States of amino acid residues in proteins

BIOCHIMICA ET BIOPHYSICA ACTA

BBA

257

25 4OO

STATES OF AMINO ACID R E S I D U E S IN P R O T E I N S VII. H Y D R O G E N B O N D I N G OF T Y R O S I N E R E S I D U E S IN T H E I N S U L I N MOLECULE MASATERU AOYAMA, KENZO KURIHARA AND KAZUO SHIBATA The Tokugawa Institute for Biological Research, Mejiromachi, Tokyo*, and Tokyo Institute of Technology, Meguroku, Tokyo (Japan)

(Received November 27th, 1964)

SUMMARY

Cyanuric fluoride, which is a reagent employed to differentiate between free and bound or buried tyrosine residues in proteins, reacts with one of the two tyrosine residues in each peptide chain, A or B, of insulin. By digestion of cyanuric fluoridetreated insulin with trypsin (EC 3.4-4-4) and with chymotrypsin (EC 3-4-4-5), the two tyrosine residues not reacting with cyanuric fluoride were located in the amino acid sequence. The AI 4 and B26 tyrosine residues were thus found to be the nonreactive type, and the AI 9 and B I 6 residues were the reactive type. The non-reactive B26 residue was transformed into the reactive type b y tryptic digestion of native insulin, while the AI4 residue remained non-reactive or bound after tryptic or chymotryptic digestion. From these effects of digestion on the reactivity as well as on the rate of ionization of the tyrosine residues, the intra-chain hydrogen bonding between the side chains of the B26 tyrosine and the B22 arginine and the inter-chain hydrogen bonding between the side chains of the AI 4 tyrosine and the B I 3 glutamic acid were suggested.

INTRODUCTION Amino acid residues in proteins are in a variety of states depending on the location of each residue in the structure of protein and other amino acid side chains in the vicinity of the residue. Some residues m a y be exposed and free to react with chemicals, and others m a y be embedded in the interior of molecule or bound by hydrogen or hydrophobic bonding to other residues Since little is known of the pairs of amino acid residues hydrogen-bonded in proteins, various reagents were studied in this series of investigations 1-3 to differentiate between free and bound or buried residues chemically in terms of the reactivities of residues with the reagents. CyF, a reagent discovered for tyrosine residues, reacts moderately with free and exposed tyrosine residues, and the ultraviolet absorption band of tyrosine disappears during Abbreviations: CyF, cyanuric fluoride; DHT, diazonium-I-H-tetrazole; CyF-insulin, CyFtreated insulin. * Postal address. Biochim. Biophys. Acta, lO7 (1965) 257-265

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M. AOYAMA, K. KURIHARA, K. SHIBATA

reaction 1. The moles of tyrosine residues not reacting with the reagent can therefore be determined simply by absorption photometry and, in addition, the non-reactive tyrosine residues can be located in the amino acid sequence by photometry of the peptides obtained by enzymatic digestion or by cleavage of disulphide bonds of CyF-treated protein. Using CyF, the four tyrosine residues in the insulin molecule were successfully classified into three types ~. In the native state at neutral pH, two of these four residues were reactive with CyF and the other two were not reactive. By treatment with alkali, one of the non-reactive residues was transformed instantaneously into the reactive type, while it took about 2 h of alkali denaturation for the remaining residue to become reactive. This accords with the spectrophotometric titration data 4 that one of the four tyrosine residues ionized slowly with alkali while the other three ionized instantaneously. Cleavage of the disulphide bonds of CyF-treated insulin 1 revealed that the two non-reactive residues are located, one in the A chain and the other in the B chain. The present paper describes: (a) the more precise location of these non-reactive residues, which of the two tyrosine residues in each chain is the non-reactive type, and (b) the change in their reactivity and ionization rate by digestion with trypsin (EC 3-4.4.4) and with chymotrypsin (EC 3.4.4.5). Hydrogen bonding of these non-reactive residues is discussed from the results. Throughout this paper, the moles of tyrosine (histidine or arginine) residues reacted or not reacted with CyF per mole of protein is expressed by n.

EXPERIMENTAL

Materials Crystalline zinc beef insulin supplied by Shimizu Pharmaceutical Co. was recrystallized by the method of ROMANDS, SCOTT AND FISHER 5 as modified by SAWADA, SHIBATA AND ITANIe. CyF was prepared in the same manner as described previously 1. CyF-insulin was prepared b y the following procedure. CyF in dioxane (75 ml) was added to a solution of insulin (200 rag) in I.O M bicarbonate buffer (600 ml) of p H 9-7. The concentration of CyF in the reaction mixture was 16. 7 mM, and the mixture was left standing at room temperature for 3 h, during which approximately two moles of tyrosine residues per mole of insulin reacted with CyF as demonstrated previously 1. CyF-insulin was precipitated with I.O M trichloroacetic acid, and washed several times with I.O M trichloroacetic acid solution and then with ether.

Digestion with trypsin A solution of trypsin (I mg) in I ml of dilute HC1 solution with 2 mM CaCI~ was added to 9 ml of a solution of CyF-insulin (50 mg) in 0.2 M acetate buffer (pH 8.0). The digestion was carried out at 23 ° for IOO h. Trypsin (Sigma Chemical Co., Type I) used for the digestion was pretreated with 0.063 N HC1 for 24 h at 37 ° to minimize chymotryptic activity. The digest was applied to a Sephadex GM-25 column, 200 m m long and 23 m m in diameter. On development with distilled water at a flow rate of 0.5 ml/min, 4-ml fractions were obtained on a fraction collector, and the contents of tyrosine and histidine in each fraction were determined. Native insulin was digested with trypsin under the same experimental conditions. Biochim. Biophys. Acta, lO 7 (1965) 257-265

STATES OF TYROSINE RESIDUES IN INSULIN

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Digestion with chymotrypsin CyF-insulin (50 rag) in I I ml of o.I M phosphate buffer (pH 8.0) was digested at 25 ° with I mg of a-chymotrypsin, a-Chymotrypsin was obtained b y the slow activation 7 of chymotrypsinogen prepared b y the method of KUNITZ AND NORTHROP8 and was recrystallized with ammonium sulfate. During the digestion, an aliquot (2 ml) was taken at intervals from the digestion mixture, and I ml of 3 M trichloroacetic acid solution was added to each aliquot to precipitate the chymotryptic core. The contents of tyrosine, histidine and arginine in the supernatant were determined and plotted against digestion time. Native insulin as well as the tryptic core derived from CyF-insulin was digested with chymotrypsin for IO h under the same experimental conditions.

Assay of amino acids The concentration of tyrosine residues not reacted with CyF was estimated b y the same procedure as described previously 1,4 from the change, AA295mv, in absorbance at 295 m/~ on addition of alkali to the sample solution. The concentration of histidine residues was determined b y the use of DHT, a new coupling reagent for histidine ~. Bis-coupling of D H T to histidine residues was achieved at p H 9.2, and the absorbance reading, A480m#, at 480 m/~ for histidine-bis(azo-i-H-tetrazole) residues thus formed was reduced to the molar histidine concentration with aid of the coefficient, e~t = 168o0 M-~ cm -~ (see ref. 2). Arginine concentrations were determined b y the Sakaguchi reaction with 8-oxyquinoline and N-bromosuccinimide 9-n. The heptapeptide released by the tryptic digestion of CyF-insulin and eluted from a Sephadex column was identified from its amino acid composition b y two-dimensional paper chromatography with Toyo No. 51 paper after hydrolysis with 6 N HC1. Development in one dimension was effected with butanol-acetic a c i d - w a t e r (4:1:2, v/v) and in the other dimension with water-saturated phenol. RESULTS

The bound tyrosine residue in the B chain Curve A in Fig. I is a reproduction of the curve for native insulin in Fig. 6 in the previous paper 1, and shows that two out of the total four tyrosine residues in the insulin molecule react with CyF in the native states. Curve B obtained for the tryptic digest is remarkably different, and rises to a higher plateau of n ---- 3.1. This indicates that one of the two non-reactive residues is transformed b y the digestion into the reactive type. The digest was made alkaline to see whether the remaining non-reactive residue was of the rapidly or slowly ionizing type. The value of AA295m/~ read immediately after exposure to alkali corresponded to n---- 3.2, and the value increased slowly to the level of n = 4.0 during incubation for 2 h. It is, therefore, evident that the remaining non-reactive residue is the slowly ionizing type. The four tyrosine residues are located at the positions AI4, AI9, B I 6 and B26 in the amino acid sequence 12 as shown in Fig. 2. The above result strongly suggests that the non-reactive residue transformed b y tryptic digestion into the reactive type is the B26 residue, since the digestion releases the B3o alanine and the heptapeptide containing the B23 to B29 residues (see dashed arrows with Tr in Fig. 2) 1.-~8. In order to determine the distribution of the non-reactive tyrosine residues between the Biochim. Biophys. Acta, lO 7 (1965) 257-265

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M. AOYAMA, K. KURIHARA, K. SHIBATA

heptapeptide containing the B26 tyrosine and the tryptic core containing the other three tyrosine residues, native insulin was pretreated with 16. 7 mM CyF, and the CyF-insulin thus obtained was digested with trypsin. The sample of CyF-insulin contained 1. 9 and 0.8 moles of intact tyrosine and histidine per mole of protein, Tryptic digest C

ve~nsulin

0-0

10

20

30

[CyF] (mM) Fig. I. The value of n (moles of tyrosine residues reacted with CyF per mole of insulin) plotted against CyF concentration; Curve A, native insulin; Curve B, tryptic digest of native insulin.

respectively. Fig. 3 shows the distributions of these intact tyrosine (Curve A) and histidine (Curve B) in the fractions obtained b y chromatographic separation of the digest on a Sephadex column. The first component between tubes IO and 17 contained both tyrosine and histidine, while the second component above tube number 20 contained no histidine. Since histidine is contained in the tryptic core and not in the heptapeptide, the first component must be the tryptic core and the second component m a y be the heptapeptide and the B3o alanine. In fact, the two-dimensional paper chromatogram of the hydrolysate of the second component showed the spots of the amino acids in the heptapeptide and alanine without any trend of contamination with other amino acids. Referring to the distribution of histidine, Curve A for the tyrosine distribution was analyzed into component curves to estimate the relative peak areas.The relative tyrosine contents thus estimated for the first and the second components were in the ratio of 1.o:o.7, indicating that the non-reactive tyrosine residues are distributed almost evenly between the tryptic core and the heptapeptide. This reveals that the B26 tyrosine residue is one of the non-reactive residues. Since the other non-reactive residue was identified previously in the A chain 1, the B I 6 residue must be of the reactive type. The bound tyrosine residue in the A chain The chymotryptic core contains the AI 4 and B I 6 tyrosine residues, and the Biochim. Biophys. Acta, lO7 (1965) 257-~65

STATES OF TYROSINE RESIDUES IN INSULIN

261

AI 9 and B26 residues are in other part of peptide (see the arrows with CTr in Fig. 2). The non-reactive tyrosine residue in the A chain was, therefore, determined by the following experiment: measuring the distribution of the non-reactive residues between the chymotryptic core and other peptides derived from CyF-insulin. The sample of CyF-insulin containing 2.3, I.I and 0.6 moles of intact tyrosine, histidine and arginine, S--S

CTr

A Cho n 5 10 G l y . I r e " V ( : I G l u - G l n . C y s - C y $ , A i o - S e r . V o f . C y s . See. L e u . TL/~j. G I n - L e U . G I u . A S n . [ T y r ) ' C y s . A s t l - C O O H

Phe.Vol-Asn.GIn-His. Leu-Cys Gly. Ser.His - Leu.Vol • Glu.AIo Leu,~.Leu.Vol.Cys.Gly.Glu.Arg-Gly-Phe,Phei[~.The.Peo.Lys~Aio-COOH 5 10 5 k /~ ~0 ! 25 ~ 130 r I I I ' t I I i i i I i CTP Tr CTP C T r TP

Fig. 2. H y d r o g e n bonding of the tyrosine residues in the insulin molecule. Tyr in a circle and in a square indicate CyF reactive and non-reactive tyrosine residues, respectively; solid arrows indicate the inter-chain and the intra-chain hydrogen bonding of the non-reactive tyrosine residues. Dashed arrows indicate the peptide bonds hydrolyzed with trypsin (Tr) and with c h y m o t r y p s i n (CTr).

respectively, was digested with chymotrypsin, and I.O M trichloroacetic acid was added to each aliquot taken at intervals during 9 h of digestion, to measure the distributions of these amino acids between the precipitate and the supernatant. The curves in Fig. 4 show the fractions in per cent of these amino acids in the supernatant. According to the amino acid sequence, the chymotryptic core should contain histidine but no arginine, while other peptides released by the digestion should contain arginine but no histidine. The presence of only 13 % (n -----o.I) of the intact histidine and more than 95 % (n---- 0.6) of the intact arginine identified in the supernatant after 6 h digestion indicate that the crude way of separation with trichloroacetic acid was nearly complete. As seen from Curve B, 54 % (n ---- 1.2) of the intact tyrosine residues were found in the supernatant, and the remaining 46 % (n = I.I) were in the pre-

A

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u

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Fig. 3. The distributions of intact tyrosine (Curve A) and histidine (Curve B) residues in t h e efttuents obtained by chromatographic separation of the tryptic digest of CyF-treated insulin on a Sephadex column. CyF-insulin before tryptic digestion contained I. 9 and o.8 moles of intact tyrosine and histidine per mole of insulin, respectively.

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M. AOYAMA, K. KURIHARA, K. SHIBATA

cipitate. This indicates t h a t the A I 4 tyrosine residue is the non-reactive t y p e because the B I 6 residue was f o u n d above to be the reactive t y p e . The presence of one mole of i n t a c t t y r o s i n e in the s u p e r n a t a n t is consistent w i t h the a b o v e o b s e r v a t i o n t h a t the B26 residue is the n o n - r e a c t i v e type. 100-

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= 0.6)

80

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Fig. 4- Chymotryptic digestion of CyF-treated insulin containing 0.6, 2. 3 and I.I moles of intact arginine, tyrosine and histidine residues per mole of insulin, respectively. Curves A, ]3 and C show the changes during digestion of the percent distributions of these intact amino acids in the supernatant obtained by addition of I.O iV[ trichloroacetic acid to an aliquot taken from the digestion mixture. This conclusion was reconfirmed in a different m a n n e r b y c h y m o t r y p t i c digestion of the t r y p t i c core d e r i v e d from CyF-insulin. The same sample of CyF-insulin as used for the above e x p e r i m e n t was digested w i t h t r y p s i n , a n d t h e t r y p t i c core was p r e c i p i t a t e d w i t h I.O M trichloroacetic acid. The t r y p t i c core, which should contain the residues A I 9 a n d B I 6 r e a c t e d w i t h C y F a n d also the i n t a c t A I 4 residue according to the a b o v e conclusion, was n e x t digested w i t h c h y m o t r y p s i n . T a b l e I shows the d i s t r i b u t i o n of i n t a c t t y r o s i n e b e t w e e n the p r e c i p i t a t e a n d t h e s u p e r n a t a n t o b t a i n e d b y a d d i t i o n of I.O M trichloroacetic acid to the digest, a n d indicates t h a t almost all of t h e i n t a c t t y r o s i n e is p r e s e n t in the p r e c i p i t a t e c o m p o s e d m o s t l y of t h e c h y m o t r y p t i c core. The d i s t r i b u t i o n s of histidine a n d arginine shown in the t a b l e i n d i c a t e n e a r l y complete s e p a r a t i o n of the core from o t h e r p e p t i d e s a n d vice versa b y the a d d i t i o n of trichloroacetic acid. I t was t h u s confirmed t h a t the A I 4 residue is the non-reactive t y p e , so t h a t the r e m a i n i n g A I 9 residue should be the reactive t y p e . As described above, one of the non-reactive residues is t r a n s f o r m e d b y t r y p t i c digestion into the reactive t y p e , while the o t h e r r e m a i n s n o n - r e a c t i v e after t h e digestion a n d requires a few hours of Biochim. Biophys. Acta, lO7 (1965) 257-265

STATES

OF TYROSINE

RESIDUES

IN

263

INSULIN

alkali denaturation to ionize or to become reactive. Considering the peptide bonds hydrolyzed with trypsin, the residue freed by the digestion might be the B26 residue rather than the A I 4 residue. More conclusive evidence for this view was obtained by chymotryptic digestion of native insulin, in which the B26 tyrosine residue should be completely freed by the hydrolysis of the B25-26 and B26-27 peptide bonds. TABLE

I

THE PERCENT DISTRIBUTIONS OF INTACT TYROSINE, HISTIDINE AND ARGININE IN THE PRECIPITATE AND THE SUPERNATANT OBTAINED BY ADDITION OF I.O 1~ TRICHLOROACETIC ACID TO THE CHYMOTRYPTIC DIGEST OF THE TRYPTIC CORE DERIVED FROM C Y F - I N S U L I N

Tyrosine Histidine Arginine

Precipitate

Supernatant

9o 86 23

IO 14 77

Native insulin was digested with chymotrypsin for 9 h, and the digest was made alkaline to observe the ionization rate of the tyrosine residues. Here again, the 2A295m# value read immediately after exposure to alkali corresponded to n ---- 3.1, and the remaining o. 9 mole of tyrosine residue ionized slowly at approximately the same rate as observed for native insulin as well as for the tryptic digest. The A I 4 residue was thus proved to be the one which ionizes and becomes slowly reactive with alkali. DISCUSSION

In the present study, the three types of tyrosine residues in the insulin molecule were located in the amino acid sequence. They were the AI 9 and B I 6 residues which are reactive with CyF and ionize rapidly with alkali, the B26 residue which is not reactive but ionizes rapidly, and the AI 4 residue which is not reactive and ionizes slowly. As reviewed previously 1, SCHERAGAet al. 17-19 made an interesting observation which m a y be correlated with our results. They found a shift of the tyrosine band toward shorter wavelengths on tryptic digestion of native insulin and also on acidifying the tryptic digest. They concluded from these results that there exist two bound tyrosine residues in the insulin molecule; the B26 residue hydrogen-bonded to a non-ionizable acceptor and freed by tryptic digestion, and another residue hydrogenbonded to an ionizable acceptor and freed at an acidic pH. These results are parallel with our results on the reactivities with CyF and their changes by digestion. On the other hand, the reactivities with iodine studied by DE ZOETEN et al.~°, 2~ were rather intricate and can hardly be correlated with our results as reviewed previously 1. The lack of the reactivity with CyF m a y be interpreted in two ways; the effect of hydrogen bonding of the tyrosine residue or the steric hindrance by other residues. Of these possibilities, the authors would propose the former mechanism as being more likely in this case of insulin for the following reasons. i. The blue shift observed b y SCHERAGA et al. indicated the abnormality of the B26 residue and a residue in the tryptic core, this result being consistent with the lack of reactivity found for the B26 and AI 4 residues. The lack of reactivity may, Biochim. Biophys. Acga, IO7

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M. AOYAMA, K. KURIHARA, K. SHIBATA

therefore, arise from an abnormal state of the tyrosine residue and not from the effect of another residue not interacting with the tyrosine residue. 2. Dissociation of a small hydrogen ion from a tyrosine residue may be less affected sterically than the accessibility of CyF to the tyrosine residue. The slow ionization of the AI 4 residue in native insulin as well as after chymotryptic digestion suggests hydrogen bonding of the AI 4 residue. 3- The steric effect may be less important in the insulin molecule than in a globular protein containing many residues in the interior of molecule. Hydrogen bonding in the tertiary structures of protein may be classified into two types; inter-chain hydrogen bonding between residues in different peptide chains, and intra-chain hydrogen bonding between nearby residues in a single peptide chain. Intra-chain bonding of a residue in an a-helical peptide structure is possible not only with its two neighboring residues but also with the 3rd and 4th residues, because one turn in the helical coiling brings the 3rd and 4th residues close to the residue one helical pitch behind. If the above inference of hydrogen bonding of the AI 4 and B26 residues is correct, the counterparts of these residues in hydrogen bonding may be deduced as follows. I. The hydrophilic residues, which are located at the Ist, 3rd or 4th position from the B26 residue, are the B22 arginine, the B29 lysine and the terminal carboxyl group of the B3o alanine. The transformation of the non-reactive B26 residue into the reactive type by tryptic digestion and the lack 17 of the blue shift on hydrolysis of the B29-3o bond with carboxypeptidase eliminate the possibilities of bonding to the B29 lysine and to the terminal carboxyl group, respectively. The remaining possibility is, therefore, bonding to the guanidyl group of the B22 arginine. 2. The fact that the AI 4 residue is strongly bound even in the chymotryptic core free from the neighboring AI5 glutamine, the 3rd AI 7 glutamic acid and the 4th AI8 asparagine, eliminates intra-chain hydrogen bonding to these residues. The remaining possibility is, therefore, inter-chain hydrogen bonding with a residue in the B chain, and bonding with the BI 3 glutamic acid may be most probable as judged from its position. This inference is supported by the view of SCHERAGA et al. that the counterpart of the residue causing the blue shift on acidifying the tryptic digest is an acidic residue. These possibilities of hydrogen bonding are indicated by solid arrows in Fig. 2. REFERENCES I K. KURIHARA, H. HORINISHI AND K. SHIBATA, Biochim. Biophys. Aeta, 74 (1963) 678. 2 H. HORINISHI, Y. HACHIMORI, K. KURIHARA AND R. SHIBATA, Bioehim. Biophys. Acta, 86 (1964) 477. 3 Y- HACHIMORI, H. HORINISHI, K. KURIHARA AND K. SHIBATA, Biochim. Biophys. Acta, 93 (1964) 346 . 4 Y. INADA, J. Biochem. Tokyo, 49 (1961) 217. 5 m. G. ROMANS, D. A. SCOTT AND A. M. FISHER, Ind. Eng. Chem., 32 (194 o) 908. 6 T. SAWADA, T. SHIBATA AND A. ITANI, Kagakuto Kogyo Tokyo, I I (1958) lO 9. 7 Y. INADA, M. KAMATA, A. 1V[ATSUSHIMA AND K. SHIBATA, Bioehim. Biophys. Acta, 81 (1964) 323 . 8 M. KUNITZ AND J. H. NORTHROP, J. Gen. Physiol., 18 (1935) 433. 9 S. SAKAGUCHI, J. Biochem. Tokyo, 5 (1925) 25i o S. SAKAGUCHI, J. Biochem. Tokyo, 37 (195o) 231. i i I. SZlLOGYI AND I. SZABO, Nature, 181 (1958) 52. 12 A. P. RYLE, F. SANGER, L. V. SMITH AND R. KITAI, Biochem. J., 6o (1955) 541. 13 F. SANGER AND FI. TtlpPY, Biochem. J., 49 (1951) 463 •

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F. SANGER AND H. TUPPY, Biochem. J., 49 (1951) 481. F. SANGER AND E. O. P. THOMPSON, Biochem. J., 53 (1953) 353. F. SANGER AND E. O. P. THOMPSON, Biochem. J., 53 (1953) 366. M. LASKOWSKI Jr., S. J. LEACH AND H. A. SCHERAGA, J. Am. Chem. Sot., 82 (196o) 571. S. J. LEACH AND H. A. SCHERAGA, J. Am. Chem. Sot., 82 (196o) 479oM. LASKOWSKI Jr., J. M. WIDOM, M. L. McFADDEN AND H. A. SCHERAGA, Biochim. Biophys. Acla, 19 (1956) 581. 2o L. W . DE ZOETEN AND O. A. DE BRUIN, Rec. Tray. Chim., 80 (1961) 907. 2I L. W. DE ZOETEN AND E. HAVlNGA, Rec. Tray. Chim., 80 (1961) 917. 14 15 16 17 18 19

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