let us discriminate them, by the positions of the minima (pH 2.2 and 3.5) in the log k,-pH curves. Arginine sidechain has two kinds of labile hydrogen atoms, that involved in - C-NH-C group and those involved in the terminal C-NH2 groups. The rate constant with the minimum at pH 3.5 is assigned to the latter hydrogen atoms, because the hydrogendeuterium exchange rate constant of guanidinium ion gives a minimum at pH 3.3 (see Fig. 7). The assignment is further supported by an examination of the hydrogendeuterium exchange reactions of N-methyl-
PH
2
3
4
FIG. 6. The rate constants k. of the hydrogendeuterium exchange reactions of L-arginine plotted on a logarithmic scale against pH of the solution at 39.5% (0) Attributable to the terminal C z NH, groups of the sidechain. (0) Attributable to the C-NH z c group.
246
TAKAHASHI.
NAKANISHI.
guanidinium ion. This shows two exponential decay curves at pH 3.2. The faster and slower rate constants are found to be 1.0 and 0.15 s-l at 39.5”C, and these are close, respectively, to the values, 1.0 and 0.2 s-l of the corresponding rate constants of arginine at pH 2.9. In addition, the extrapolation of the straight line corresponding to the slower reaction to zero time gives a AE, value which is again 0.33 times as great as the Act value obtained by the extrapolating for the faster reaction. The hydrogen exchange rates of arginine was also examined by a 15N-magnetic resonance method (5). The results of the two different methods are in agreement with each other. In Table 1, the catalytic constants, kH+ and koH-, estimated from the pH dependence (see Fig. 6) of the exchange rates are given for arginine at 20°C. The rate constant k, of the hydrogendeuterium exchange reaction assignable to the terminal NH, groups of L-arginine is plotted in Fig. 4, (C) on a logarithmic scale against reciprocal absolute temperature at pH 2.1. From the slope of this Arrhenius plot, the activation energy (AH+) of the reaction is given as 11.9 kcal /mol at pH 2.1. Histidine
In Fig. 8, absorption spectrum of imidazole is shown. On deuteration, a time-dependent change (he) in the molar extinction co-
AND TSUBOI
FIG. 8. Lower: Absorption spectrum of imidaxole in aqueous solution at pH 8. Upper: The amount of AC (difference in the molar extinction coefficient caused by 50% deuteration is given by (x) as a function of the wavelength. The differential coefficient of the absorption curve is also shown by (0) and (O), for comparison.
efficient (E) is found, and its amount at zero time is plotted against wavelength. As may be seen in the figure, this is nearly coincidental with the differential curve of the absorption curve. This fact may be taken as indicating that the deuteration of imidazole causes a blue shift of its absorption profile by about 0.15 nm. Lysbze, Serine, Threonine, and Cysteine
PH
3
4
FIG. 7. The rate constant k, of the hydrogendeuterium exchange reaction of the guanidinium chloride plotted on a logarithmic scale against pH of the solution at 392°C.
None of these give time-dependent absorbance change in the 200- to 280~nm region, when their Hz0 solutions are rapidly mixed with D20. This means that their deuteration rate is higher than 1000 s-l or their deuteration does not cause any appreciable absorbance change in the spectral range exmained.
HYDROGEN-DEUTERIUMEXCHANGEBYULTRAVIOLETSPECTROSCOPY
. \ I.: 01 . :0
247
248 Tryptophan
TAKAHASHI,
NAKANISHI,
AND
TSUBOI
TABLE2
and Tyrosine
Tryptophan has a few absorption bands in the 250- to 300-nm region and tyrosine has an absorption band at 275 nm. By the use of the deuteration effects on those absorptions, we previously established a useful method of tracing the time course of the hydrogen-deuterium exchange reactions in the tryptophan and tyrosine residues in proteins [6,7]. These residues have, however, other absorption bands in the 200- to 230-nm region, too, as shown in Fig. 9. Deuteration of the tryptophan residue causes a time-dependent change not only in the 2% to 300-nm region (6) but also in the 200- to 230-nm region. Therefore, a stoppedflow ultraviolet absorption experiment was newly made at each wavelength in the 200to 230-nm region. The, rate constant obtained from the semilogarithmic kinetic plot was found to be equal to what was previously obtained in the longer wavelength region. The amount of the change (Ae) in molar extinction coefficient (e) at zero time was estimated by an extrapolation, and this is plotted against the wavelength in Fig. 9. This may be useful to estimate the singal level in planning a stopped-flow ultraviolet absorption measurement of a system involving tryptophan residues. For tyrosine, a similar examination was made also in the shorter wavelength region (200-240 nm) and the result is shown also in Fig. 9.
RATE CONSTANTS (k) OF THE DEUTERATION REACTIONS OF
DISCUSSION
One may suspect that the Ae values of the sidechain residues in a protein would be greatly different from those given in Table
Rate constant k/k* (at 20°C pH7Y
Residue Arginine
-NH-
AP 20
NH, -+c
100
40
1 Fast and/or
30
NH, Asparagine Cysteine Glutamine Histidine Lysine Serine Threonine Tryptophane Tyrosine
-NH* -SH -NH2 -NH-NH,+ -OH -OH -NH-OH
AE= 0 0.5
10 Fast and/or AC= 0 Fastand/or AC= 0 Fast and/or AC= 0 1.3 300
n k* = the rate constant for poly-oL-alanine = 4.6 s-l. b be for 509k deuteration, i.e., H -) (H + D)/2 = 33 for hly-m-alanine).
25 30
20 20
(AC
absorption method is applicable to a protein study. The Ae Values-a Comparison of the Mainchain and Sidechain Hydrogens
In Table 2 (last column), the amount of the change (AE) in the molar extinction coefficient (E) caused by the deuteration of each sidechain residue at 206 nm is given. Polypeptides Arginine residue has a greater value of be A detailed description on our hydrogen than that of peptide group (he = 33 for exchange study of poly-D,L-alanine has poly-r>L-alanine), but it has fived exchangebeen made in our previous paper (2). A able hydrogen atoms. As long as the AE similar work has been made of poly-L- value (at 206 nm) per one exchangeable glutamic acid and poly+lysine at various hydrogen atom is concerned, the peptide pHs and at various temperatures. The cata- group is the highest. lytic constants k OH-determined at 20°C are given in Table 1, with the he values ob- Possible Shifts of the Absorption Bands served at 206 nm. in Proteins Let us now examine how the stoppedflow ultraviolet (at 206 nm, for example)
HYDROGEN-DEUTERIUM
EXCHANGE
2, because these values were obtained for free amino acids whereas in a protein the absorption bands are often shifted depending upon the intramolecular environments in which the amino acid residues are located. Nakanishi and Tsuboi (7) found, for example, that the be value is greatest at 285 nm for free L-tyrosine whereas at 290 nm for the tyrosine residues in ribonuclease A. In general, however, the amount of the shift of the absorption band of an aromatic ring (when it is brought from a free amino acid into a protein molecule) is smaller in the 210-nm region than in the 280-nm region (8). Deuteration rates-a Comparison of the Mainchain and Sidechain Hydrogens
In Table 2, the ratio of the rate constant (k) of the deuteration reaction of each sidechain residue over the rate constant (k*) of the peptide group is given. As may be seen here, k’s are much greater than k* for all the sidechain residues except for asparagine, glutamine, and tryptophan. This fact gives a support to an analysis (9) of the slower hydrogen-exchange reactions of proteins on an assumption that they are mostly due to the peptide groups rather than to the sidechain residues. It is true, however, that a certain allowance should be made for a contribution from the asparagine-, glutamine- and/or tryptophan-hydrogens in the analysis. A Possible Error in Tracing the Deuteration Reaction of the Peptide Groups in a Protein by Absorbance Measurement at 206 nm
On the basis of the results of the examination so far described, the contributions of
BY ULTRAVIOLET
SPECTROSCOPY
249
the sidechain residues to the AC values at 206 nm are summarized as follows: (i) Asparagine, glutamine, and tryptophan.
Each one of these residues gives a contribution comparable to that of one peptide group which is exposed to the solvent on the surface of the protein molecule (fast peptide group). (ii) Arginine, histidine, and tyrosine. If these residues in the protein are involved in hydrogen bonds with some other residues, so that their deuteration rates are greatly lowered, they may cause a slight disturbance in examining the fast peptide groups. Otherwise, these should not cause any error in an estimation of the rate of the peptide deuteration. Actual application to the stopped-flow 206-nm spectrophotometry to proteins will be detailed elsewhere, with a quantitative estimation of errors. REFERENCES 1. Englander, J. J., Calhoun, D. B., and Englander, W. (1979) Anal. Biochem. 92, 517-524. 2. Takahashi, T., Nakanishi, M., and Tsuboi, M., (1978)Bull. Chem. Sot. Japan. 51, 1988-1990. 3. Molday, R. S., Englander, S. W., and Kallen, R. G. (1972) Biochemistry 11, 150- 158. 4. Blomberg, F., Maurer, W., and Riiterjans. H. (1976) Proc. Nat. Acad. Sci. USA 73, 14091413. 5. Yavari, I., and Roberts, J. D. (1978) Eiochem. Biophys. Res. Commun. 83, 635440. 6. Nakanishi, M., Nakamura, H., Hirakawa, A. Y., Tsuboi, M., Nagamura, T., and Saijo, Y. (1978) J. Amer. Chem. Sot. 100, 272-276. 7. Nakanishi, M., and Tsuboi, M. (1978) J. Amer. Chem. Sot. 100, 1273-1275. 8. Donovan, J. W. (1%9) Physical Principles and Techniques of Protein Chemistry, part A, pp. lOl- 170, Academic Press, New York. 9. Englander, J. J., and Englander, S. W. (1977) Nature (London) 265,658-659.
BIOCHEMISTRY
Hydrogen-Deuterium
110,
242-249 (1981)
Exchange Study of Amino Acids and Proteins by 200- to 230~nm Spectroscopy
TETSUO TAKAHASHI,MAMORU Faculty
of Pharmaceutical
Sciences,
NAKANISHI, AND MASAMICHI TSUBOI
University
of Tokyo,
Hongo,
Bunkyo-ku,
Tokyo
113, Japan
Received June 9, 1980 Deuteration of asparagine, glutamine, arginine, tryptophan, and tyrosine has been found to cause a small but appreciable spectral change in the 200- to 230-nm region. By the use of a stopped-flow ultraviolet absorption method, the rates of deuteration of these amino acids have been determined. On the basis of these results, and on the basis of our previous study on the hydrogen-deuterium exchange of the peptide groups, a few comments are given as for how to apply the method to protein study.
In a recent issue of this journal, Englander et al. (1) described that hydrogen-deuterium exchange behavior of peptide bond-containing molecules can be measured in an extremely simple manner by ultraviolet spectrophotometry. This is just what we had shown in our earlier paper (2). There, we showed that a time-dependent decrease in absorbance at 227 nm was observed when N-methylacetamide or poly-D,L-alanine was suddenly brought into a D,O medium by a stopped-flow device. The work described in this paper forms a part of our effort to bring this stopped-flow 200- to 230~nm spectrophotometry method into a form directly applicable to an actual protein study. Some of the sidechains of proteins have absorptions in the 200- to 230nm region, and their intensities may change on deuteration; these may interfere with the kinetic measurement of the mainchain deuteration. Therefore, we should first examine in this respect all the sidechains which have labile hydrogen atoms and have ultraviolet absorptions in the 200- to 230-nm region. They are asparagine , glutamine, arginine, histidine, lysine, serine, threonine, cysteine, tryptophan, and tyrosine. We can then fix a proper wavelength to use for the 0003-2697/81/010242-08$02.00/O Copyright 0 1981 by Academic Press, Inc. AU rights of reproduction in any form reserved.
242
mainchain deuteration kinetic measurement that gives minimum interference and error. It is also expected that, in such examinations, a method might be newly established to trace some of the sidechain deuteration reactions without interference with the mainchain kinetics. MATERIALS
AND METHODS
Imidazole was purchased from Nakarai Company, Ltd.; phenol, L-tryptophan, and L-tyrosine were from Wako Pure Chemical Industries, Ltd.; L-lysine, L-serine, Lthreonine, L-cysteine, L-glutamine, Lasparagine, and L-arginine, from Kyowa Hakko Company, Ltd. ; and deuterium oxide (99.75%) from CEA-CEN-SACLAY. The hydrogen-deuterium exchange reactions were traced with a Union Giken stoppedflow spectrophotometer RA-401. This is equipped with a rapid mixing device with a dead time of 0.5 ms; the ultraviolet spectrophotometer has a focal length of 25 cm, a sensitivity of 0.0001 O.D. units (absorbance), and a response time of 0.1 ms. The optical path length of the cell is 10 or 2 mm. This equipment was connected to a UnionGiken data processor RA-450, a monitor
HYDROGEN-DEUTERIUM
Wavelength
1 “In
EXCHANGE
)
FIG. 1. Absorption spectra of L-asparagine (-), L-glutamine (- - -), and L-arginine (- - -). The amount of he (difference in the molar extinction coefficient) which is considered to be solely caused by the deuteration of asparagine (0), glutamine (O), or arginine (A) is also given as a function of the wavelength.
scope, and an XY plotter. Ultraviolet absorptions and difference spectra were observed by use of Hitachi EPS-3 spectrophotometer. The hydrogen ion concentration of the solution was measured with a HitachiHoriba MSS pH meter. In this paper, we use the notation pH even for the deuterium ion concentration of a deuterium oxide solution, and pH meter readings are always given without any corrections.
BY ULTRAVIOLET
243
SPECTROSCOPY
sorption coefficient is observed at any of the wavelengths in the 200- to 220-nm region. An example of the recorded curve in such an experiment is shown in Fig. 2. A replot of such a curve with a logarithmic scale along ordinate gives a straight line; the deuteration proceeds as a single first-order reaction at every temperature in the IO-40°C range, and at every pH in the 3.5-6.5 range. Extrapolation of such a straight line to zero time gives a total amount of Ae (where E is the molar extinction coefficient) which is attributable to deuteration (note that this is 50% deuteration because the ultimate solvent is 1: 1 H,O + D,O). This be value is plotted against the wavelength of the ultraviolet light used for the kinetic measurement as shown in Fig. 1 with white and black circles (for L-asparagine and L-glutamine, respectively), In Fig. 3, the deuteration rate constants (k,) are plotted along a logarithmic scale against pH. As is seen here, asparagine shows a minimum at pH 4.4, while glutamine at pH 4.8. In both acidic and alkaline sides of these minima, the relations are given nearly by straight lines with slope l/l. Thus, the k,-pH relation is given by k, = k,+[H+]
+ k&OH-]
+ k,,,
[1]
where k. is a constant independent of [H+] and [OH-]. The catalytic constants kH+ and kOH-are estimated to be 2200 M-’ s-l and 2.2 Y
f A =r
RESULTS Asparagine and Glutamine
Both of these amino acids show a sharp rise of their absorption coefficients on changing the wavelength from 210 to 200 nm (see Fig. 1). When a H,O solution of asparagine or glutamine is rapidly mixed with DzO, a time-dependent decrease of the ab-
TIME
(SEC
1
FIG. 2. A recorded curve in a stopped-flow ultraviolet absorption study of the H -+ D exchange reaction of L-asparagine. At 18.5”C, L-asparagine in H,O (1 mg/ml in HpO) was rapidly mixed with D,O (1: 1 in volume), so that the final concentration of L-asparagine was 0.5 mg/ml. A time-dependent decrease in absorbance at 202 nm was observed and the data were stored. A 2-mm cell was used.
244
TAKAHASHI,
NAKANISHI,
AND TSUBOI TABLE
HYDROGEN-EXCHANGE AT 20°C) AND
koH-,
MOLAR CAUSED
AMOUNT
1
RATE CONSTANTS OF CHANGE
EXTINCTION COEFFICIENT BY DEUTERATION
(E)
h+ Sample
o.tk , PH
. 4
,
, 4
5
6
FIG. 3. Hydrogen-deuterium exchange rate constant for r-asparagine (0) and for L-glutamine (0) at 39.W plotted against pH. The continuous lines show calculated relations on an assumption thatk,, = 2200 and k,,a- = 2.2 X 108 M-’ s-r for L-asparagine, and k,, = 5500 and kc,,- = 1 x lo8 M-’ s-* for L-glutamine.
lo8 M-’ s-l, respectively, for asparagine, and 5500 M-’ s-l and 1 X lo8 M-’ s-l, respectively, for glutamine, both at 395°C. The catalytic constant values for 20°C are given in Table 1. In Fig. 4, a few Arrhenius plots for the deuteration rate constants k, are illustrated. Here, a straight line is obtained in every case, and from its slope activation energy (AH+) of each deuteration reaction is estimated. It is 13.5 kcal/mol for asparagine at pH 2.7 and 19 kcal/mol at pH 6.5. For asparagine and glutamine sidechains, Molday et al. (3) examined tritium (T-H)exchange rates at 0°C. The T-H rate constant for poly-o,L-asparagine, for example, is estimated, from their Fig. 5, to be about 6 min-’ at pH 6.5 and at 0°C. This is nearly equal to what is expected from our Arrhenius plot (Fig. 4A); at 0°C (and at pH 6.5) the hydrogen deuterium exchange rate of asparagine comes out to be about 0.1 s-l (=6 min-‘). The values of the activation energy given above are also in agreement with what was obtained by Blomberg et al. (4) (17.7 kcal/mol at pH 4.8) with the 15Nmagnetic resonance method. x
Arginine
The absorption spectrum of L-arginine is shown in Fig. 1. When L-arginine in H,O is rapidly mixed with D,O (at pH 2.95 and
(M-’
L-Asparagine L-Glutamine L-Arginine -NH-NH*
AT
nm
206
km-
S-‘)
s-‘)
(M-l
530
1300 2 220
POly-DL-akWIiIK.
Poly-rglutamic Poly-L-lysine
(kH+ AND IN THE
(de)
acid
AC
8.0 x 10’ 3.6 x 10’
30 25
3.9 x 108 1.9 x 100 9.3 x 10’ 2.0 x lo8 4.3 x 108
20 20
33 28
40
a 50% deuteration (see text). I
I
I
I
I
(A) 1
I
\
3k---. 32
I
40
I
3.3
35 3’0 Temperature
l/T
I
3-4 x lo-’
d5 2’0 (“C 1
FIG. 4. (A) The rate constant k, of the hydrogendeuterium exchange reaction of L-asparagine at pH 6.5 plotted on a logarithmic scale against reciprocal absolute temperature (Arrhenius plot). (B) A similar Arrhenius plot for L-asparagine at pH 2.8. (C) A similar Arrhenius plot for L-arginine (terminal C Y-Y NH,) at pH 2.1.
HYDROGEN-DEUTERIUM
EXCHANGE
s< 0
10
20 TIME
30 ( SEC
40
!iO
1
FIG. 5. The time dependence of the decrease in absorbance at 202 nm observed when L-arginine dissolved in H,O (1 mg/ml) is mixed with DIO (1: 1 volume; final concentration of L-arginine is 0.5 m&ml at pH 2.95 and 395°C. This is a photographic reproduction of the curve recorded on the plotter connected with a Union Giken stopped-flow spectrophotometer RA-401 and a data processor RA-450.
39.5”C, the final concentration is 0.5 mg/ml), a time-dependent decrease of the absorbance at 202 nm is observed as shown in Fig. 5. A replot of such data, as illustrated also in Fig. 5, shows that the absorbance decrease takes place as two first-order processes, fast and slow ones. By extrapolating the straight line corresponding to the faster reaction to zero time, we are able to determine the total change Act in the molar extinction coefficient associated to the deuteration. The Act value measured in this way is plotted against wavelength in Fig. 1. When the straight line corresponding to the slower reaction is extrapolated to zero time, the change (he,) in the extinction coefficient corresponding to the slower reaction is obtained. It was found that Ar, = 0.33 Act for every wavelength examined. The rate constants of the fast and slow reactions are plotted on a logarithmic scale against pH of the solution at 39.5”C in Fig. 6. The rate constant for the slow reaction shows a minimum at pH 2.2, while that of the fast reaction seems to come at about 3.5. In the pH region of 3.5 to 4.5, the estimation of the two reaction rates could not
BY ULTRAVIOLET
245
SPECTROSCOPY
straightforwardly be made. Here, the first guess was made by drawing a straightline in the log k,-pH space (Fig. 6) from the point corresponding to the slower rate constant at pH 3.0 with the slope of l/l, and by assuming that one of the two rate constants is on this straight line at every pH in the pH 3.5-4.5 region. After such a first guess a small adjustment of the rate constants caused a fit of the experimental kinetic data. Such an estimation shows that, on going from lower pH region to higher, the order of the rate constant values seems to be inverted at pH 3.5. Therefore, it is not proper to call the two reactions “fast” and “slow” ones. Instead, let us discriminate them, by the positions of the minima (pH 2.2 and 3.5) in the log k,-pH curves. Arginine sidechain has two kinds of labile hydrogen atoms, that involved in - C-NH-C group and those involved in the terminal C-NH2 groups. The rate constant with the minimum at pH 3.5 is assigned to the latter hydrogen atoms, because the hydrogendeuterium exchange rate constant of guanidinium ion gives a minimum at pH 3.3 (see Fig. 7). The assignment is further supported by an examination of the hydrogendeuterium exchange reactions of N-methyl-
PH
2
3
4
FIG. 6. The rate constants k. of the hydrogendeuterium exchange reactions of L-arginine plotted on a logarithmic scale against pH of the solution at 39.5% (0) Attributable to the terminal C z NH, groups of the sidechain. (0) Attributable to the C-NH z c group.
246
TAKAHASHI.
NAKANISHI.
guanidinium ion. This shows two exponential decay curves at pH 3.2. The faster and slower rate constants are found to be 1.0 and 0.15 s-l at 39.5”C, and these are close, respectively, to the values, 1.0 and 0.2 s-l of the corresponding rate constants of arginine at pH 2.9. In addition, the extrapolation of the straight line corresponding to the slower reaction to zero time gives a AE, value which is again 0.33 times as great as the Act value obtained by the extrapolating for the faster reaction. The hydrogen exchange rates of arginine was also examined by a 15N-magnetic resonance method (5). The results of the two different methods are in agreement with each other. In Table 1, the catalytic constants, kH+ and koH-, estimated from the pH dependence (see Fig. 6) of the exchange rates are given for arginine at 20°C. The rate constant k, of the hydrogendeuterium exchange reaction assignable to the terminal NH, groups of L-arginine is plotted in Fig. 4, (C) on a logarithmic scale against reciprocal absolute temperature at pH 2.1. From the slope of this Arrhenius plot, the activation energy (AH+) of the reaction is given as 11.9 kcal /mol at pH 2.1. Histidine
In Fig. 8, absorption spectrum of imidazole is shown. On deuteration, a time-dependent change (he) in the molar extinction co-
AND TSUBOI
FIG. 8. Lower: Absorption spectrum of imidaxole in aqueous solution at pH 8. Upper: The amount of AC (difference in the molar extinction coefficient caused by 50% deuteration is given by (x) as a function of the wavelength. The differential coefficient of the absorption curve is also shown by (0) and (O), for comparison.
efficient (E) is found, and its amount at zero time is plotted against wavelength. As may be seen in the figure, this is nearly coincidental with the differential curve of the absorption curve. This fact may be taken as indicating that the deuteration of imidazole causes a blue shift of its absorption profile by about 0.15 nm. Lysbze, Serine, Threonine, and Cysteine
PH
3
4
FIG. 7. The rate constant k, of the hydrogendeuterium exchange reaction of the guanidinium chloride plotted on a logarithmic scale against pH of the solution at 392°C.
None of these give time-dependent absorbance change in the 200- to 280~nm region, when their Hz0 solutions are rapidly mixed with D20. This means that their deuteration rate is higher than 1000 s-l or their deuteration does not cause any appreciable absorbance change in the spectral range exmained.
HYDROGEN-DEUTERIUMEXCHANGEBYULTRAVIOLETSPECTROSCOPY
. \ I.: 01 . :0
247
248 Tryptophan
TAKAHASHI,
NAKANISHI,
AND
TSUBOI
TABLE2
and Tyrosine
Tryptophan has a few absorption bands in the 250- to 300-nm region and tyrosine has an absorption band at 275 nm. By the use of the deuteration effects on those absorptions, we previously established a useful method of tracing the time course of the hydrogen-deuterium exchange reactions in the tryptophan and tyrosine residues in proteins [6,7]. These residues have, however, other absorption bands in the 200- to 230-nm region, too, as shown in Fig. 9. Deuteration of the tryptophan residue causes a time-dependent change not only in the 2% to 300-nm region (6) but also in the 200- to 230-nm region. Therefore, a stoppedflow ultraviolet absorption experiment was newly made at each wavelength in the 200to 230-nm region. The, rate constant obtained from the semilogarithmic kinetic plot was found to be equal to what was previously obtained in the longer wavelength region. The amount of the change (Ae) in molar extinction coefficient (e) at zero time was estimated by an extrapolation, and this is plotted against the wavelength in Fig. 9. This may be useful to estimate the singal level in planning a stopped-flow ultraviolet absorption measurement of a system involving tryptophan residues. For tyrosine, a similar examination was made also in the shorter wavelength region (200-240 nm) and the result is shown also in Fig. 9.
RATE CONSTANTS (k) OF THE DEUTERATION REACTIONS OF
DISCUSSION
One may suspect that the Ae values of the sidechain residues in a protein would be greatly different from those given in Table
Rate constant k/k* (at 20°C pH7Y
Residue Arginine
-NH-
AP 20
NH, -+c
100
40
1 Fast and/or
30
NH, Asparagine Cysteine Glutamine Histidine Lysine Serine Threonine Tryptophane Tyrosine
-NH* -SH -NH2 -NH-NH,+ -OH -OH -NH-OH
AE= 0 0.5
10 Fast and/or AC= 0 Fastand/or AC= 0 Fast and/or AC= 0 1.3 300
n k* = the rate constant for poly-oL-alanine = 4.6 s-l. b be for 509k deuteration, i.e., H -) (H + D)/2 = 33 for hly-m-alanine).
25 30
20 20
(AC
absorption method is applicable to a protein study. The Ae Values-a Comparison of the Mainchain and Sidechain Hydrogens
In Table 2 (last column), the amount of the change (AE) in the molar extinction coefficient (E) caused by the deuteration of each sidechain residue at 206 nm is given. Polypeptides Arginine residue has a greater value of be A detailed description on our hydrogen than that of peptide group (he = 33 for exchange study of poly-D,L-alanine has poly-r>L-alanine), but it has fived exchangebeen made in our previous paper (2). A able hydrogen atoms. As long as the AE similar work has been made of poly-L- value (at 206 nm) per one exchangeable glutamic acid and poly+lysine at various hydrogen atom is concerned, the peptide pHs and at various temperatures. The cata- group is the highest. lytic constants k OH-determined at 20°C are given in Table 1, with the he values ob- Possible Shifts of the Absorption Bands served at 206 nm. in Proteins Let us now examine how the stoppedflow ultraviolet (at 206 nm, for example)
HYDROGEN-DEUTERIUM
EXCHANGE
2, because these values were obtained for free amino acids whereas in a protein the absorption bands are often shifted depending upon the intramolecular environments in which the amino acid residues are located. Nakanishi and Tsuboi (7) found, for example, that the be value is greatest at 285 nm for free L-tyrosine whereas at 290 nm for the tyrosine residues in ribonuclease A. In general, however, the amount of the shift of the absorption band of an aromatic ring (when it is brought from a free amino acid into a protein molecule) is smaller in the 210-nm region than in the 280-nm region (8). Deuteration rates-a Comparison of the Mainchain and Sidechain Hydrogens
In Table 2, the ratio of the rate constant (k) of the deuteration reaction of each sidechain residue over the rate constant (k*) of the peptide group is given. As may be seen here, k’s are much greater than k* for all the sidechain residues except for asparagine, glutamine, and tryptophan. This fact gives a support to an analysis (9) of the slower hydrogen-exchange reactions of proteins on an assumption that they are mostly due to the peptide groups rather than to the sidechain residues. It is true, however, that a certain allowance should be made for a contribution from the asparagine-, glutamine- and/or tryptophan-hydrogens in the analysis. A Possible Error in Tracing the Deuteration Reaction of the Peptide Groups in a Protein by Absorbance Measurement at 206 nm
On the basis of the results of the examination so far described, the contributions of
BY ULTRAVIOLET
SPECTROSCOPY
249
the sidechain residues to the AC values at 206 nm are summarized as follows: (i) Asparagine, glutamine, and tryptophan.
Each one of these residues gives a contribution comparable to that of one peptide group which is exposed to the solvent on the surface of the protein molecule (fast peptide group). (ii) Arginine, histidine, and tyrosine. If these residues in the protein are involved in hydrogen bonds with some other residues, so that their deuteration rates are greatly lowered, they may cause a slight disturbance in examining the fast peptide groups. Otherwise, these should not cause any error in an estimation of the rate of the peptide deuteration. Actual application to the stopped-flow 206-nm spectrophotometry to proteins will be detailed elsewhere, with a quantitative estimation of errors. REFERENCES 1. Englander, J. J., Calhoun, D. B., and Englander, W. (1979) Anal. Biochem. 92, 517-524. 2. Takahashi, T., Nakanishi, M., and Tsuboi, M., (1978)Bull. Chem. Sot. Japan. 51, 1988-1990. 3. Molday, R. S., Englander, S. W., and Kallen, R. G. (1972) Biochemistry 11, 150- 158. 4. Blomberg, F., Maurer, W., and Riiterjans. H. (1976) Proc. Nat. Acad. Sci. USA 73, 14091413. 5. Yavari, I., and Roberts, J. D. (1978) Eiochem. Biophys. Res. Commun. 83, 635440. 6. Nakanishi, M., Nakamura, H., Hirakawa, A. Y., Tsuboi, M., Nagamura, T., and Saijo, Y. (1978) J. Amer. Chem. Sot. 100, 272-276. 7. Nakanishi, M., and Tsuboi, M. (1978) J. Amer. Chem. Sot. 100, 1273-1275. 8. Donovan, J. W. (1%9) Physical Principles and Techniques of Protein Chemistry, part A, pp. lOl- 170, Academic Press, New York. 9. Englander, J. J., and Englander, S. W. (1977) Nature (London) 265,658-659.