Volume 12, number 2

MOLECULAR • CELLULAR BIOCHEMISTRY

August 30, 1976

KINETICS OF HYDROGEN-DEUTERIUM EXCHANGE OF TRYPTOPHAN AND TRYPTOPHAN PEPTIDES IN DEUTERO-TRIFLUOROACETIC ACID USING PROTON MAGNETIC RESONANCE SPECTROSCOPY* Raymond S. N O R T O N ? and J. Howard B R A D B U R Y ~

Chemistry Department, Australian National University, Canberra, A.C.T., Australia (Received February 27, 1976)

Summary The methods which have been used for the observation and assignment of resonances in the NMR spectra of proteins are reviewed. One such method, the selective deuteration of the aromatic protons of tryptophyl residues, is studied by NMR spectroscopy in model compounds in this paper, and in proteins in the following paper. On the basis of a reassignment of the PMR spectrum of the aromatic protons of Ltryptophan, the relative rates of H - D exchange in deutero-trifluoroacetic acid (d-TFA) are H2 > H-5 > H-6 > H - 4 - - H-7. The energies of activation for the first order exchange of both the H-2 and H-5 protons is 12 k.cal.mol -~. The rate constant for exchange of the H-2 protons of tryptophyl residues in peptides is much greater than in the amino acid itself and 5-10 times that for exchange of the H-5 protons. This suggests that the method can be used to label tryptophyl residues in proteins rapidly and specifically.

Introduction High resolution nuclear magnetic resonance (NMR) studies of native proteins in solution are capable of providing detailed information about * An invited article. ? Present address: Chemistry Department, Indiana University, Bloomington, Indiana 47401, U.S.A. :~To whom all correspondence should be directed.

protein structure and function when it is possible to observe resolved resonances from individual nuclei, and assign these resonances to particular nuclei in the protein. In the proton magnetic resonance (PMR) spectra of proteins it is possible to observe individual resonances from a number of proton types in favourable cases. These include the H-2 and H-4 resonances of histidine 1-4, the aromatic resonances of tyrosine 5-9, tryptophan 7'1° and in some cases phenylalanine, the N - H resonances of tryptophan 11'12, histidine a3-~8, arginine ~9 and some slowly exchanging amide groups 12, the methyl resonances of all the aliphatic amino acids a° including methionine 1°'2°'21, a - C H resonances of N-terminal amino acids 1°, the E-CH2 resonance of lysine z2'23, and various resonances which are shifted by interactions with ring currents from aromatic amino acid side chains 24-26 or with paramagnetic metal ions 27. The resolution in ~3C NMR spectra of proteins is superior to that observed in P M R spectra, because all ~3C resonances are converted to singlets as a result of proton decoupling, and because the range of a3C chemical shifts is considerably greater than that of protons 28. The region of aromatic carbons 29-37, provides the greatest potential for observation of resolved single carbon resonances, because the quaternary carbons yield sharp resonances which cover a large range of chemical shifts 35-37. The upfield region of the spectrum, which contains resonances from aliphatic side chain carbons, may yield single carbon resonances in spectra recorded at high magnetic field strength.

Dr. W. Junk b.v. Publishers - The Hague, The Netherlands

103

However, 13C N M R studies of native proteins suffer from the disadvantage that the natural sensitivity of the 13C nucleus is considerably less than that of protons. Therefore, in order to obtain spectra with sufficient signal-to-noise ratios to permit the detailed study of single carbon sites, it is necessary to employ long accumulation times and large sample tubes. In order to assist in the observation and identification o f resonances, in both PMR and 13C NMR spectra of proteins, a number of methods have been developed. These include (1) selective decoupling 6'7'37, (2) chemical shift changes on titration of residues ~ 10,13,15,18,30 32,37, (3) chemical reactions such as H - D exchange3,4,38 41, methylation of lysine 22"23'42'43, reaction of SCH3 methionine 2°'44'4s, (4) synthesis of selectively deuterated proteins 46-48 or proteins selectively enriched with 13C31'49, (5) difference spectroscopy 4'1°'12"31'37'42'43'5°'51, (6) use of differences in T1 values 36'37, special pulse sequences s2'53 and nuclear Overhauser effects 54. The assignment of a particular resonance to a specific nucleus in the protein is a matter of importance and also considerable difficulty. The classic example of the difficulties involved concerns the assignment of the four histidine H-2 resonances of ribonuclease-A 3,4,38,ss-s7. Two of the four resonances were correctly assigned to residues 48 and 105 at the first attempP s, but the other two resonances have only now been correctly assigned 3"56"57eight years later. The importance of a correct assignment in this case need hardly be emphasised as the two resonances concerned arise from the two histidine residues involved in the active site of the enzyme and inferences have been made about its mode of action s8, based on the incorrect assignment. If there is more than one residue of a particular type present in a protein molecule, then the assignment of the resonances derived from them has been made by chemical procedures such as H - D exchange 3"11"38'39'41'55'57, differential rates of exchange of NH protons with water u, selective chemical modification 37'51 and selective perturbation of residues by binding of ligands 1°'5°. Alternative procedures rely on the knowledge of the three dimensional structure of the molecule obtained from X-ray crystallography. On the basis of the known distances between nuclei and other groups in 104

the molecule that cause shifting or broadening of resonances (aromatic residues 1°'24-26 paramagnetic metal ions 1°J2'27"37'42 or spin labels 59"6°) it is possible to calculate the extent of the effect and hence correlate the shifted or broadened resonance with a particular nucleus in the molecule. It is advisable to use more than one method of assignment for each resonance whenever this is possible. We have been involved with the development of methods,for the observation of histidine a,4,21,38,39,51,57, tyrosine 34, lysine 23,42,43, methionine 2°'21'44, tryptophan 33'41 and arginine 19 residues in proteins and for the assignment of resonances to particular nuclei 38"42"51'57'61. One technique which we have found to be particularly useful is the selective deuteration of specific residues (histidine 3m39,51,s7, tyrosine 4°, tryptophan 41) of a protein and the use of PMR difference spectroscopy to observe the amount of deuteration. In this and the following paper 41 we report our studies on the deuteration of tryptophan residues in deutero-trifluoroacetic acid (d-TFA). The deuteration of tryptophan in d-TFA was reported by BAK et al. 62, and the reaction has been employed to label specifically tryptophan residues in glucagon 63"64 and various proteins 65. The kinetics of the reaction have not been examined in detail, but it was concluded 62 that C-2 protons exchange most rapidly followed by C-6 protons, while exchange of the C-4, C-5, and C-7 protons is much slower (the numbering system is shown in I). In this paper, we describe

H

H

H H

H

(I)

the kinetics of deuteration in the free amino acid and in peptides and in the following paper 41, the application of the method to the deuteration of proteins. A preliminary report of some of these results has been presented 66. Materials and Methods

All amino acids, amino acid derivatives and small peptides were commercial products and were used without further purification. Their purity was checked by PMR spectroscopy. Melittin from bee venom was obtained from Nutritional Biochemicals. All samples were lyophilized from D 2 0 at least once before use. D e u t e r o - T F A ( > 98% deuterated, spectroscopic grade) and dz-formic acid ( > 99% deuterated, spectroscopic grade) were obtained from Merck. The d-TFA was supplied in 1 ml ampoules and was used immediately after the ampoules were opened. The water content of the acid is specified to be no more than 0.2%. D20 ( > 99.8%) was supplied by the Australian Atomic Energy Commission. PMR spectra were obtained at 100 MHz using a Jeol J N M - M H - 1 0 0 spectrometer, and at 220 MHz using a Varian H R - 2 2 0 instrument. Both spectrometers were operated in the continuous wave mode. Probe temperatures were measured from the chemical shifts of the resonances of methanol and/or ethylene glycol, both of which were dried over Molecular Sieve Type 4A before use. Quoted temperatures are accurate to +1 °C. ~3C NMR spectra were obtained as described previously 33. Rate constants were measured from the timedependent changes in heights or areas of proton resonances. Heights were always corrected for any changes in instrumental resolution during the course of an experiment. First order rate constants were calculated from the slopes of plots of logao (area or height) against time. G o o d straight lines were obtained in all cases. The first order rate constant (kl) for exchange of H-5 could also be determined from the rate of emergence of the H-4 singlet (see below) using the standard type of equation, viz. (h~-ho~ In \ h - ~ - h t / = kit, where ho and h~ are the initial and final heights respectively, of the H-4 singlet, and ht is the

height at any time t. Since the accurate determination of ho and h= is difficult in this case (see below), we have used the method of GUGGENHEIM 67 to analyse the data. Values of ht and ht+At are calculated, where At is a constant time interval, usually about one half the reaction time, and a plot of loglo (ht+At--ht) against t gives a straight line of slope - k l / 2 . 3 0 3 . Results

(1) Assignment of the aromatic proton resonances

of L- tryptophan The 220 MHz PMR spectrum of L-tryptophan in d - T F A is shown in Figure 1. Figure 1A shows some evidence of H - D exchange, but L-tryptophan in H - T F A (where there is no exchange) gives a spectrum (not shown) which contains a broad resonance from the a-amino group, that overlaps the upfield part of the spectrum. The assignment of the resonances in Figure 1 is different from that given earlier 62 and is based on 13C NMR evidence. In the assignment of the 13C NMR spectrum given previously 33, the C-7 ~3C resonance was assigned quite unequivocally on the basis of model compound studies. Since irradiation of the upfield doublet in the PMR spectrum in 10% and 100% T F A caused decouplihg of the C-7 resonance in the ~3C N M R spectrum, we assigned the upfield doublet to H-7. This was confirmed by a second experiment in which the downfield doublet was irradiated. This caused decoupling of a 13C resonance in the C-2/C4/C-5/C-6 region, thus establishing the C-4 assignment as well as verifying that the downfield P M R doublet corresponds to H-4. The assignment of the two doublets in the PMR spectrum is supported by an examination of the PMR spectra of 4-methyl and 6-methyl substituted tryptophan in H - T F A . Furthermore, it is consistent with previous assignments in D2 O68'69. Since the downfield doublet is converted to a singlet as a result of exchange of the upfield triplet (see Fig. 1), we assign the latter to H-5. The assignment at 220 MHz is, therefore, H-4 downfield doublet, H-7 upfield doublet, H-6 downfield triplet, H-2 singlet, H-5 upfield triplet. (2) Exchange of L-tryptophan in d-TFA The effect of H - D exchange in d - T F A on the 105

H-2 H-7

H-d

H-5 I-6

J

I I

11 mi

B 35min.~

22hr I

I

1

I

7.6

7.4

7.2

7.0

ppm

f r o m TMS

Fig. 1. PMR spectra of L-Tryptophan in d-TFA at 220 MHz and 18 °C. Each spectrum is a single scan recorded at various times after mixing. The amplitudes of A, B and C vary slightly.

220 MHz spectrum of tryptophan is shown in Figure 1. Even after 11 rain at 18 °C, significant H-2 exchange has taken place, as shown by area measurements. Exchange of this proton does not affect the other aromatic resonances. The H-5 proton exchanges more slowly, and as the area of the H-5 triplet decreases, the H-4 doublet is converted to a singlet (barely visible in Figure 1A) and the H-6 "triplet" is converted to a doublet (split only by H-7). The 106

upfield half of the emerging H-6 doublet is obscured by the H-2 singlet at 220 MHz, but they are separate at 270 MHz (spectra not shown). These changes go almost to completion before H-4, H-6 and H-7 exchange becomes evident, but eventually all 5 indole protons exchange (Fig. 1C). Complete exchange is not possible in one exchange cycle because of the accumulation in the solvent of protons from the solute. It is possible to determine rate constants from area measurements at 220 and 270 MHz, but not at 100 MHz. Two corrections are necessary. The area of H-2 must be corrected for the upfield half of the emerging H-6 doublet at 220 MHz, and the areas of H-5 and H-6 must be corrected for the broad underlying a-NH~resonance which can be seen in Figure 1C. Rather than attempting to estimate an arbitrary value for the area of the a - N H ~ peak, the Guggenheim method 67 was used, in which loglo(areat-areat+~t) v s time was plotted. The rate constants for H-5 and H-6 exchange w e r e calculated in this way. No correction was possible for the small increase in area of the a-NH~peak with time as protons from the solute accumulated in the solvent and exchanged onto the amino group. The first order rate constant for H-2 exchange shown in Table 1, is more than twice that for H-5 exchange. The rates of H-4, H-6 and H-7 exchange are almost an order of magnitude lower than that of H-5 exchange, with H-6 being slightly faster than H-4 and H-7. Since access to the 220 MHz spectrometer was limited, most of the kinetic data have been obtained at 100 MHz where the H-2, H-5 and H-6 peak areas cannot be measured accurately because of peak overlap. The H-2 exchange rate at 100 MHz was determined, therefore, by measuring the height of the H-2 singlet above the H - 5 / H - 6 envelope and plotting loglo(htht+At) against time. This eliminates the need to choose a baseline such that h~ = 0. Usually two different baselines were chosen, with reasonable agreement being obtained between the two calculated rate constants. At 100 MHz the rate of H-5 exchange could be determined only from the height of the H-4 singlet. Errors in the quoted values of rate constants are 10-20%. The effect of temperature on the H-2 and H-5 exchange rates is shown in Table 2 and

Table

1

• , 100 MHz oo 2 2 0 ,,

-0.8

Rate constants L-tryptophan

for deuteration of in d-TFA at 18°C

-\ -1.2

Indole

proton

k 1 (min - 1 )

H-2

5.4

H-5

2.6

x 10 2 * A

"T E: "~ -1.6

2.4 ** H-4

0.25

H-6

0.46

H-7

0.29

*

Obtained from area measurements in 220 MHz PMR spectra unless stated otherwise. ** Calculated from the height of the H-4 singlet using the Guggenheim method.b7 Figure 2. The rate constants for H - 2 and H-5 exchange determined at 220 M H z agree within experimental error with the data at 100 MHz, indicating that the latter values are acceptable, despite the limitations imposed by peak overlap.

Table 2 Effect of temperature on exchange of L-tryptophan in d-TFA*

Temperature (O°C)

k I (H-2) min-I x I02

kl (H-5) min-I x 102

0

l.O

0.44

15

6.8

2.5

23

9.7

3.0

26

8.8

3.5

27

8.7

3.4

31

9.7

4.9

35 * All

14

4.7

rate constants were determined from I00 MHz PHR spectra.

H-2 H-..=

O Gr) O m_2.0

-2.4

I

3.2

1

I

I

3.4

I

3.6

I

3.8

1 T (°K) ' 1 0 3 Fig. 2. Arrhenius plots for H-D exchange of H-2 and H-5 protons of L-tryptophan in d-TFA, using rate constants from Tables 1 (open symbols) and 2 (closed symbols). From Figure 2 the energies of activation are found to be 12 k.cal.mo1-1 for both H - 2 and H-5 exchange. Since these values are the same within experimental error, it is evident that t e m p e r a t u r e variation is not a suitable means of altering the ratio k l ( H - 2 ) / k l ( H - 5 ) , in favour of H - 2 exchange. The effect of addition of D 2 0 on the exchange of H - 2 and H-5 is shown in Table 3. Quite large decreases occur in the rates of exchange of both protons, with the effect on H - 2 being greater than that on H-5, such that in 89.6% d - T F A the rates of exchange of H - 2 and H-5 are equal. (3) Exchange of peptides in d-TFA The rates of H-2 exchange of glycyl-Ltryptophan and glycyl-L-tryptophyl-glycine in 100% d - T F A are too fast to measure by conventional continuous wave P M R spectroscopy, even at 0 °C. The rate constants obtained for H-5 exchange at 0 °C are 0.028 and 0.023 min -1 for the dipeptide and tripeptide, 107

Table 3

Table

Effect of D20 and CCI 4 on exchange of L-tryptophan in d-TFA at 26°C

d-TFA

(% v/v)* I00

D20 or CCl 4

k I (H-2) x lO 2

(% v/v)*

min-1

4

E x c h a n g e of t r y p t o p h a n - c o n t a i n i n g p e p t i d e s in 89.6% d - T F A at O°C *

k I (H-5) x lO 2

min-1

Peptide

k I (H-2) x 102 min -I

k I (H-5) x lO 2 min-l

0

8.8

3.5

96.0

6.8 D20

4.5

2.4

L-Trp

O. 14

92.9

I0.4 D20

3.7

1.9

N-acetyl -L-Trp

2.5

89.6

14.3 D20

2.4

2.5

Gly-L-Trp

3.2

0.5

89.6

14.3 D20

Gly-L-Trp-Gly

1 .8

0.21

L-AI a-L-Tr p

3.0

O. 37

L-Leu-L-Trp

3.2

0.32

0.14'*

0.14'*

95.4

3.9 CCI4

6.8

1.6

86.5

11.8 CC14

2.9

1.5

Volume in ml of one component per lO0 ml of mixture. Because of an appreciable volume change on mixing the two components, the values in columns I and 2 do not sum to I00%. ** Determined at 0°C.

O. 14 **

*

respectively. The addition of a small amount of D20 was chosen as the means for reducing the H-2 exchange rate to a conveniently measurable value. This also had the advantage of removing the broadening of resonances sometimes observed with peptides in 100% TFA. The extent of broadening was variable and depended on the type of compound (some tryptophyl peptides gave broadening but phenylalanyl and tyrosyl peptides gave no broadening in 100% H - T F A of d-TFA) and the solvent. The rate constants obtained are presented in Table 4 and the important features are summarized as follows: (a) replacement of the a-amino group of L-tryptophan by an amide bond greatly increases the rate of exchange, (b) replacement of the a-carboxyl group of L-tryptophan by a peptide bond slows down exchange, (c) a neighbouring aromatic amino acid reduces the exchange rate but a neighbouring bulky aliphatic side chain has little effect, (d) a neighbouring positively charged side chain reduces the exchange rate. The exchange of melittin, a naturally occurring polypeptide containing 26 amino acids, has also been examined. Melittin gives quite a sharp spectrum in 100% H-TFA but its spectrum in 89.6% d-TFA at 0 °C is very broad. Nevertheless, it was possible to follow the exchange of the C-2 proton of its single tryptophan residue and a rate constant of 0.72 x 10 -2 min -1 was found at 0 °C. This is less than half that of 108

L-TrPA-L-TrPB

A 0.I0

***

B 2.0

L-Phe-L-Trp

":~**

I .8

L-Tyr-L-Trp

0.28 ***

0.27

L-Hi s-L-Trp

l.l

0.14

L-Arg-L-Trp

1 .7

0.20

L-Lys-L-Trp

2.0

0.40

L-Lys-L-Trp-L-Lys

0.98

0.46

*

All rate c o n s t a n t s w e r e d e t e r m i n e d from spectra recorded at I00 MHz. ** H-4 and H-7 r e s o n a n c e overlap. *** R e s o n a n c e o v e r l a p s w i t h other a r o m a t i c resonances.

glycyl-L-tryptophyl-glycine but only slightly less than that for L-lysyl-L-tryptophyl-L-lysine (see Table 4). Discussion

Isotopic exchange at the 2, 4, 5, 6 and 7 positions of the indole ring of tryptophan probably occurs by addition of a deuteron to the uncharged ring, followed by loss of a proton from the same position (see for example, ref. 70). The rate-limiting step is assumed to be the addition of a deuteron to the ring, since the charged intermediates are unstable and rapidly lose a proton (leading to exchange) or a deuteron (to regenerate the original species). Stable protonation of the indole ring 71-73 can be ruled out from an analysis of the PMR spectra of L-tryptophan and tryptophan-containing peptides in TFA 74. The faster exchange at the 2-position compared with positions 4-7 (Table 1) is in accord with theoretical predictions of the

reactivity of the indole ring to electrophilic substitution7°; the relative reactivities of positions 4 to 7 vary with the electrophile. Replacement of the a-NH~- group of Ltryptophan by an amide bond greatly increases the rates of H-2 and H-5 exchange. At first sight this can be explained by removal of the positive charge, which hinders the approach of a positively charged ion to the indole ring and destabilizes the positively charged ring in Ltryptophan. However, the situation is complicated because the amide bonds of small amides and peptides are charged in strong acid 75-82, although the extent of charging of polypeptides is still a matter of controversy83'84. A positive charge on a peptide bond would be delocalised over the oxygen and the nitrogen atoms (the O-protonated form has been shown to predominate over the N-protonated form) 74-76 and hence its affect in decreasing the rate of reaction would be less than that of the a-NH~. Some support for charging of the peptide bond comes from the fact that the indole rings in the tryptophyl peptides are not charged in TFA, as shown by their PMR spectra (since charging at the 3-position) 7°-72 would be detected by the presence of a peak corresponding to the 3-proton and by additional splitting of the /3-CH2 resonance, neither of which are observed). On the other hand, 3-methyl indole, indole-3-acetic acid, and 4-(3-indolyl) butyric acid, all of which lack a positive charge on the substituent at the 3-position, acquire a positive charge at the 3-position in TFA, and subsequently dimerize 85, as shown by their PMR spectra TM. The replacement of a carboxyl group (almost certainly uncharged in d-TFA) by a positively charged peptide group causes a reduction in the rate of exchange (compare Gly-L-Trp and GlyL-Trp-Gly in Table 4). The same effect is observed for the rate of H-2 exchange in 89.6% d-TFA at 26 °C of L-Trp and L-Trp-Gly in which the rate constants are 0.024 rain -1 and 0.019 min -1 respectively. The presence of a nearby positively charged side chain also decreases the rate of exchange (see Table 4) with histidine being more effective than arginine and lysine. The order of effectiveness here is probably related to the proximity of the charged group to the tryptophan ring and also (in the case of histidine) to the fact that the rate is

reduced by the presence of a nearby aromatic residue. Since a bulky aliphatic substituent has no effect on the exchange rate, it seems likely that an adjacent aromatic ring decreases the rate of exchange (see Table 4) by a partial "stacking'of the two rings, which could hinder the attack of the reagent on one side of the indole ring. The single tryptophan residue of melittin exchanges slightly more slowly than that of L-Lys-L-Trp-L-Lys but not as slowly as L-tryptophan itself. This slow rate of exchange may be due to the occurrence of four positively charged side chains in close proximity in the sequence to the tryptophan residue, i.e. -SerTrp-Ile-Lys-Arg-Lys-Arg-86 The rate of exchange of L-tryptophan is reduced considerably by the addition of D20 or CCl4 to the d-TFA (see Table 3). The same effect is observed with tryptophyl peptides, since we find that the half times for H-2 exchange of Gly-L-Trp and Gly-L-Trp-Gly in d-TFA at 0 °C are less than 1.0 and 1.6 min respectively, whereas in 89.6% d-TFA (Table 4) the corresponding values are 22 and 39 rain respectively. The addition of carbon tetrachloride or D20 to the d-TFA causes reductions in rates which far exceed that expected simply on the basis of the reduction of the concentration of d-TFA. Carbon tetrachloride can probably be considered as an inert diluent, the effect of which is to reduce the concentration of deuterons solvated by TFA. The latter is probably the active reagent in the rate determining step of the reaction. On addition of D20 to d-TFA, reaction (1) occurs to an appreciable degree, and this causes a considerable reduction of the rate of exchange, which indicates that CF3COOD+D20 ~ C F 3 C O O - + D 3 0 + (1) D 3 0 + is not as active in promoting H-D

exchange as CF3COOD~ and other ions of this type. At any particular concentration of d-TFA the relative effectiveness of CC14 and D20 in reducing the rate of exchange of the H-2 proton is different from that of the H-5 proton (see Table 3). This could result if the addition of f E E 4 to d-TFA favoured a particular conformation of L-tryptophan in which H-2 exchange is facile, whereas addition of D20 to d-TFA favours a conformation in which H-5 exchange is slightly preferred. 109

In 1 0 0 % d - T F A the r a t e of H - 2 e x c h a n g e of L - t r y p t o p h a n is a b o u t twice the r a t e of H - 5 e x c h a n g e ( T a b l e 1) w h e r e a s on a d d i t i o n of 14.3% D 2 0 t h e rates of e x c h a n g e a r e e q u a l i s e d ( T a b l e 3). This c o u l d b e d u e to a c o n f o r m a t i o n a l c h a n g e which brings the s - a m i n o g r o u p closer to t h e 2 - p o s i t i o n in the p r e s e n c e of D 2 0 . It is also i n t e r e s t i n g to n o t e t h a t the rates of H - 2 and H - 5 e x c h a n g e in 8 9 . 6 % d - T F A , 14.3% D 2 0 for L - t r y p t o p h a n are equal, w h e r e a s in p e p t i d e s of the t y p e X - L - T r p in the s a m e solvent, the r a t e of H - 2 e x c h a n g e is 5 - 1 0 t i m e s faster t h a n t h a t of H - 5 e x c h a n g e . W e h a v e a l r e a d y n o t e d t h a t the positive charge on the p e p t i d e g r o u p a p p e a r s to be less effective in r e d u c i n g t h e r a t e of r e a c t i o n t h a n t h a t on the a m i n o g r o u p a n d this m a y e x p l a i n the o b s e r v e d differences. In a d d i t i o n , the p o s h i v e c h a r g e on t h e p e p t i d e g r o u p m a y be r e l a t i v e l y f u r t h e r f r o m the 2 - p o s i t i o n a n d closer to t h e 5 - p o s i t i o n t h a n is the c h a r g e on the s - a m i n o group. In conclusion, t h e m u c h m o r e r a p i d e x c h a n g e of the a r o m a t i c p r o t o n s of t r y p t o p h y l r e s i d u e s in p e p t i d e s t h a n in L - t r y p t o p h a n itself m a k e s it p o s s i b l e to e x c h a n g e t h e t r y p t o p h a n r e s i d u e s in p r o t e i n s with o n l y v e r y s h o r t e x p o s u r e to d - T F A . F u r t h e r m o r e , b e c a u s e the r a t e of H - 2 e x c h a n g e is c o n s i d e r a b l y faster t h a n t h a t o.f H - 5 e x c h a n g e in p e p t i d e s , it s h o u l d be p o s s i b l e to e x c h a n g e the t r y p t o p h a n C-2 p r o t o n s of a protein without much interference from H-5 e x c h a n g e . T h e a p p l i c a t i o n of this e x c h a n g e p r o c e d u r e to p r o t e i n s will b e d e s c r i b e d in the following paper.

Acknowledgments W e wish to t h a n k D r . M. C. M c I v o R for access to the Science R e s e a r c h C o u n c i l 220 M H z P M R s p e c t r o m e t e r w h e n it was l o c a t e d at Runcorn, Cheshire and the Australian Res e a r c h G r a n t s C o m m i t t e e for financial s u p p o r t of the p r o j e c t .

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