Magnetic circular dichroism studies

ANALYTICAL

48,

BIOCHEXISTRY

Magnetic XIX.

Dichroism

Studies

of the Tyrosine:Trytophan BARTH, AND

Department

(1972)

Circular

Determination GUNTER

471-479

of Chemistry,

Ratio

in Proteins

EDWARD BUNNENBERG, CARL DJERASSI

Stanford Received

University, January

Stanford,

California

9@05

5, 1972

Recently (a), we reported the use of magnetic circular dichroism (MCD) for the determination of tryptophan in intact proteins (1). We pointed out that, in comparison to other methods currently in use, MCD has the unique advantage of giving unambiguous results even in cases in which the tyrosine:tryptophan ratio is high, that conformational and environmental effects do not interfere with the accuracy of the method, and that only small amounts of sample are required. Here, we wish to report an important refinement in the procedure that enables the technique to be extended to samples which, due to the presence of unknown quantities of water or inorganic salts, are not of analytical purity. This modification makes use of the fact that, in contradistinction to tryptophan, the quantitative determination of tyrosine by the usual chromatographic procedure subsequent to hydrolytic cleavage of the protein does not present serious difficulties. Consequently, the spectroscopic determination of the tyrosine: tryptophan ratio has the advantage that, given knowledge of the tyrosine content, accurate values for the tryptophan content can be obtained with only rough knowledge of the protein concentration. Thus, tyrosine serves as an internal st.andard in the determination of tryptophan by the MCD technique. In addition to enabling tryptophan determinations to be made with increased accuracy, this modification illustrates the advantages inherent in a spectroscopic method in which signals may occur with either positive or negative sign. EXPERIMENTAL

Magnetic circular dichroism spectra were obtained on a Japan Spectroscopic Co. spectropolarimeter (Durrum JASCO Model ORD-UV-5) which had been modified for CD and to accept a superconducting magnet built by Lockheed Palo Alto Research Laboratories (model OSCM-103) 471 Q 1972 by

Academic

Press,

Inc.

472

BARTH,

BCNNENBERG,

AND

DJERASSI

(3). The magnetic field used for all measurements was 49.5 kgauss. Absorption spectra were taken on a Gary 14 apectrophotometer, and a Metrohm pH meter E 30C1equipped with an EA 120 X electrode was used for pH measurements. The purity of the reference compounds L-tyrosine (Eastman Kodak), L-leucyl-L-tyrosine amide hydrochloride monohydrate (Cycle), and glycyl-oL-tryptophan (puriss., Fluka) was confirmed by TLC and by elemental analysis, and they were used without further purification after drying over P,@, for 12 hr. Guanidine hydrochloride, ultrapure grade, was obtained from Schwarz/Mann. The following proteins were used without verifying their purity: albumin (bovine) cry&, homogeneity estimated to be 100% by electrophoresis (S&ware/Mann) ; trypsin (bovine pancreas) 2 X cry&. (Wort’hington) ; ovalbumin 2X cry&. (Schwarz/Mann) ; lysozyme (egg white) 6X tryst. dehydrogenase (yeast) 2X cry&. (Miles-Seravac) and lyophilized (Boehringer-Mannheim) . In the modified procedure, the proteins were dissolved in 6 M guanidine hydrochloride solution which had been adjusted t,o pH 6.0, and MCD measurements were made after the solutions had been allowed to stand at room temperature for 12 hr. Next, the pH was adjusted to 11.7 with 5 1V NaOH, and the MCD spectrum was measured again. [ 01, values are expressed in deg cm? dmole-l for a magnetic field of 49.5 kgauss. RESULTS

AND

DISCUSSION

Proteins containing tyrosyl residues exhibit, in solution at neutral pH, negative MCD bands which correspond to the lowest energy absorption band of the phenol chromophore. When tryptophanyl groups are also present, the tyrosyl MCD band at 276 nm ([Q], = -5.5 X 103) is strongly overlapped by the more intense ( [Q], = -3.08 X 104) negative MCD band of t’he indole chromophore at 269 nm. This unfavorable ratio between the intensities of the tyrosine and tryptophan MCD bands, the wavelength separation of 7 nm between their band maxima, and the necessity of correcting the tyrosine MCD bands for contributions from overlapping cystine MCD bands (6%) are factors that preclude the accurate determination of tyrosine using neutral solution MCD spectra alone. These difficulties are readily overcome, however, by recourse to a procedure developed by Edelhoch (4) for the determination of tyrosine by absorption spectroscopy. The modified procedure makes use of the well-known fact that the ionization of tyrosine to form the phenolate ion is accompanied by a bathochromic shift of its lowest energy absorption band; however, in contrast to the situation obtaining in the absorption spectrum where

TYROSINE:

TRYPTOPHAN

RATIO

IN

473

PROTEINS

7

FIG. 1. MCD (upper curvrs) and UV amide hydrochloride monohydrate at guanidine hydrochloride solution.

(lower curves) pH 6.0 (--)

spectra of L-leucyl-L-tyrosine and pH 11.7 (- --) in

6M

the extent of overlap of two absorption bands is merely altered, in the MCD spect,rum the negative MCD band of the internal standard, tyrosine, is shifted in wavelength to a position that is nearly coincident with the positive MCD band of tryptophan. The spectral changes accompanying the ionization of tyrosine are shown in Fig. 1. In basic solution a new absorption band appears at 294 nm; t.he same feature is observed in the MCD spectrum. In addition, the intensities of the absorption and MCD bands in basic solution are nearly doubled as compared to the neutral solution. Tryptophan, on the other hand, exhibits only a small diminution in the intensity of the 278 nm absorption band (c = 5550 at pH 1 and 6 = 5430 at pH 13 (5) ) . This change is also reflected in the MCD spectrum by a 12% hypochromic effect. The position as well as the intensity of the 292 nm positive MCD remain unchanged in this pH range, e.g., glycyl-nn-tryptophan in 6 M guanidine hydrochloride solution has the constant magnetic ellipticity of 3.70 X lo4 throughout this pH range. Consequently, in proteins con-

474

BARTH,

BUNNENBERG,

BND

DJERASSI

taining both amino acids, the decrease in intensity of the positive MCD band at 292 mn in alkaline as coml)arctl to neutral solution might he expected to be proportional to the tyrosillc content. The validity of this statement depends on several factors. In reference 2, we discussed the conditions necessary for adclitivity of tryptophan contributions regardless of the particular protein under measurement. These conditions must, of course, hold for tyrosine as well. That the magnetic ellipticities measured for L-tyrosine ( [O],V = -5.6 X 10” at pH 6.0 and [0],,1 = - 1.08 X lOA at pH 11.7) are within 8% of the values measured for the dipeptide L-leucyl-L-tyrosine amide (see Fig. I ) shows that the mode of binding of the amino and carboxyl groups do not affect the observed magnetic ellipticity to an appreciable extent. A more likely source for error comes from the possibility that some of the tyrosyl residues may not be readily accessible to the solvent. This has been observed in numerous cases (6,7) and is commonly indicated when the tyrosine ionization is time-dependent. It is for this reason that we have made all of our measurements in 6 n/1 guanidine hydrochloride solution and then only after the solution has remained at room temperature for 12 hr. It, has been well established (8,9) that guanidine hydrochloride is a more effective denaturing agent than is urea. There were, however, two possible drawbacks to using guanidine hydrochloride. The pR value of the phenolic group in this system was observed to be 10.0 (4). Consequently, one would normally ensure that ionization was complet,e by using solutions of pH greater than 12. However, due to the weakly acidic properties of guanidine hydrochloride, large quantities of sodium hydroxide would be required to obtain pH values in this range. Furthermore, it is known that guanidine hydrochloride becomes unstable under these conditions (10). Fortunately, a careful study of several systems indicatecl that almost no further increase in the MCD or absorption curves could be noted above pH 11.7 (which corresponds to a 0.1 M NaOH solution). For an equimolar mixture of tyrosine and tryptophan, the ratio of the intensity of the 292 nm band at neutral pH t,o the observed intensity decrease at alkaline pH is 3.20. Consequently, the tyrosine: tryptophan ratio for an unknown mixture of these amino acids can be calculated quite simply from t.he relationship: tyrosine tryptophan

= 3w2o AAN - AAalk

where AA is the observed difference and illk stands for pH 11.7.

AAN

in absorbance,

N stands for pH 6.0,

TYROSIKE

: TRYPTOPHAN

TABLE Tyrosine:Tryptophan

No.

Protein Albumin (bovine) Trypsin (bovine) Ovalbumin (hen egg) Lysozyme (hen egg) Pepsin (swine stomach) Alcohol dehydrogenase (yeast’)

MW 64,000 23,560 46,000 14,300 36,970 150,000

ty’ 17.9 10 9.4 3 1S :x5 9 48

RATIO

1 Ratios

IN

475

PROTEINS

in Proteins

of tyr and trp (lit. valttes) trp I .s3 4 2.71 6 5 27.0 13

res.

ratio 9 7 2 5 :3 J 0.5 3 .6 2. 1 3 7

Ilef.

I: u b b c d

Tyr: trp ratdo as det,d. by JICI)

x0. of t,rp res. as calcd. from the ratio

10.3 3.53 3.36 0.51 3 50 2.94

1.75 3.95 2.80 5.90 5.15 19 0

a G. R. Tristam and R. H. Smith, Advan. Protein Chcm. 18, 227 (1963). b hI. 0. Dayhoff and R. V. Eck, “Atlas of Protein Sequence and Structure,” Vol. 4. The National Biomedical Research Foundation, Silver Spring, Maryland, 1969. c Ei. Wallenfels and A. Arens, Biochem. Z. 332, 217 (1960). d P. J. G. Butler and J. I. Harris, FEBS 5th Meeting, Prague, 1968, Abstr. NO. 714.

FIG. 2. MCD (upper curves) and UV (lower curves) at pH 6.0 (--) and pH 11.7 (---) in 6 M guanidine concentration (1.03 mg/ml) is uncorrected for water

spectra of albumin (bovine) hydrochloride solution. The content.

476

BARTH,

BUNNENBERG,

AND

DJERASSI

In order t,o evaluate the method in practice, we have carried out the determination of the tyrosine:tryptophan ratio for five proteins (see Table 1). The UV and MCD spectra of two of these, albumin and lysozyme, are also presented in Figs. 2 and 3 as examples of proteins with high and low tyrosine:tryptophan ratios, respectively. The values obtained are in good agreement with t.hose given in the literature with the exception of alcohol-dchydrogenase. The amino acid analysis of this enzyme has been made by Wallenfels and Arens and by But,ler and Harris (see references c and rZ of Table I). Both groups report different values for the tyrosine and tryptophan content with the discrepancy between their findings of the number of tryptophan residues being particularly large. Analysis of the tryptophan content by MCD (2) on protein samples from two different. sources gave the same number of residues, namely 12, a value which is in good agreement with the number given by Butler and Harris (reference d, Table 1). On the other hand,

l 4 x 7. E

FIG. 3. MCD (upper curves) and UV (lower curves) spectra of lysozyme at pH 6.0 (-) and pH 11.7 (- - -) in 6 AI guanidine hydrochloride solution. centration (0.285 mg/ml) is uncorrected for water content.

(hen The

egg) con-

TYROSINE:

Tryptophan

Analysis

TRYPTOPHAN

of Some

T.4BLE Prot.eins

RATIO

IN

477

PROTEIKS

2 by Magnetic

Circular

1 jichroism Trp

content, as detd. by MCIY

Protein Cow brain protein Pig brain protein” Ovomucoid (chicken)d Ovoinhibittrr (rhicken)“’

27,01)1) 4S,O’Y)

s :; 2.6 4.1 7

0 iFi I

0 cc;

:i I’* 0 !P’ 0 0 s.;

0 (S) 1 (F)

I (AH) Immunoglobiu L-chain
-

!). s

x .5’1

-

6 L

I)

-

10 7

-

11 .7” 1.i .) lrn

h)

0 $1 0.s I 1 “1

F, fluorescence spectroscopy. S, spectrophotometric. C, calorimetric. AH, alkaline hydrolysis. a& (l%, 1 cm) values are those obtained for the solutions on which MCD measurements have been carried out. * Measurements have been carried out in 0.01 M phosphate buffer at pH 680 unless indicated otherwise. c M. L. Kornguth, L. G. Tomasi, D. L. Keges, and S. E. Kornguth, B&him. Biophys. Acta 229, 167 (1971). d J. G. Davis, C. J. Mapes, aud J. W. Donovan, Biochemislry 10, 39 (1971). e J. C. Zahnley and J. G. Davis, Federution. Proceedings 30, 1234 Abs (1971). 1 W. H. Liu, G. E. Means, and R. E. Feeney, Biochirn. Biophys. Acta 229, 176 (1971). 0 J. Mestecky, J. Zikan, and W. T. Butler, Science 171, 1163 (1971). h H. Maeda, K. Kumagai, and K. N. Ishida, J. Antibiot. Ser. A 19, 253 (1966); H. Maeda and J. Meienhofer, Int. J. Protein Rcs. II, 1970, 135. ’ B. Muller-Hill, K. Beyreuther, and G. Gilbert, Mel/&s Enzytn. 21-D, 483 (1971). j A. T. Tu, B. Hong, and T. N. Solie, Biochemistry 10, 1285 (1971). k A. L. Schade and R. W. Reinhart, Biochem. J. 118, 181 (1970). r J. G. G. Schoenmakers and H. Bloemendal, Biochenl. Biophys. Res. Commun. 31, 257 (1968); J. G. G. Schoenmakers, J. T. T. Gerding, and H. Bloemendal, Eur. J. Riothem. 11, 472 (1969). m Measurement carried out in 6 2M guanidine hydrochloride solution at pH 6.8. ‘I Value was calculated for a molecular weight of 106.

478

BARTH,

BUNNENBERG,

AND

DJERASSI

our value for the tyrosine:tryptophan ratio imI)lies then the presence of 34 tyrosine residues which is lower than given in the literature. Because of t,he good agreement, that, WC have obtained for those enzymes listed in Table 1 we feel that, our value is correct and we therefore suggest a reexamination of the tyrosine content by other methods. Table 1 lists, in addition, the tryptophan content as determined from the tyrosine: tryptophan rat,io a,.q well as the tyrosine content, the latter values being accessible when the sample purity is known. These values are also in good agreement with the literature values. It should be pointed out, however, that the direct tryptophan determination by MCD will yield more accurate values if the protein concentration is precisely known, and t.hat the method described here should serve primarily as an independent check for the tyrosine and trypt,ophan values obtained by other methods. Although not directly connect’ed with the subject of this publication we wish to report analytical data that we have accumulated during recent months for several proteins whose tryptophan content was either unknown or else appeared to be uncertain. The data are summarized in Table 2. A particularly interesting example, and one that exemplifies the advantages of the MCD method, is chicken ovoinhibitor (see references e and f in Table 2) for which conventional methods have as yet not been able to ascertain whether tryptophan is present or not. Because of the large number (17 tyr) of tyrosine residues (11)) tryptophan was not detected by the usual spectrophotometric method. The same result was obtained using a calorimetric method (12). The only positive-result (one residue, references e and f, Table 2) report was based on the fluorescence spectrum and the amino acid analysis after hydrolysis in the presence of thioglycolic acid (13). These findings are supported by our MCD results, which demonstrate waequivocnlly that chicken ovoinhibitor contains one tryptophan residue. Similar ambiguous results were reported for neocarzinostat’in, for which the calorimetric method (13) gave a lower tryptophan content than either the hydrolytic or the spect’rophotometric procedures (see reference h, Table 2). Our value of 1.7 residues per mole is in close agreement wit.h the value obtained by the latter method. In summary, the availability of an independent. and very rapid method for tryptophan and tyrosine analysis is clearly advantageous. In view of the fact that most modern instruments designed for ordinary circular dichroism measurements are now equipped to accept magnets in their cell compartments, it becomes a relatively simple matter to carry out MCD measurements routinely.

TYROGINE:TRYPTOPHAN

RATIO

IN

PROTEISS

479

ACKNOWLEDGMENTS We gratefully acknowledge financial support by the Stanford Center for Materials Research and the National Institutes of Health (Grant AM-12758). For the generous supply of protein samples we wish to thank Professor H. Bloemendal (Department of Biochemistry, University of Nijmegen, The Netherlands), Professor J. Mestccky (Department of Microbiology, Unirersit,y of Alabama, Birmingham), Professor S. E. Iiornguth (Department, of Neurology and Physiological Chemistry, University of Wisconsin), Professor R. E. Feeney (Department of Food Science and Technology, University of California, Davis), Dr. J. W. Donovan (Department of Agriculture, Berkeley), Dr. A. L. Schnde (National Institutes of Health, Bethesda), Dr. H. Macda (Children’s Cancer Research Foundation, Inc., Boston), Profcssoi A. T. Tu and M. Reymond (Department of Biochemistry, Colorado State University), and Dr. H. Thielmann (Department of Pharmacology, Stanford University, Stanford). We are thankful to Mrs. Ruth Records, who carefully cnrricd out the measurements. REFERENCES 1. For

Part

A. Moscow~z, G. BARTH, E. B~NNENBERG, Sot. (submitted for publication). G. BARTH, W. VOELTER, E. BVNNENBEHG, ANII C. DJERASSI, J. Amer. C’hem. Sot. 94, 1293 (1972). G. BARTH, R. RECORDS. W. VOELTER, E. BUXNENBERG, AND C. DJERASSI, .I. Amer. Chem. sot. 93, 2545 (1971). S. R. HAWKINS AID J. H. HARSHMaN, Rev. fki. Instr. 38, 50 (1967). H. EDELHOCII, Biochemisfq 6, 1948 (1967). T. IV. GOODWIN AND R. A. MORTON, Biochem. J. 40, 625 (1946). G. H. BEAVAN AKD E. R. HOLIDAY, Adva~ Pro&u Chem. 7, 319 (1952). C. TANFORD, Advan. Protein Chem. 1’7, 69 (1962). D. B. WETLAUFER, Aduan. Protein Chem. 17, 303 (1962). C. TANFORD, Ii. KAWAKARA, AND S. L.~P.~NJE, J. Amer. Chem. sot. 89, 729 (1967). H. EDELHOCH AND R. F. STEINER, Biopol?Jmers 4, 999 (1966). P. r\‘oz.4~1 AND C. TANFORD, J. Amer. Chem. Sot. 89, 736 (1967). J. G. DAVIS, J. C. ZAHSLEY, AND J. W. DONOVAN, Biochemistry 8, 2044 (1969). G. R. SPIES AND D. C. CHAMBERS, Anal. Chem. 21, 1249 (1949). H. MATSUBARA AND R. M. SASAKI, Biochem. Biophys. Res. Commun. 35, 175 (1969). AND

2.

3.

4. 5. 6. 7.

5. 9. 10. 11. 12. 13.

C.

XVIII.

DJERAZSI,

see L. HK:~M.~Ns, J. Amer. Chetrr.