Proton magnetic resonance studies of human hemoglobin: Histidine titrations

BIOCHIMICAET BIOPHYSICAACTA

~87

BBA 36O39 PROTON MAGNETIC RESONANCE S T U D I E S OF HUMAN H E M O G L O B I N HISTIDINE TITRATIONS

NORMA J. G R E E N F I E L D AND MYRA N. WILLIAMS

Department of Biophysics and Pharmacology, Merck Institute for Therapeutic Research, Rahway, N.J., 07o65 (U.S.A.) (Received August 24th, 1971)

SUMMARY

The ioo-MHz proton magnetic resonance (PMR) spectra of human hemoglobin in the oxy, deoxy, and met forms are reported from 680 to 930 Hz downfield from external tetramethylsilane. In this region the spectra show 5 to 9 titratable, occasionally overlapping resonances that can be assigned to the C-2 protons of histidines. The peaks with a line width of approximately IO Hz, shift upfield about IOO Hz upon deprotonation of the histidine rings. In oxy- and methemoglobin the observed histidines exhibit similar p K values. Deoxyhemoglobin, when compared to oxyhemoglobin, has several histidine residues which show changes in p K values.

INTRODUCTION

During the past twenty years there has been considerable speculation on the nature of the residues responsible for the difference in the proton titration behavior of oxy- and deoxyhemoglobin. At alkaline p H deoxyhemoglobin binds more hydrogenion than oxyhemoglobin, a phenomenon referred to as the alkaline Bohr effect. Theoretical calculations of the titration curves of individual residues indicate relatively few of the acidic or basic amino acids of hemoglobin are exposed to the solvent and contribute to the proton titration behavior 1-9. These calculations implicate imidazole, carboxyl, and a-amino groups as the functional groups responsible for the alkaline Bohr effect. From analysis of X - r a y crystallographic data, PERUTZ el al. 1° concluded that the imidazole groups of the C-terminal histidines of the fl chains together with the N-terminal amino groups of the a chains were responsible for most of the differences in the alkaline portion of the pH titration curves of oxy- and deoxyhemoglobin. In oxyhemoglobin these groups were suggested to be free while in deoxyhemoglobin they were interacting with the carboxyl groups. In deoxyhemoglobin the a amino groups of each a chain are linked to the C-terminal carboxyl group of the other a Biochim. Biophys. Acta, 257 (1972) 187-197

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N. J. GREENFIELD, M. N. WILLIAMS

chain and the C-terminal imidazole of histidine HC 3 (146) of each fi chain is linked to the carboxyl group of aspartate FG I (94) of the same fi chain. PERUTZ11 subsequently noted that, in oxybemoglobin, histidine H 5 (122 a) is close to the guanidinium group of arginine B 12 (3o fl) and that in deoxyhemoglobin it is close to the carboxyl group of aspartate H 9 (126 a). This change in position would tend to raise its p K in deoxyhemoglobin and lower its p K in oxyhemoglobin. Thus both histidine 146 fl and 122 a could be involved in the alkaline Bohr effect. Before the development of high resolution proton magnetic resonance (PMR), there was no generally applicable method for direct determination of the titration curves of individual amino acid residues within a protein, although some attempts were made to isolate tyrosine contributions by various spectrophotometric techniques (for examples, see ORTUNGg). Now, high resolution PMR permits the direct observation of the titration curves of individual histidine residues by monitoring the chemical shifts of the C-2 protons as a function of pH 1~. Until recently la, this technique had been useful only with proteins of molecular weight less than 2o ooo since the spectral line width in diamagnetic macromolecules is determined primarily by the rotational correlation time of the molecule 14. Thus large macromolecules exhibit slower tumbling and correspondingly broader nuclear resonances. The molecular weight of hemoglobin (64 ooo) would tend to make its associated proton magnetic resonances broad and poorly resolved ~'~. Previous PMR studies on contact shifted resonances15,16 and exchangeable N H resonances of tryptophan 17 in hemoglobin showed protons exhibiting line width of approx. 4 ° Hz. We investigated the possibility that the external titratable histidines would rotate relatively freely compared to the bulk of the polypeptide chain, and thus would exhibit narrower resonances. In this paper we report the histidine titration curves of human hemoglobin in the oxygenated, deoxygenated and oxidized (met) forms. We also report preliminary attemps to identify one of the residues. MATERIALS AND METHODS

Hemoglobin preparation H u m a n hemoglobin was prepared from washed red blood cells drawn the previous day by two procedures. Either the method of CAMERON AND GEORGE18 was used, or the cells were lyzed via sonication, centrifuged and dialysed against o.I M NaC1. Methemoglobin was prepared by oxidation with a 3-fold excess of NaNO 2 followed by dialysis against o.I M NaC1.

Preparation of solutions for P M R spectroscopy Solutions of oxy- and methemoglobin were prepared by dialyzing aliquots of stock solutions against o.I M deuterated phosphate buffers in 99.7% 2H~O of appropriate pH. I f necessary the pH was adjusted with o.I M NaOZH. Oxyhemoglobin was run at heine concentrations ranging from 5 to IO raM. Methemoglobin was run at a heine concentration of 7 raM. Solutions of deoxyhemoglobin were prepared by flushing oxyhemoglobin in o.I M deuterated phosphate buffer with nitrogen and by reducing it with an approximate 2-fold excess of sodium dithionite. Solutions of deoxyhemogloLin were run at a heme concentration of I O - I I raM.

Biochim. Biophys. Acta, 257 (I972) I87-I97

PMR

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S T U D I E S OF H U M A N H E M O G L O B I N

Preparation of hemoglobin cleaved with carboxypeptidase A 6 mg of carboxypeptidase A (Worthington D F P treated) were slurried into 0.25 ml 1% NaHCO 3 and a minimum of o.I M NaOH was added to dissolve the enzyme. The solution was added to IOO ml of hemoglobin (0.5 mM/heme) and the pH was adjusted to 8 with o.I M NaOH. Digestion proceeded for 24 h at 25 °. The release of amino acids was monitored by taking io-ml aliquots, precipitating the protein with dichloroacetic acid and extracting the supernatant with 6 washes of ether to remove the dichloroacetic acid. The aqueous solution was then identified via thinlayer chromatography. The total concentration of amino acids released was determined by ninhydrin analysis as described by ROSEN 19 using histidine as a standard. The reaction appeared to cleave the histidine and tyrosine completely and no significant amounts of other amino acids were noted. The digested hemoglobin was dialyzed against water and concentrated to 1.25 mM in an Amicon ultrafiltration unit (5 ° m l capacity) on a UM-Io Amicon filter at 30 lb/inch 2. The digestion procedure led to formation of some methemoglobin. To regenerate oxyhemoglobin an aliquot of the digested solution was treated with sodium dithionite, immediately put through a Sephadex G-25 M column to remove the dithionite, and reconcentrated by ultrafiltration. The solutions for PMR spectroscopy were prepared as for undigested hemoglobin and spectra were taken in o.I M deuterated phosphate buffer in 99.7% *H20 immediately following ultrafiltration.

pH measurement The p H of hemoglobin solutions was measured with a Radiometer pH meter model 26. The pH and p K values reported are uncorrected for the effect of deuterium ; the p K readings are applicable to water as well as deuterium oxide 2°.

P M R spectroscopy PMR spectra were obtained on a Varian HA-ioo spectrometer at 32 °. Spectra were obtained at a sweep rate of o. 5 Hz/sec over a 25o-Hz range and the spectra were usually averaged for 1-2 h (6-15 sweeps) with a Varian C-Io24 computer of average transients. Wilmad precision bore NMR cells were used with coaxial inserts containing the external standard tetramethylsilane. Spectra are referenced to the ex-ternal tetramethylsilane in Hz or parts per million (ppm), with downfield shifts assigned positive values.

RESULTS AND DISCUSSION

The PMR spectra of oxy-, deoxy- and methemoglobin all show considerable detail in the 7.5 to 9.5 p p m range. Five to nine peaks are resolvable in most of the spectra in the pH range of 6- 9. These resonances move upfield with increasing p H with a total shift of about I.O p p m upon deprotonation. The resonances have a line width at half-maximum of about io Hz. From the chemical shift and its pH dependence, the range of observed p K values, and the line width, we suggest that these resonances m a y be assigned to the C-2 protons of the imidazole ring of external histidines. Typical spectra of the three forms of hemoglobin at p H 5.9 and 7.2 are shown in Figs. I and 2.

Biochim. Biophys. Acta, 257 (1972) I87-197

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N . J . GREENFIELD, M. N. WILLIAMS

IO0-MHz SPECTRA

'

I 930

[ 880

I 830 CHEMICAL SHIFT Hz

I 780

I 730

Fig. I. loo-MHz P M R spectra of oxy-, deoxy- and methemoglobin from 73 ° to 93 ° Hz downfield from external t e t r a m e t h y l s i l a n e ; o.i M deuterated p h o s p h a t e buffer, p H 5.9, 320. Heine concent r a t i o n : oxy, 5 mM, 15 scans; deoxy, lO raM, 5 scans; met, 7 raM, 7 scans.

IO0-MHz SPECTRA

/ W

pH 7.2

I 930

I 880

I 830 CHEMICAL SHIFT Hz

780

I 730

F i g . 2. Ioo-MHz P M R spectra of oxy-, deoxy- and m e t h e m o g l o b i n from 73 ° to 93 ° Hz downfield f r o m external tetramethylsilane; o.i M deuterated p h o s p h a t e buffer, p H 7.2, 32°. Heine concent r a t i o n : oxy, 5 mM, 9 scans; deoxy, IO mM, IO scans; met, 7 mM, 14 scans.

Biochim. Biophys. Acta, 257 (1972) 187-197

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191

OXYHEMOGLOBIN (IOO MHZ) 32" pK VALUES AI

7,3

7 8

2 3 7 3 7 N 880 -l-

8

u_ 3~ 09

u

830

780

I 6

I 7

1 8

I 9

pH

Fig. 3. Chemical shifts of oxyhemoglobin histidine C-2 proton peaks as a function of pH at 320 in o.I M deuterated p h o s p h a t e buffer. The broken curve appears to be missing or broadened in hemoglobin cleaved with carboxypeptidase A.

The change in chemical shift of the C-2 protons with p H of the resolvable histidines of the three forms of hemoglobin, are shown in Figs. 3, 4 and 5- The chemical shifts reported were all obtained at or near the same hemoglobin concentration for each form of hemoglobin, in order to minimize uncertainties arising from the use of an external reference. Resolution of the titration curves is complicated because there are eight to nine overlapping curves in each form. Moreover, the titrating peaks are superimposed on the broad aromatic envelope which consists of of resonances corresponding to protons on internal histidine residues which do not titrate, on non-exchangeable N H groups in the interior of hemoglobin, and on other aromatic residues. Thus, the titration curves depicted in Figs. 3-5 are not a unique fit. The reported curves are the best fit assuming normal titration curves with a pHdependent chemical shift closely appproximating that of free histidine (done under analogous experimental conditions) though altered in position and pK. The estimated accuracy for the p K values is about ± 0.2 p H unit. The p K values were determined by comparing the experimental curves with standard titration curves. Biochim. Biophys. Acta, 257 (i972) 187-i97.

192

N.J.

GREENFIELD,

M. X. W I L L I A M S

MET HEMOGLOBIN (I00 MHZ} 32 =

930

DEOXYHEMOGLOBIN 32 °

pK At 7 3

(100 MHz)

90 ~

Az ~

b

'

.

~

Bz 7 1

B~ 7.3

\-~

B4 79

\\

C r 68

88

"&l o

B

85!

83C Bi

B2

o m z 83(

<2

_o

8 0 .=

780

7BC

73C

~

7

8

9

6

7

e

9

pH F i g . 4' Chemical shifts of methemoglobin histidine C-2 proton peaks as a function of pH a t 3 2o in o . i M deuterated phosphate buffer. F i g . 5. Chemical shifts of deoxyhemoglobin histidine C-2 proton peaks as a function of pH at 32o i n o . I M deuterated phosphate buffer.

Oxyhemoglobin The titration curves for the C-2 protons of the external histidines of oxyhemoglobin are shown in Fig. 3. O x y h e m o g l o b i n shows 5-8 titrating peaks at various pH values. These can be resolved into 9 titration curves. The titration curves have arbitrarily been classed into three groups, A, B and C according to their chemical shifts. Group A has two curves with pK values of A 1 ~--- 7.2 and A 2 = 7.7. Group B has four peaks with pK values of B 1 = 6.8, B e = 7.2, B 3 = 7.3 and B 4 = 7.7. Group C has three curves which collapse to two curves below pH 7. C1 has a pK of 6.3, Ca a p K of 6. 7 and Ca a p K of 6.8. In Group C it is difficult to decide which branch of each C curve above pH 7 corresponds to which branch below pH 7- Mismatching the branches would lead to p K errors of about ~- o.2 p H unit (the p K values are tabulated in Table I).

Biochym. Biophys. Acta,

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STUDIES OF HUMAN HEMOGLOBIN

193

TABLE I THE p t ( VALUES OF RESOLVABLE HISTIDINE RESIDUES OF HEMOGLOBIN DETERMINED BY THE CHANGE IN CHEMICAL SHIFT OF THE C-2 PROTONS WITH p H

Curve

Oxy

Met

Deoxy

A1 A2 B1 B2 Bz B4 C1 C2 Ca

7.3 7.7 6.8 7.2 7.3 7.7 6.3 6.7 6.8

7.3 7 .8 7.0 7.1 7.3 7.9 6.8 6.7

7-3 8.1 8.1 7.2 7-o 7-7 7.1 7.0 6.8

Methemoglobin The titration curves for methemoglobin are shown in Fig. 4. The spectra are somewhat less complicated than those for oxyhemoglobin possibly due to the fact that some of the resonances may be shifted or broadened as a result of interaction with the unpaired electrons associated with the heine and thus do not contribute to the histidine C-2 region. The resonances of methemoglobin are more widely separated and do not exactly correspond with those of oxyhemoglobin. However, when the curves are grouFed in the same way as oxyhemoglobin there are striking similarities. A comparison of the pK values of all three forms is shown in Table I. Curve A 1 of methemoglobin has a pK = 7.3, A2 = 7 .8, B1 = 7.o, B2 = 7.1, Ba -- 7.3, B4 = 7.9, C1 = 6.8 and C2 = 6.7. All these pK values are within o.2 pK unit of corresponding curves of oxyhemoglobin; thus they are within the limit of experimental accuracy. The C1 curve of oxyhemoglobin has no corresponding C curve in methemoglobin. The apparent absence of the curve has several possible explanations. The first is that the absence is real and that oxy- and methemoglobin have different conformations that result in a change in pK for one of the histidine residues. However, such conformational differences are not apparent in X-ray diffraction data n. A second, more likely explanation, is that the curve may simply be obscured. The C-2 proton peaks of histidine in the "C" curves of methemoglobin overlap the main aromatic envelope of the spectra much more than in oxyhemoglobin. A third explanation is that the curve is displaced and broadened via pseudocontact interactions with the paramagnetism of the heine. This explanation requires that the heme area be vicinal to a titrating, freely rotating histidine.

Deoxyhemoglobin The titration curves for the C-2 protons of the external histidines of deoxyhemoglobin are shown in Fig. 5. When the curves are grouped in the same manner as those of oxy- and methemoglobin it is apparent that there are real differences among the forms, although there is considerable overlap of the resonances in the deoxy form. There are three curves with p K values near 8 and the rest are very close to pH 7Curve A 1 is 7.3 as it is in oxy- and methemoglobin but A 2 is 8.1. This is somewhat high compared to 7.7 and 7.8 in oxyhemoglobin and methemoglobin, but is still within experimental error. Tile B group shows the biggest differences in that it has Biochim. Biophys. Mcta, 257 (1972) 1 8 7 - 1 9 7

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N . J . GREENFIELD, M. N. WILLIAMS

lost one curve with a p K close to 7 and gained one curve with a p K close to 8. Thus the p K of B 1 is 8.1, B 2 is 7.2, B 3 is 7.0 and B 4 is 7.7. The C group has three merging curves with a p K near 7 and thus deoxyhemoglobin, like methemoglobin, does not have an external histidine with a "low" pK. The p K ofC 1 is 7.1, C~ is 7.0 and C3 is 6.8.

Hemoglobin carboxypeptidase A It is possible to group the titration curves so that they correspond to one another in the three forms of hemoglobin. One would like to say that the noted changes in p K for certain external histidines between oxy- and deoxyhemoglobin correspond to histidines involved in the alkaline Bohr effect. Unfortunately, there is no evidence that the above indicated correspondence is indeed the correct assignment. We attempted to identify one histidine by cleaving the C-terminal histidine of the fi chain of hemoglobin with carboxypeptidase A, as described b y ANTONINI et al. 21. This procedure cleaves the C-terminal histidine and tyrosine of the fl chain of hemoglobin. When we did this we found that the peak corresponding to the B 1 curve of oxyhemoglobin appeared to be missing in the cleaved hemoglobin as shown in Figs. 6 and 7. However, when we examined the deoxyhemoglobin spectra we could find no clear evidence of a missing peak, although there were slight spectral differences. The resonances overlap, however, and we are unwilling to assign small changes in area and position of the peaks to a missing residue. Spectra of deoxyhemoglobin

,

I

I

930

905

I

[

880 855 CHEMICAL SHIFT Hz

I

I

830

805

Fig. 6. Ioo-MHz N M R spectra of oxyhelnoglobin and oxyhelnoglobin cleaved w i t h c a r b o x y p e p tidase A from 805 to 93 ° Hz downfield froln external t e t r a m e t h y l s i l a n e in o.i M deuterated phosp h a t e buffer p H 5.9, 320. Heine concentration: oxyhemoglobin, IO inM, I scan; oxyhelnoglobin cleaved w i t h c a r b o x y p e p t i d a s e A (CPA), 5 raM, 6 scans. The arrow indicates the peak missing in o x y b e l n o g l o b i n cleaved w i t h c a r b o x y p e p t i d a s e A.

t3iochim. Biopt;ys. Acta, 257 (1972) 187-197

PMR STUDIES OF HUMAN HEMOGLOBIN

I95

IO0-MHz SPECTRA pH 7.2

OXY

I 930

I 905

i 880

t 855

f 830

I 805

CHEMICAL SHIFT Hz

Fig. 7. Ioo-MHz NMR spectra of oxyhemoglobin and oxyhemoglobin cleaved with carboxypeptidase A from 805 to 93 ° Hz downfield from external tetramethylsilane in o.I M deuterated phosphate buffer, p H 7.2, 32°. Heme concentration: oxyhemoglobin, IO mM, 9 scans; oxyhemoglobin cleaved with carboxypeptidase A (CPA), 5 raM, 13 scans. The arrow indicates the peak missing in oxyhemoglobin cleaved with carboxypeptidase A.

cleaved with carboxypeptidase A are shown in Figs. 8 and 9. PERuTzll finds, on the basis of X - r a y diffraction studies, that deoxyhemoglobin has its C-terminal histidine residue in the fl chain linked to both aspartate 94 (of the fl chain) via its imidazole ring and to the epsilon amino group of lysine 4 ° (a chain) via its C-terminal carboxyl. It is possible that the histidine residue is thus so tightly bound to deoxyhemoglobin that the nuclear resonance of its C-2 proton is broadened to 4 ° Hz. In this case the C-terminal histidine of the/5 chain would not give a visible peak in the first place and cleavage with carboxypeptidase A could not cause any visible change in the spectrum of deoxyhemoglobin. An alternative explanation is that the resonance is obscured. The B and C groups of curves of deoxyhemoglobin show more overlap than those of oxy- and methemoglobin in the titration data. I t is possible that one of the curves in these groups is indeed missing in the cleaved homoglobin but that it is obscured by overlapping peaks. The problems alluded to above might be resolved on an instrument of higher resolution. Also, some of the other residues might be assigned by examining hemoglobin mutants. CONCLUSIONS

The histidine titration curves of human oxy-, deoxy-, and methemoglobin have been obtained by following the change in chemical shift of the C-2 protons with Biochim. Biophys. Acta, 257 (1972) I87-I97

196

N . J . GREENFIELD, M. N. \VILLIAM,~

IO0-MHz

SPECTRA

PHIYIox] / ~ ,

I

I

I

I

[

905

880

855

830

805

Fig. 8. i o o - M H z N M R s p e c t r a of d e o x y h e m o g l o b i n a n d d e o x y h e m o g l o b i n cleaved w i t h carb o x y p e p t i d a s e A from 805 to 93 ° Hz downfield f r o m e x t e r n a l t e t r a m e t h y l s i l a n e in o.i M d e u t e r a t e d p h o s p h a t e buffer, p H 5.9, 320. H e i n e c o n c e n t r a t i o n : d e o x y h e m o g l o b i n , IO mM, 5 s c a n s ; d e o x y h e m o g l o b i n cleaved w i t h c a r b o x y p e p t i d a s e A, io raM, IO scans. IO0-MHz SPECTRA

pH 6.7

DEOXY CPA •

I

I

I

I

I

905

880

855

830

805

Fig. 9- i o o - M H z N M R s p e c t r a of d e o x y h e m o g l o b i n a n d d e o x y h e m o g l o b i n cleaved w i t h c a r b o x y p e p t i d a s e A f r o m 805 to 93 ° Hz downfield f r o m e x t e r n a l t e t r a m e t h y l s i l a n e in o.i M d e u t e r a t e d phosp h a t e buffer, p H 6.7, 32°. Heine c o n c e n t r a t i o n : d e o x y h e m o g l o b i n , io raM, 24 scans; d e o x y h e m o globin cleaved with c a r b o x y p e p t i d a s e A, io raM, io scans.

Biochim. Biophys. dcta, 257 (I972) 187-197

PMR STUDIES OF HUMAN HEMOGLOBIN

197

pH via IOO MHz NMR spectroscopy. The three forms show eight or nine titration curves per dimer in reasonable agreement with several theoretical studiesS,L 8. The C-terminal histidine of the fl chain of oxyhemoglobin appears to have a pK of approx. 6.8 ± 0.2 at 32°. The histidines of met- and oxyhemoglobin appear to have homologous pK values although the PMR spectra of the aromatic region of the two forms differ greatly. Deoxyhemoglobin appears to have gained one histidine residue with pK near 8 and lost a histidine residue of pK near 6.3, when compared to the other forms. Absolute assignment of these residues in human hemoglobin remains to be accomplished. Since certain histidine resonances are resolvable by PMR, they may serve as a useful probe of the interaction of hemoglobin in solution, (e.g. interaction with diphosphoglycerate). Previously, such studies have mainly been limited to contact shifted15, 2~ and exchangeable protons TM. ACKNOWLEDGEMENT

We wish to thank Dr. D. Meadows and Dr. O. Jardetzky for suggesting this problem and Dr. R. G. Shulman and Dr. M. Poe for helpful discussions. REFERENCES 1 2 3 4 5 6 7 8 9 io Ii 12 13 14 15 16 17 18 19 20 21 22

WYMAN, J. Biol. Chem., I27 (I939) 581. B. CONANT,Harvey Lect., 28 (i932) i59. D. CORYELL AND L. PAULIi','G, J. Biol. Chem., 132 (194 o) 769. ANTONINI, J. WYMAN, M. BRUNORI, C. FRONTICELLI, E. B u c c I AND A. ROSSI--FANELLI, J. Biol. Chem., 240 (1965) lO96. C. TANFORD AND Y. NOZAKI, J . Biol. Chem., 241 (1966) 2832. J. t~ILMARTIN AND L. ROSSI-BERNARDI, Nature, 222 (1969) 1243. W. H. ORTUNG, J. Am. Chem. Soe., 91 (1969) 162. S. H. DE ]3RUIN, L. H. M. JANSSEN AND G. A. J. VAN Os, Biochim. Biophys. Acta, 188 (1969) 207 . W. H. ORTUNG, Biochemistry, 9 (197 o) 2394. M. FERUTZ, H. MUIRHEAD, L. MAZZARELLA, R. A. CROWTHER, J. GREEN AND J. V. KILMARTIN, Nature, 222 (1969) 1241. M. PERUTZ, Nature, 228 (197 o) 734. D. H. MEADOWS, L. J. MARKLEY, J. S. COHEN AND O. JARDETZKY, Proc. Natl. Acad. Sci U.S., 58 (1967) 13o7. R. W. KING AND G. C. }(. ReBERTS, Biochemistry, io (1971 ) 558 . A. ABRAGAM, The Principles of Nuclear Magnetism, Oxford University Press, London, 1961. I4~. WUTHRICH, ]:{. G. SHULMAN AND T. YAMANE, Proe. Natl. Acad. Sci. U.S., 61 (1968) 1199. D. G. DAVIS, N. L. MOCK, V. R. LAMAN AND C. HO, J. Mol. Biol., 4 ° (1969) 311. D. J. PATEL, L. I4~AMPA, R. G. SHULMAN, T. YAMANE AND M. FUJIWARA, Biochem. Biophys. Res. Commun., 4 ° (197 ° ) 1224 . ]3. F. CAMERON AND P. GEORGE, Bioehim. Biophys. Acta, 194 (1969) 16. H. RosEN, Arch. Biochem. Biophys., 67 (1957) IO. G. C. K. ROBERTS, D. H. MEADOWS AND O. JARDETZKY, J. Am. Chem. Soc., 9 ° (1968) lO42. E. ANTONINI, J. WYMAN, R. ZITO, A. ROSSI-FANELLI AND A. CAPUTO, J. Biol. Chem., 236 (1961) 60. R. G. SHULMAN, S. OGAM,TA, I~. VXTUTHRICH, T. YAMANE, J. PEISACH AND ~V'. BLUMBERG, Science, 165 (1969) 251. J. J. C. E.

Biochim. Biophys. Acta, 257 (1972) I 8 7 - I 9 7