THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 237, No. 6, June 1962

Printed in U.S.A

Nuclear Magnetic Resonance Studies of Proteins

ARTHUR KOWALSKY

From the Department of Physiological Chemistry, University of Minnesota, Minneapolis 14, Minnesota

(Received for publication, July 24, 1961)

Although nuclear magnetic resonance has shown itself to be a powerful tool in the investigation of the structure and behavior of small molecular weight compounds, little work has as yet been done to explore its use in the investigation of protein structure. The technique, which allows the investigator literally to focus his attention on a particular structural segment, seems to offer an entirely new approach to the study of proteins. It is not to be expected that the same detailed and precise characterization typical of the small molecular weight compounds should be ob- tained. But it should be possible to gain information, if only in a very gross sense, about molecular motion and protein con- formation which is not available through any of the existing methods.

The first proton resonance spectrum of a protein, ribonuclease, was published by Saunders, Wishnia, and Kirkwood (1) in 1957. Jardetzky and Jardetzky (2, 3) have reported on the spectra of amino acids and have interpreted the ribonuclease spectrum on the basis of the amino acid composition. Bovey and Tiers (4) have also examined amino acids, although in trifluoroacetic acid solution, and Bovey, Tiers, and Filipovich (5) have investigated the spectra of high polymers, including proteins, mostly in tri- fluoroacetic acid but also in aqueous solution. Saunders and Wishnia (6) have also reported limited studies on the proton magnetic resonance of proteins in aqueous solution.

The work reported here is a survey of the proton magnetic resonance spectra of a number of proteins under different con- ditions and of the possibilities that appear to be inherent in the technique. A preliminary report of this work has appeared (7).

EXPERIMENTAL PROCEDURE

Materials and Methods

Ribonuclease was obtained from several sources. The major source was Armour and Company; others were Pentex, Inc. and the Sigma Chemical Company. No important difference was noted in the behavior of the various products. Three times crystallized ovalbumin, crystalline bovine serum albumin, three times crystallized pepsin, pepsinogen, and three times crystal- lized P-lactoglobulin were obtained from Pentex, Inc. Cyto- chrome c from horse heart, type III, was obtained from Sigma Chemical Company. Chymotrypsin was a three times crystal- lized preparation of the Worthington Biochemical Corporation. Insulin was obtained through a generous gift of Eli Lilly and Company. Myoglobin was a gift of Dr. R. Lumry. Crystalline yeast alcohol dehydrogenase was obtained from the California Corporation for Biochemical Research. Aldolase was prepared from rabbit muscle (8). 2,2-Dimethyl-2-silapropanol and 4,4-dimethyl-4-silapentane-1-sulfonic acid, sodium salt,l were

1 The preparation of this compound is given in (9).

gifts of Dr. G. V. D. Tiers of the Minnesota Mining and Manu- facturing Company. Deuterium oxide was purchased from the Stewart Oxygen Company and was distilled before use. Deute- rium chloride, solution in DzO, was obtained from Merck and Company, Inc. Urea-D1 was prepared by exchanging urea, which had been previously recrystallized from ethylenediamine- tetraacetic acid solution, with DzO three times.

Oxidation of insulin and fractionation into A and B chains were carried out according to the procedure of Sanger (10). Residual acetate was removed from the A chain preparation by heating in a vacuum.

Measurements of pH were carried out with a Beckman model G pH meter. Acidities in DzO are reported in terms of pH. For purposes of comparison, the pD was taken as pD = pH + 0.4 (11). Absorption spectra were observed on a Cary model 11 recording spectrophotometer.

Spectra were observed by means of a Varian high resolution nuclear magnetic resonance spectrometer, V-4311, operating at 56.4 mc, and were recorded on a Varian recorder G-10. The spectrometer was equipped with a flux stabilizer V-K3506, a Hewlett Packard wide range oscillator, BOOCDR, and a Hewlett Packard electronic counter 521C. Samples were examined in the presence of air, except where noted. No temperature con- trol was used, but the temperature in the air gap remained at approximately 20” and did not vary more than 1t2”. A field of high homogeneity, as judged by the line width of the absorptions of the methyl hydrogens of acetaldehyde, was desirable for maximal resolution but was not always obtainable. In such a field, spectra were usually similar with or without spinning of the sample. In less homogeneous fields, spinning increased resolu- tion with most samples, but with some protein solutions, spinning invariably gave erratic results. In later phases of the work use of a Varian Field Homogeneity Control Unit V-4365 considerably reduced, but did not eliminate, the need for spinning. Compari- sons of various spectra are made, therefore, as far as possible between samples observed under identical conditions (spinning or nonspinning).

Spectra were recorded at a sweep rate of about 3 milligauss per second or less. The radio-frequency power level was usually set at about 5 microwatts. Saturation of the protein absorptions did not occur until power levels of 5 milliwatts were reached. The frequency scale was calibrated with the side band technique of Arnold and Packard (12).

Samples of cytochrome c were also examined at 40 mc through the courtesy of Dr. G. V. D. Tiers of the Minnesota Mining and Manufacturing Company.

General Procedure for Observations of Spectra-Spectra were ob- served in DzO following the procedure of Saunders, Wishnia, and Kirkwood (1). In most cases, the preliminary dialysis of the

1807

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1808 Nuclear Magnetic Resonance Studies of Proteins Vol. 237, No. 6

protein did not affect the spectra obtained, and consequently it was eliminated. The dried protein, obtained in this form if necessary by lyophilization, was dissolved in DzO, and the pH of the solution was measured and adjusted if necessary to the required value. The solution was then lyophilized, and the solid was redissolved in DzO for exchange twice more with DzO. The times allowed for this exchange were of the order of 5 to 10 minutes at room temperature. The protein was then dissolved in approximately 0.50 to 0.35 ml of DzO. The solution was cen- trifuged if necessary and then transferred to the sample tube, which was a suitable length of 5-mm O.D. Pyrex tubing sealed at one end. Concentrations were in the range of 10 to 20%. However, solutions of 5% concentrations could be studied, and a 1 y0 solution of ribonuclease gives an observable signal. Yeast alcohol dehydrogenase was observed in DzO without the pre- liminary exchange and lyophilization.

There were usually residual water protons left in the sample as prepared by this procedure. At the radio-frequency power levels used in these experiments, the absorption of these protons appeared as a definite sharp spike at 57 (see below for definition of tau). This peak could be eliminated by use of higher radio- frequency intensities which saturated it only. However, it proved convenient to have this residual proton peak present be- cause its sharpness made it a convenient marker for locating the spectrum and qualitatively identifying various peaks and for use in calibration of the frequency scale.

Cytochrome c and myoglobin samples were examined in the absence of air. Ferricytochrome c (the commercial sample was treated with ferricyanide and dialyzed before use) was exchanged with DzO at the desired pH, dissolved in D20, and placed in the sample tube which was sealed off under vacuum. Ferrocyto- chrome c samples were prepared by reduction of the ferri-form with a very small amount of sodium dithionite. The pH was adjusted to the desired value and the sample was transferred to the sample tube which was sealed off under vacuum. Ferromyo- globin was prepared in the same way, except that the sample tube and sample were flushed with nitrogen, and the tube was sealed off under nitrogen at atmospheric pressure.

&ferencing-Since shifts of the peaks in the magnetic reso- nance spectrum of a protein are very small, an internal reference is desirable. Such shifts with an external reference might be obscured by the correction necessary for differing bulk magnetic susceptibility of reference and sample. For general work, not involving extremely precise measurements, an external reference may possibly be more convenient and preferable because of the sensitivity of proteins to small molecular weight compounds either by way of reaction, complexing, or by induced configura- tional changes.

The internal references used were 2,2-dimethyl-2-silapropanol and 4,4-dimethyl-4-silapentane-1-sulfonic acid, sodium salt (13). Both compounds possess a trimethylsilyl group that gives rise to a very narrow high field absorption. The alcohol has a methylene group whose absorption falls in a region of protein absorptions. The absorptions of the methylene groups of the sulfonic acid salt combine to form a low, broad band which does not interfere with the protein spectrum.

The reference compounds have surface active properties and do cause changes in the ultraviolet spectra of various proteins over a period of time. However, there was no immediate effect on ultraviolet spectra, and the magnetic resonance spectrum of the proteins studied was always observed in the absence and in

the presence of these compounds and found to be the same. The position of the prominent methyl hydrogen peak relative to that of the reference compound was measured, and in a separate ex- periment without the reference compound, this methyl hydrogen absorption was used as a reference for the other peaks.

Tetramethylammonium ion as a reference (13) might elimi- nate the danger of structural changes in the protein, since the reference compound is usually present to approximately 1 y0 by weight which would make the tetramethylammonium ion about 0.07 M, but it would also necessitate two measurements since the peaks due to the tetramethylammonium ion appear in the center of the protein absorption.

The positions of the various absorptions are expressed in terms of r units similar to the scheme of Tiers (14). Tau is defined:

7 = 10.00 - l-H,--1 106 L Hr J

where H, is the resonant field for the reference absorption and H, is the resonant field for the absorption being studied. The reference absorption is arbitrarily assigned the value of lO.OOr. Peak positions could be measured to within a standard deviation of 0.01 to 0.027 except for the very broad peaks. In one case, (myoglobin) tetramethylammonium bromide was used as a reference, the position of its resonance having been determined as 6.867.

Assignment of Amino Acids-The work of Jardetzky and Jardetzky (2) and of Bovey and Tiers (4) was used as a basis for the assignment of amino acids to the various absorption peaks. Although the use of positions in one solvent as a basis for assign- ments in another solvent, in this case water and trifluoroacetic acid, can be misleading, a comparison of the positions of the various absorptions in these two solvents shows that in general they run parallel. In those cases where data obtained from aqueous solutions were not available and the data obtained in trifluoroacetic acid seemed inapplicable, observations were made in aqueous solution to determine the point. Assignments mere made on the basis of position only.

RESULTS

General-An observable spectrum has been obtained for all proteins examined, ranging in character from broad, low, un- resolved bands to well defined peaks with incipient structure and, in certain cases, with sharply defined structure. As would be expected the peaks in native proteins are broad in comparison with the absorptions of the individual amino acids, the major well defined protein absorption band having a width of approxi- mately 100 c.p.s. Under various denaturing conditions the peaks become sharper. The positions of the maxima of the absorp- tions range from 9r to 17, the range expected from the absorp- tions of the individual amino acids.

Changes in position of the absorptions of the various hydro- gens occurred when proteins were subjected to varying condi- tions. In most cases these changes are associated with the resolution of an absorption into two or more components. The presence of oxygen and salt also seemed to shift the absorptions, but very slightly. Since these latter effects were so small, they were disregarded. In the spectra recorded here no quantitative importance should be attached to the height of the residual pro- ton peak. The sharpness and amplitude of this absorption will depend to a far greater degree than the protein absorptions on the homogeneity of the magnetic field.

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Ribonuclease ........... Ribonuclease 8 M urea. .. Oxidized ribonuclease. ... p-Lact.oglobulin Pepsin. .................. Ovalbumin ............. Aldolase 8 M urea ........ Insulin (base). ......... Insulin (A chain). ....... Ferricytochrome c. ...... Ferrocytochrome c ....... Myoglobin .............. Metmyoglobin. ..........

TABLE I

Tau values of magnetic resonance absorptions of proteins

1

9.07 9.14 9.12 9.16 9.19 9.15d 9.14 9.26 9.12

8.90 9.10

2

8.54 8.64 8.64 8.58

8.63 8.56 8.50 8.54 8.54 8.49 8.76

- 3

8.17 8.17

8.04 i.87 7.95 8.06

4

7.09 7.12 6.92 7.14 7.06 7.14 7.14

7.29 7.01 6.68 7.20 7.15 7.11 7.4

Peaks

5.54 5.66

6.02 5.68 5.79 5.63 5.92 5.74 6.01

6.13 5.84 6.16 6.05 5.96 6.12

T _- wb

5.2

5.33 5.32

5.22 5.25 5.30 5.29 5.18 5.21

-7

UC

4.26

4.27

I

3.05 3.13 3.15

3.13 3.04

3.13 3.27 3.17 2.95 2.94

8

2.77 2.77 2.73

2.87 2.88 2.97

2.75

9

1.4 1.52 1.67 1.3 1.86

2.30 0.89 1.6 1.5s

a For native proteins, Peaks 5 and 6 are fused and almost indistinguishable. b Residual HDO. c Urea. d Assumed as 9.15 7 for reference.

In the presentation of the spectra the following system has been adopted. Absorption peaks are numbered, the numbers referring to the spectrum of osidized ribonuclease at pH 6.5. Absorptions of reference compound, water, and urea are labeled R, W, and U, respectively, and anomalous absorptions are in- dicated in the text. Assignments of amino acids to the various absorption peaks and the approximate positions are as follows.

1. Methyl hydrogens of valine, isoleucine, and leucine, 9.2r to 8.87.

2. Methyl, methylene, and methine hydrogens of isoleucine, methyl hydrogens of alanine and threonine, methylene hydrogens of leucine, lysine, and methine hydrogen of leucine, 3% to 8.3~.

3. Methyl hydrogens of methionine, methylene hydrogens of proline and arginine, 8.27 to 7.97.

4. “Adjacent methylene” hydrogens (methylene hydrogens in which the carbon atom is adjacent to a nitrogen oxygen, sulfur atom, a carboxyl group, or an aromatic ring), 7.87 to 6.47.

5. Hydrogens alpha to a carboxyl group in valine, histidine, leucine, alanine, methionine, lysine, tryptophan, glutamic acid, phenylalanine, and tyrosine, 6.3~ to 5.5~.

6. Hydrogens alpha to a carboxyl group of aspartic acid,

I I I 0.0 5.0 10.0

TAU

arginine, proline, cysteine, serine, and threonine, 5.47 to 5.1~. FIG. 1. Proton magnetic resonance spectra of ribonuclease.

7. Ring hydrogens of tyrosine, as well as tryptophan ring and Solid line, native ribonuclease; C, 20%; pH 6.5; s.~ Broken line,

amide proton absorptions, 2.97 to 2.7-r. ribonuclease in 8 M urea solution; C, 20%; pH 6.5; s.

8. Ring hydrogens of .phenylalanine and one histidine ring hydrogen, 2.6-r to 2.4~.

9. One ring hydrogen of histidine and amino, and peptide hydrogens, 2.3~ to 0.5~.

The positions of the absorption peaks for the various proteins

observed are listed in Table I. Ribonuclease-The spectrum of native ribonuclease and ribo-

nuclease in 8 M urea are shown in Fig. 1. Less detailed spectra have been reported before (I, 5-7). The assignment of amino acids to the various peaks follows that of Jardetzky and Jar- detzky (3) with the exception of the absorptions in the region of 2r to 07.

Peak No. 9 includes a histidine ring hydrogen but arises mainly

from residual hydrogens attached to amino, amide, and peptide nitrogens. These absorptions may possibly arise from the rela- tively slowly exchanging hydrogens investigated by Grunwald, Loewenstein, and Meiboom (15). They may also arise from hydrogens sterically hindered from exchange with the medium (16). Ribonuclease does possess exchangeable hydrogens not accessible to the solvent at room temperature (17). A calcula- tion with the aromatic amino acid absorption as a standard shows that the number of these hydrogens is approximately 23. A

2 The abbreviations used are: C, concentration; s, sample ob- served with spinning; n.s., sample observed without spinning. In the various spectra, R, W, and U indicate the absorptions of the reference compound, residual HDO, and urea, respectively.

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1810 Nuclear Magnetic Resonance Xtudies of Proteins Vol. 237, No. 6

8 7 A

9 \

h--.2- I 1 I

0.0 5.0 10.0

TAU FIG. 2. Proton magnetic resonance spectrum of performic acid-

oxidized ribonuclease. C, 20%; pH 6.5; s.

H- FIG. 3. Proton magnetic resonance spectrum of amino acid mix-

ture of the same composition as ribonuclease. C, 10%; pH 10; s.

similar result has been obtained by Saunders and Wishnia (6) and by Schildkraut and Scheraga and Hermans and Scheraga (17). The details of this low field absorption in ribonuclease and other proteins will be presented in a later publication.

The spectrum of ribonuclease at pH 9 was found to be closely similar to that at pH 2. In particular, the absorption of the difficulty exchangeable hydrogens is still present, although it does not seem to be as prominent as in the spectrum of the acid solution.

In the presence of 8 M urea the absorptions of ribonuclease become sharper and new peaks appear. Peak No. 1 is now clearly separated from Peak No. 2. Peak No. 3 at 8.OOr, occur- ring at the approximate position of the methyl hydrogen ab- sorption of methionine, is new. Bovey, Tiers, and Filipovich (5) found that pepsin and hemoglobin after several days in trifluoroacetic acid at 5” exhibit a prominent absorption in this region. The absorption of the “adjacent methylene” hydrogens, Peak No. 4, is considerably sharper, as is the absorption of the cu-hydrogens, Peak No. 5. Peaks Nos. 7 and 8, arising from aromatic hydrogens, are just beginning to be separated. Peak No. 8 at approximately 1.57 is attributed to a histidine ring hydrogen.

For further comparison, a sample of ribonuclease was oxidized with performic acid according to the procedure of Harrington and Schellman (18). Comparison of the ultraviolet absorption spectrum of this sample with that previously obtained indicated that the protein was not completely oxidized. Examination of the magnetic resonance spectrum, Fig. 2, showed enough change to satisfy our purpose; the sample had 11 discernible absorptions, neglecting those of the reference and water. In this spectrum the aromatic hydrogen absorptions are almost resolved.

Some comparison was made of the spectra of ribonuclease with that of a mixture of amino acids of the same composition as the protein (Fig. 3). Limited amino acid solubilities made necessary a strongly acidic or basic solution. Although it proved im- possible to spin the amino acid solutions, those spectra obtained suffice to show, even in the absence of optimal resolving condi- tions, that the approximate shapes of the absorptions to be expected in a mixture of amino acids roughly resemble those of oxidized ribonuclease. An attempt to observe the oxidized protein under acid conditions failed because of gelation of the sample.

Bovine Serum Albumin--k pattern similar to that of ribonucle- ase is seen in the behavior of bovine serum albumin (Fig. 4). The native protein exhibits only three low broad peaks, the com- bined methyl and methylene hydrogen absorptions, the ab- sorptions of the ar-hydrogens and those of the aromatic hydro- gens. In 8 M urea, Fig. 4, absorptions due to methyl, methylene, ‘adjacent methylene,” CX- and aromatic hydrogens can be ob-

V’ I

5.0 10.0

TAU FIG. 4. Proton magnetic resonance spectra of bovine serum al-

bumin. Solid line, protein in 8 M urea solution; C, 10%; pH 5.7; n.s. Broken line. native serum albumin: C, 10%; pH 5.2; n.s.

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0.0 5.0 10.0 TAU

FIG. 5. Proton magnetic resonance spectra of insulin. Solid line, insulin; C, 10%; pH 10; s. . .

Broken lane, insulin; C, 10%; pH 2; n.s.

0.0 5.0 10.0 IS.0

TAU FIG. 6. Proton magnetic resonance spectrum of insulin. C, 20%; pH 2; n.s.

served. Similar patterns have been observed by Bovey, Tiers, sociated forms of insulin are probably being observed.3 A peak and Filipovich (5). In the presence of urea and excess sulfite at assignable to “adjacent methylene” hydrogens cannot be ob- pH 6, conditions which are known to split the disulfide bonds served. The absorptions of the aromatic hydrogens are very (19), there is only a slight change in the appearance of the spec- prominent, insulin possessing four tyrosines and three phenyl- trum from that of Fig. 4. alanines per 6000 molecular weight unit. The spectrum of the

Insulin-The spectra of insulin and insulin derivatives under acid solution is similar to that of the basic solution except for various conditionsare shown in Figs. 5 through 8. Because of its insolubility at neutral pH, this protein was observed under acid

3 The pH 2 solution was observed with sample spinning, ac-

and basic conditions. At the high concentrations used, the as- counting for the difference in widths of the residual proton peak, W.

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I 1 I I

0.0 5.0 10.0

TAU

FIG. 7. Proton magnetic resonance spectrum of performic acid oxidized insulin. C, 10%; pH 10; n.s.

ii . . :: :: :: . . : : . . 3

I I 1 I 0.0 5.0 10.0

TAU FIG. 8. Proton magnetic resonance spectra of insulin A and B chains. Solid line, insulin A chain; C, 10%; pH 10; n.s. Broken line,

insulin B chain; C, 10%; pH 10; n.s.

the presence of Peak No. 9 (Fig. 5). In Fig. 6, insulin at 20% concentration in acid solution, a marked broadening and loss of resolution in the spectrum has occurred. The absorptions of hydrogens on nitrogen, however, are still visible. When the solution is diluted to lo’%, the spectrum of Fig. 5 is again ob- tained. Per-formic acid oxidized insulin, Fig. 7, shows a spec- trum which is similar to that of the original protein but in which the individual absorptions, except for the aromatic hydrogen

absorptions, are clearly much narrower. This spectrum repre- sents a minimum in the degree of resolution since the sample could not be spun. Peak Nos. 2 and 3 are clearly separate in contrast to the native insulin spectrum where they are fused and broad. The peak labeled 4 is new. There is less resolution in the aromatic absorptions, Nos. 7 and 8.

The spectra of the A and B chains are shown in Fig. 8. Con- tamination of the A chain preparation with acetate ion was

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June 1962 A. K ‘owalsky 1813

probably responsible for a narrow absorption at 8.077. This spectrum is of a nonspinning sample, but since the sharp acetate peak does not appear in the spectrum of a spinning sample, Peak No. 3 arises from the A chain. The spectrum is far more re- solved than that of native insulin. Peak No. 1, representing the methyl hydrogens of valine and leucine, is very sharp and prominent. The aromatic absorptions appear to be somewhat broader than in the native insulin spectrum and are not as clearly resolved. No hydrogens that are difficult to exchange are observed.

The spectrum of the B chain consists of three broad peaks similar to those of bovine serum albumin, except that here there seems to be incipient structure. Again, no difficult-to-exchange hydrogen absorptions are observed.

Aldolase-The spectra of native aldolase and of urea denatured aldolase are shown in Fig. 9. The native protein shows a broad unresolved line, independent of the concentration. In 8 M

urea, four peaks besides the urea peak are clearly evident, and a fifth one can be discerned as a shoulder on the residual proton absorption.

Cytochrome c-Cytochrome c in both its oxidized and reduced forms was examined (Fig. 10). A striking feature of these spectra is the presence of two broad absorptions at very high fields; at 10.157 and 12.55~ for ferricytochrome c and at 10.437 and 13.207 for reduced cytochrome c. The 13.207 absorption is very weak and is not clearly evident in the spectrum shown here. However, it has been observed in other spectra. Other points of difference between these two spectra are:

1. The reduced form possesses two shoulders, Nos. 1 and 3, on either side of the main peak. The oxidized form has no shoulder

: ! W

j; ji :; :: : : : : : I i: j : . : : : : : : : : : ; : : ; ; : ! :

FIG. 9

at Peak No. 1, but it does have one at Peak No. 2. This shoul- der may reflect the methylene hydrogens of lysine, of which cytochrome c possesses 18 per molecule.

2. The relative heights of the “adjacent methylene” hydrogen absorptions and those of the cr-hydrogens are different in the two forms.

3. The low field absorptions, region 4r to 17, in ferrocyto- chrome c seem to follow the usual protein pattern, that is, an aromatic peak and a low shoulder tailing off. In contrast, ferricytochrome c possesses a bewildering array of weak absorp- tions in this region, at 3.98r, 3.177, 2.307, and 0.89~.

Ferricytochrome c was investigated with respect to the effect of salt and oxygen. Apparent shifts of, at the most, 0.17 were noted; these factors were therefore disregarded. Because of autoxidation, ferrocytochrome c was examined in the absence of air. The material received from the supplier contained approxi- mately 20 To autoxidizable protein, and the proportion of autoxi- dizable component increased as the protein was subjected to operations such as freezing, thawing, or lyophilization.

In some samples of ferricytochrome c, a sharp absorption was observed in the region of Peak No. 3 (approximately 8.07). The amplitude of this peak seemed to vary with the lot number of protein used. The spectrum shown here does not possess this absorption.

Myoglobin-Myoglobin (Fig. ll), presumably the oxygen-free form because dithionite was present and the gas phase was Nz, shows a relatively well resolved spectrum. The spectrum is similar to that of ribonuclease, having absorptions corresponding to Peaks 1, 2, 4, 5-6, 7-8, and 9. There are noticeable absorp- tions between 8.49~ and 7.117. The myoglobin spectrum is

I I I

0.0 5.0 10.0

TAU 0.0 5.0 10.0 IS.0

TAU

FIG. 10 FIG. 9. Proton magnetic resonance spectra of aldolase. C, 10%; pH 6.3; n.s. Solid line, native protein; broken line, protein in 8

M urea. FIG. 10. Proton magnetic resonance spectra of cytochrome c.

ferricytochrome c; C, 20%; pH 4.6; ns. Solid line, ferrocytochrome c; C, 20%; pH 5.0; n.s. Broken line,

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Nuclear Magnetic Resonance Xtudies of Proteins Vol. 237, No. 6

: 695

0.0 5.0 TAU

10.0 15.0

FIG. 11. Proton magnetic resonance spectra of myoglobin. Solid line, myoglobin; C, 16%; pH 5.5; n.s. Broken line, metmyoglobin; C, 16%; pH 5.5; n.s.

similar also to that of cytochrome c in that there are unexplained high field absorptions at 10.67 and possibly at 11.8~ and a low field absorption at 3.857.

The spectrum of metmyoglobin (Fig. 11) is somewhat differ- ent. The aromatic absorptions merge with the absorptions of the difficult-to-exchange hydrogens so that the two are indis- tinguishable. There is only one prominent aliphatic hydrogen peak. The peak of the cr-hydrogens at 6.127 may not be as broad as is suggested in the figure because of the presence of a high concentration of residual protons in the medium. Metmyo- globin also possesses extreme high field absorptions. They are not as noticeable as those in the spectrum of the reduced form and are most easily detected by sighting along the curve. They are reproducibly evident and appear at 9.737 and 10.507 in one sample and at 9.657 and at 10.427 in another sample.

Metmyoglobin in acid showed a spectrum somewhat similar to that of myoglobin in neutral solution. The absorptions ap- peared broad, but two peaks were present in the region of 8~ to 97. The absorptions of the “adjacent methylene” hydrogens were more pronounced than those of either myoglobin or met- myoglobin at higher pH. Observation of this sample was diffi- cult, since acid solutions of metmyoglobin become extremely viscous and relatively low concentrations of protein (5%) were necessary. The differences in the spectra were shown not to arise from the differences in concentration.

The visible absorption spectrum of metmyoglobin was ob-

served in 0.1 M KC1 at pH 6.8 (maxima at 630, 502, and 407 mp) and at pH 3.2 (maxima at 645, 513, and 367 mp).

Other representative spectra are presented in Figs. 12 and 13: native chymotrypsin and chymotrypsin in urea, pepsin, and pepsinogen. Chymotrypsin was observed in acid solution. The presence or absence of 0.1 M KC1 did not alter the spectrum. Urea, 8 M, had the effect of causing new peaks to appear. Chy- motrypsinogen showed a spectrum similar to chymotrypsin. In contrast, pepsinogen exhibits a much broader and less re- solved spectrum than pepsin. Pepsin could not be observed at acid pH’s because of its low solubility in DzO. Other proteins which have been examined are ovalbumin, P-lactoglobulin, lysoeyme, and yeast alcohol dehydrogenase.

DISCUSSION

Correlation of Spectra and Amino Acid Content

Jardetzky and Jardetzky (3) have shown that the amino acid composition of ribonuclease can be correlated with its proton magnetic resonance spectrum. This is not true for all the pro- teins studied here, as for example, native serum albumin and aldolase. Only those proteins showing well-defined absorptions allow such correlations. Thus, cytochrome c possesses a high percentage of lysine, which can account for the shoulder on the main peak at 8.06~. Those proteins with a high content of aromatic amino acids show a more prominent aromatic hydrogen absorption. The spectrum of performic acid-oxidized insulin

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FIG. 12

0.0 8.0 10.0

TAU

FIG. 13

FIG. 12. Proton magnetic resonance spectra of chymotrypsin. C, 10%; pH 2.6; KCl, 0.1 M. Broken line, 8 M urea solution; solid line, no urea.

FIG. 13. Proton magnetic resonance spectra of pepsin and pepsinogen. Solid line, pepsin (dialyzed at, pH 6.0); C, 200%; pH 6.0; s. Broken line, pepsinogen; C, 20%; pH 6.0; s. The sweep rates are the same and the position of the HDO absorption is assumed to be the same.

appears to be the summation of the spectra of the A and B chains. However, even in the extreme cases of highest possible resolution, e.g. in trifluoroacetic solution where the protein chains exist as a random coil (5), one does not observe separate ab- sorptions for each of the amino acids. Bovey, Tiers, and Filipo- vich (5) found nine discernible peaks for /I-lactoglobulin in trifluoroacetic acid solution. This is probably close to the maxi- mum to be expected, for a mixture of amino acids of the same composition as ribonuclease also shows nine discernible absorp- tions as does the A chain of insulin. Oxidized ribonuclease shows 11 discernible absorptions.

Assignment of amino acids to various absorption peaks in a protein spectrum on the basis of the position of the absorptions of the pure amino acid can possibly be misleading. The positions of the absorptions may be shifted far from their normal location by such a simple mechanism as an extreme local pH (2) or by a ring-current effect (20). The absorptions of the protons bonded to nitrogen or oxygen, but not involved in hydrogen bonding, lie at far higher fields than the absorptions of hydrogen-bonded pro- tons (21, 22).4 Even relatively inert hydrogens can be involved in hydrogen bonding. The chemical shift of the chloroform proton varies with the concentration of the chloroform and the nature of the solvent (23). Proteins do possess hydrophobic regions, and it is conceivable that interactions of the type men- tioned with chloroform may occur. The absorptions of the difficult-to-exchange protons lie in approximately the right region of the magnetic resonance spectrum although the width of the absorption makes anomalous shifts difficult or impossible

* R. L. Batdorf, Ph.D. thesis, University of Minnesota, 1955.

to observe. However, anomalous absorptions have been ob- served in heme proteins.

Factors Afecting Line Width5

For macromolecular compounds the possible sources of line broadening are: (a) exchange processes, (b) translational diffu- sion, as related to spin-lattice relaxation, (c) rotational diffusion, as related to spin-lattice relaxation, (d) dipole-dipole interaction, and (e) electronic relaxation (for paramagnetic metallo-proteins). Such effects as quadrupolar broadening are present in the con- stituent amino acids and would not change markedly in the protein.

5 In this discussion, the terms used are defined as follows. The nuclear magnetic moment precesses about the direction of the ap- plied magnetic field with a frequency ?, the frequency for the resonance condition. In a system of precessing dipoles, the spin- lattice relaxation time, Tr, is a measure of the rate at which the component of magnetization parallel to the applied field ap- proaches its equilibrium value; alternatively, it is a measure of the rate at which a system of excited dipoles will lose energy to its environment. The spin-spin relaxation time, Tz, is a measure of the rate at which the individual precessing moments will get out of phase with each other. It represents the decav rate of the com- ponents of magnetization perpendicular to the applied magnetic field. Both TZ and Tz have a complex dependency on the motions of the nuclei and can be expressed in terms of a time characteristic of the motion of the nuclei, t, , the correlation time. The width of the absorption may be determined by either T1 or Tz, or by both relaxation times. For a complete discussion, see Poole. J. A., Schneider, W. G., and Bernstein, H. J., High resolutibn’nuclea~ magnetic resonance, McGraw-Hill Book Company, Inc., New York, 1958, Chapters 3, 9, and 10.

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1. Exchange Processes-These do not contribute significantly. Those nuclei which are exchanging rapidly between the solvent and the protein (deuterium nuclei) are not observed. Only the secondary effects of exchange need be considered and these are limited to nearest neighbors, e.g. the methyl group resonance of ethanol is unaffected by exchange of the hydroxyl proton (24).

2. Translational Diffusion-For protein solutions, the relative motion between solute and solvent is approximated by that of the solvent. This would contribute an insignificant amount to the width. Within the protein molecule itself there would be no proton-proton effect (neglecting vibrational and rotational components). Although the inter-molecular effect falls off as the inverse first power of the distance between nuclei (25) and the solution is dilute with respect to the solute, any two protein molecules in approaching each other will be subjected to the influence of a high local concentration of protons.

Experimental observations argue against a translational diffusion effect. The observation (5) that the line width of a resonance of a polymer in solution is independent of the degree of polymerization of the polymer indicates that, for macromolecules existing as a random coil, other processes must determine the line width. The magnetic resonance spectrum of a 20 ‘% solution of ribonuclease has the same characteristics as that of a 5% solution. Bovine serum albumin in 8 M urea and metmyoglobin at pH 3 form viscous solutions which can barely be poured, yet these solutions show well resolved spectra. The spectrum of oxidized insulin appears to be a summation of the spectra of the A and B chains. Since the A chain spectrum is sharp and re- solved and the B chain spectrum is broad and diffuse, it cannot be any property of the solution or an intermolecular effect which is determining the line width, it must be an intramolecular effect.

Similar spectra (with respect to degree of resolution) have been observed for the following native proteins: ribonuclease, cyto- chrome c, insulin, pepsin, ovalbumin, lysozyme, /?-lactoglobulin, and myoglobin. Broad unresolved lines have been observed for native bovine serum albumin and native aldolase. Spectra of intermediate resolution have been observed for pepsinogen and metmyoglobin. There does not seem to be any correlation be- tween the frictional ratios of the proteins (26) and the degree of resolution of their spectra. The participation of translational diffusion in the determination of line width seems to be small.

3. Rotational Diflusion-The rotational relaxation times, as estimated from dielectric dispersion data, of ovalbumin, insulin, fi-lactoglobulin, and myoglobin vary from 2.7 X 10-T set to 1.8 X lo-* set (27). These proteins yield spectra with approximately the same degree of resolution. The lower values are fairly close to the minimum in the T1 us. t,: curve (t, = 3 x 10eg set) (25). Rotational motion probably contributes to the line width. But since this effect varies as the inverse sixth power of the distance between nuclei, the effect is completely intramolecular and only nearest neighbors contribute. When a tightly coiled molecule changes to a flexible random chain, it ceases to rotate as a molecu- lar entity. The segmental lengths of the chain now possess the random or partially random rotational motion. The question then is, how fast is this segmental rotation in comparison with the rotational diffusion of the coiled molecule?

One may calculate a maximal order of magnitude of segmental rotation, with Eyring’s equation and the assumption of an energy barrier to rotation of 4 kilocalories, to be about lo-i0 sec. Since (2avo)-l = 3 x low9 set, the change in the rotational correlation time could account for an increase in the resolution of a spectrum.

However, McCall and Bovey (28) found that the longitudinal relaxation time of the backbone protons of polystyrene in carbon tetrachloride solution is 0.033 sec. T1 for the methyl hydrogens of toluene is reported to be 9 set (29). Comparing this with polystyrene, and allowing for the difference in number of nearest neighbors, it can be estimated that the time of rotational motion of the backbone segments in polystyrene is approximately 0.01 that of the corresponding hydrogens in the monomer. For small peptides the rotational relaxation time is of the order of lo-l1 to lo-i0 set (30). This would give a maximal value of lO-g to lo-* set for the time of segmental rotation of a polymer. In this case the conversion of a coiled molecule to a random chain would lead to either little change or a broadening of the absorp- tions.

4. Dipole-dipole Interaction-Ordinarily, for small molecules this interaction is averaged out through the fast rotational and diffusional motion of the molecule. In a protein solution, where the peptide chain of the protein molecule is coiled within itself, it is quite probable that some segments of the chain are restricted in their motion and, moving slowly relative to one another, are unable to average out completely the local fields. Consequently in the movement of the protein molecule through the medium, any one type of amino acid, such as the collection of lysines, methionines, or valines, will see a range of magnetic fields rather than a single, time-averaged field, and the absorption of this collection of amino acids will then be broadened.

Considering these two contributions to the line width, the spin-lattice interaction, Ti, and the spin-spin interaction, Tz, as the segmental motions become more and more prominent through some external agency such as heat or urea denaturation and as the correlation time shortens, both T1 and T:! will change. But the effect of a change in T1 is first, to broaden the line, and then to narrow it; whereas the effect of a change in Tz is only in the direction of narrowing the line. Conceivably, as for solids, the correlation time can change and TP remain approximately con- stant. But solid polystyrene has a proton line width of 10 gauss which in solution becomes 0.005 gauss (5), comparable to the values observed for proteins. Consequently, it is justified to assume that TP for protein solutions is a variable function of t,.

Probably any change in T1 will be overshowed by a change in T2. The spectrum of insulin solutions is concentration depend- ent, being broader at higher concentrations. Insulin is exten- sively associated in solution. This must result in an increase in the various correlation times of the protein which are all close to or greater than (2nv0)-I. I f Ti were governing the line width protein association would cause the absorptions to become narrower and more resolved. If Tz is the determining factor, the peak will become broader, which is what is observed. In view of the closeness of the values of the molecular rotational times, of the segmental rotational times, and of (2svo)-1, the dipole-dipole interaction likely determines the width of the ab- sorptions in the protein solutions. Such a conclusion has also been reached by Saunders and Wishnia (6).

Quantitative assessments are difficult because of the polymo- lecular nature of the substance being studied. A prominent broadening arises simply from the superposition of a number of individual lines, each possessing a slightly different chemical shift. This summation of individual lines is then further broadened, presumably by the restriction of motion of the various different segments of the molecule. Any individual absorption contributing to the over-all width may itself have a width de-

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pending on a heterogeneous relaxation mechanism, i.e. the methyl hydrogens of an amino acid residue such as leucine will not all possess the same correlation times. In view of this, only comparisons of a qualitative nature are made.

Ribonuclease, Bovine Serum Albumin, and Other Proteins

The progressive increase in resolution in the spectra of the various forms of ribonuclease and bovine serum albumin may be explained as follows. In the native protein, the constituent amino acids are constrained in their motion and any one type of amino acid is subjected to a dipole-dipole interaction resulting in broad absorptions. In the presence of urea as H-bond and helical structure are disrupted, the segments of the molecule gain extra motional freedom increasing the efficiency of averaging of the magnetic field. When the disulfide bonds are split, the constraining forces are almost completely eliminated and the molecular components are able to move in what is essentially the random motion of a flexible coil. The time-averaging of the magnetic field becomes extremely efficient; the absorptions be- come very sharp.

Such an interpretation is supported by the viscosity studies of Harrington and Schellman (18) on ribonuclease solutions and of Kolthoff, Anastasi, and Tan (19) on bovine serum albumin solu- tions. For these proteins the intrinsic viscosity increases through the series native protein, urea-denatured protein, and disulfide-split protein, indicating a progressive unfolding of the molecule as it is first denatured and then oxidized or reduced. The optical rotatory behavior in general follows this pattern. The viscosity and sedimentation studies of ribonuclease led Harrington and Schellman to conclude that it is a flexible chain polymer with an axial ratio 20 times that of the native protein. Although the viscosity of the albumin in urea increases by 24 when the disulfide bonds are reduced with sulfite, there is no marked change in the magnetic resonance spectrum. In urea the molecule may exist as a large loose floppy ring, and reduction of the disulfide bonds may not result in any great increase in motional freedom of the segments of the chain.

Similar considerations hold for aldolase. -411 the -SH groups of aldolase are not available for reaction with p-mercuribenzoate ion unless the medium is 5 M in urea or more (31). In 6 M urea the optical levorotation of the protein increases by a factor of 4. The increased accessibility of the sulfhgdryl groups and the change in optical rotation indicate that the protein has undergone a change in structure. Aldolase is known to have at least three peptide chains (32). In 8 M urea, the protein molecule may dissociate into small fragments or it may undergo an unfolding. In either case it is reasonable that segments of the chain, which in the native molecule are prevented from moving freely on dis- sociation or expansion of the molecule, achieve a greater freedom of motion.

Under the conditions of these experiments, it is probable that it is the dimer of chymotrypsin (33) that is being observed. Since formation of the dimer does not result in any higher degree of order in the molecular structure (34) and the monomer is reported to exist as a partially unfolded molecule (34), this would account for the sharp spectrum obtained. The slight increase in resolution of the magnetic resonance spectrum of the protein in 8 M urea is consistent with the unfolding in urea at pH 3 (35). The chymotrypsinogen spectrum is quite similar to that of chgmotrypsin. It is reported that chymotrypsin and its pre-

cursor contain approximately the same number of fast exchange- able hydrogens (36).

In comparison, the pepsinogen spectrum is far broader and less well resolved than the spectrum of pepsin. Yet, the optical rotation of pepsinogen is quite close to that of pepsin (36). The pepsin molecule is thought to have very little helical content and it has been suggested that it maintains its required conformation by means of hydrophobic bonds (36), yet absorptions of hydro- gens that are slow or difficult to exchange and are attached to nitrogen, are visible in the pepsin spectrum.

Insulin

In the spectrum of the 20% solution of insulin, Peak No. 1, 9.267, appears the most drastically broadened. Waugh has observed insulin fibril formation in the presence of organic acids and urea and at low pH (37). He has suggested that the non- polar side chains of insulin interact to form aggregates and fibrils. The magnetic resonance spectrum does support the idea that at high concentrations the nonpolar side chains undergo an ex- tensive curtailment of their motion.

Insulin at acid pH at concentrations of 0.3% exists as a tetramer or possibly as a more highly associated form (38). At pH of 9, the state of insulin is uncertain, yet the spectra of acid and alkaline solutions are very similar. Optical rotation (39) and deuterium exchange studies (40, 41) indicate that insulin possesses some structure in solution and that at alkaline pH’s it undergoes an unfolding. The unfolding is suggested by the pH 10 magnetic resonance spectrum in that Peak JXo. 9 is absent. These spectra indicate that the association effect at 10% con- centration does not interfere with the internal motions of the molecule.

Optical rotation (42) and deuterium exchange studies (43) indicate that the A chain acts as a purely random coil. The sharp spectra obtained are in agreement with this. The B-chain absorptions on the other hand are very broad and resemble those of bovine serum albumin. The B chain has been shown to associate in solution and to exhibit a slow exchange of hydrogens with the solvent (43) consistent with a restricted segmental motion of the molecule.

Cytochrome c and Myoglobin

The nature of the extreme high field absorptions in cytochrome c and in myoglobin spectra is unknown. These absorptions have been observed in all samples of these proteins and were found also at 40 mc in the same positions. B possible explanation is that the structure of the protein is such that certain side chains are forced to occupy positions in a space above and within the cir- cumference of the aromatic porphyrin ring. The ring current effect would then account for such high field shifts. Such shifts have been found in the spectra of porphyrins (44). These anomalous absorptions may also arise from hydrogens on oxygen or nitrogen (nonexchangeable with the solvent) being forced into a hydrophobic region where they cannot form hydrogen bonds. The fact that similar absorptions are seen, although not as prominently in the myoglobin spectra, and are absent, as far as has been observed, in ribonuclease, insulin, ovalbumin, and pepsin spectra, lends support to the ring-current explanation. Certainly the coordinated histidine residues in cytochrome c and myoglobin must be strongly influenced by the porphyrin ring.

The absorption at 3.997 in the spectrum of ferricytochrome c

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may also be accounted for by a ring-current effect, the absorption being shifted to higher or lower fields according to the position of the group. Yet this resonance is absent in the spectrum of the reduced form. In contrast, myoglobin also possesses an absorption in this region, 3.857 which is not seen in the metmyo- globin spectrum. None of the constituent amino acids possesses an absorption in this region, although urea and methyl ureas do. Because of their nonappearance in one form of the protein, these unexplained resonances probably do not arise from the porphyrin ring.

Since ferricytochrome c is paramagnetic and ferrocytochrome c is diamagnetic (45), it might be argued that the differences in these spectra arise from the change in the magnetic properties of the iron. If the electronic relaxation were governing the line width, one would expect the oxidized form to have decreased resolution and increased line width. The peaks seem to be of the same general width. The earlier report (7) of a difference in the width of the two forms is now in question since these early observations were made on a sample of cytochrome c which later proved to be impure and to have several spurious absorptions. Furthermore, new absorptions appear in the spec- trum of the oxidized form. The presence of 0.02 M K3Fe(CN)G does not alter the spectrum of ribonuclease. Other work (46) has shown that the heme iron has little effect on the relaxation of hydration or bulk-phase water except for that in the sixth ligand position. The influence, if any, of the heme iron must be small.

The distinct differences between the spectra of the oxidized and reduced forms of the protein, if not accounted for by the change in the state of the iron, can be explained by a change in the structure of the protein moiety. Other observations support such an interpretation. Oxidized cytochrome c is strongly ab- sorbed on kaolin whereas the reduced form is not (47). The reduced form is more resistant to heat denaturation, and the oxidized form of yeast cytochrome c is more susceptible to pro- teolytic attack than the reduced form (48).

Both metmyoglobin and myoglobin are paramagnetic (49) and electronic relaxation in one or both could be influencing the line width, although not markedly. The fact that metmyoglobin in acid solution shows a magnetic resonance spectrum similar to myoglobin in neutral solution does not necessarily obviate this. Myoglobin is denatured below pH 4.4 to 4.1, and at pH 3.6 the exchange of hydrogens with the solvent is very rapid in contrast to the exchange at higher pH’s (50). The increased viscosity of metmyoglobin solutions at pH 3.2 is similar to the increased viscosity of hemoglobin solutions at pH 3.5 (51). Although the molecule may be unfolding, as hemoglobin, it is also possible that, although no organic solvent is present, the heme is dis- sociated from the protein. Reversible dissociation of heme pro- teins and heme groups in aqueous systems has been observed by Rossi-Fanelli and Antonini (52). There is some change involv- ing the heme group since the visible absorption spectrum at pH 6.8 is different from that at pH 3.2. The spectrum of ferricyto- chrome c also changes with pH (53), and magnetic susceptibility measurements confirm that there is a change in the bonding of the heme iron (54). The magnetic susceptibility of metmyo- globin might also be a function of the pH. The spectral changes observed with metmyoglobin, both visible and magnetic reso- nance, may partially reflect the change in the type of bonding of the iron. Thus, whereas for cytochrome c a change in the oxidation state of the iron results in a reorganization of the protein structure reflected in a change in the magnetic resonance

spectrum, for myoglobin an additional effect of electronic relaxa- tion on the line width is also possible.

SUMMARY

The proton magnetic resonance spectra of a number of pro- teins have been observed in DzO under various conditions. The proteins examined included ribonuclease, bovine serum albumin, fi-lactoglobulin, pepsin, pepsinogen, chymotrypsin, chymotryp- sinogen, ovalbumin, aldolase, insulin, cytochrome c, myoglobin, lysozyme, and yeast alcohol dehydrogenase.

The spectra range from low, broad, unresolved bands to sharply defined peaks with structure. The latter type of spec- trum reflects the amino acid composition of the protein. In the sequence, native ribonuclease, urea denatured protein, and performic acid oxidized protein, the spectra undergo a progressive narrowing and increase in resolution. Similar changes are ob- served between the native and urea denatured forms of bovine serum albumin, aldolase, and chymotrypsin. A sharp, well re- solved spectrum is exhibited by the A chain of insulin, but not by the B chain. Insulin itself shows a moderately resolved spectrum. The increase in resolution is attributed to an increase in the segmental motions of the peptide chain which decrease the effectiveness of dipole-dipole interactions.

Absorptions of protons bonded to nitrogen which exchange very slowly with the solvent can be observed in the spectra of ribonuclease, insulin, and other proteins.

The spectra of cytochrome c and myoglobin exhibit anomalous absorptions at very high fields and in the region of 47. These arise possibly through a ring-current effect of the porphyrin. The spectra of these two proteins change with the oxidation state of the iron. The differences that are observed probably arise from a modification in the structure of the protein moiety rather than from a change in the electronic spin relaxation time of the iron.

Acknowledgments-This investigation was supported by United States Public Health Research Grant A-2316. The author is deeply indebted to Dr. Paul Boyer, in whose laboratory this work was carried out, for support, advice, and encouragement, and to Drs. Rufus Lumry and Edward Westhead for many helpful discussions.

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