Feb. 5, 1960
TYROSYL HYDROGEN BONDSIN INSULIN
571
It might also be possible t o turn the method around, and use it t o isolate a pure protein Ag.Is Assuming that there is available a y-globulin fraction containing Ab reactive t o the desired Ag, one might thiolate it, use the thiolated Ab t o precipitate the Ag free of contaminating proteins, and then remove the thiolated Ab from the Ag by MMD precipitation a t acid pH.
We gratefully acknowledge the interest and support of Professor J. If.Sturtevant, from whose National Science Foundation grant G-2855, the postdoctoral research assistantship held by one of us (J.E.F.) was provided. This research was also supported in part by U. S. Public Health Service grants E-1204 (C2,3) to Professor S. J. Singer.
(18) S. S. Stone and R. R. Williams, Arch. Biockem. Biogkys., 71, 386 (1957).
NEWHAVEN,CONN.
[CONTRIBUTION FROM THE
DEPARTMENT OF CHEMISTRY, CORNELL UNIVERSITY ]
Tyrosyl Hydrogen Bonds in B Y M. LASKOWSKI, JR.,3a
s. J. LEACH3'
AND
H. A. SCHERAGA
RECEIVED JUNE 8, 1959 The ultraviolet absorption spectra of zinc- and zinc-free insulins a t pH 8 are modified either by acidification or by tryptic digestion. The character of the difference spectra produced by either treatment is very similar and appears to be due to the breaking of tyrosyl hydrogen bonds. Since the acid-induced shift still persists after tryptic digestion and is of undiminished magnitude, it is concluded that there are two tyrosyl residues which are hydrogen bonded as donors to two acceptor groups. The pH-dependence of the acid-induced shift suggests that one of the acceptors is a carboxylate ion side chain. In a 6000 mol. wt. insulin model, involving a-helices, the only tyrosyl-carboxylate ion hydrogen bond which can be formed is one between the B13 glutamic acid residue and the B16 tyrosyl residue. For the spectral shift induced by tryptic digestion, the B26 tyrosyl residue is implicated. I t has been shown that trypsin splits native insulin a t the same two bonds as in isolated B-chain, viz., at the B22-B23 and the B29-B30 peptide bonds. The former splitting releases one of the four tyrosyl residues in insulin as a heptapeptide fragment. The rate of splitting follows the rate of spectral change during tryptic digestion. Removal of the terminal B-30 alanyl residue by carboxypeptidase digestion has no effect on the spectrum. It is concluded that the B26 tyrosyl residue is hydrogen bonded to an acceptor which does not ionize the PH range 1.5 to 8.0. The validity of Beer's Law and the effect of dioxane and added salt on the difference spectra have been examined. I t is not yet possible t o decide whether the two tyrosyl hydrogen bonds are within the 6000 or 12,000 mol. wt. units of insulin.
titration data is considerably complicated by pHIntroduction When the amino acid sequence and the positions dependent changes in the degree of aggregation. of the disulfide cross links in a protein are known, Since titration curves provide no evidence for it should be possible t o determine the folding of the tyrosyl hydrogen bonds in insulin, the more senmolecule in solution by locating a few specific sitive and group-specific technique of difference interactions (e.g., hydrogen or hydrophobic bonds) spectrophotometry was employed in this investibetween side chains. The present work is an at- gation. Difference spectrophotometry has been used to tempt to apply this approach to the determination of the configuration of insulin4in solution by search- determine the p K values of ionizable groups in coming for possible side chain hydrogen bonds involv- pounds of tyrosine and tryptophan.'-1° It has also been used to determine the pK values of the ing tyrosyl residues as donors. Titration data6 on zinc- and zinc-free insulins acceptor groups involved with tyrosyl groups in have been interpreted to indicate that the tyrosyl hydrogen bonds in insulinll and in ribonuclease. 1 2 - 1 3 groups ionize normally. However, it should be A number of workers have also observed the difkept in mind that titration curves give only average ference spectra arising from shifts of the tyrosyl pK values over several groups and, in the case of absorption band in proteins, without investigating insulin, it has not thus far been possible to dis- the nature of the groups responsible for the perl particular it has not always tinguish between the four tyrosyl groups and the t u r b a t i ~ n . ' ~ - ~In single €-amino group6 from titration data.6 Fur(7) C. Fromageot and G. Schnek, Biochint. Biophys. Acla, 6, 113 thermore, the detailed interpretation of insulin (1950). (1) This investigation was supported by research grant No. E1473 from the National Institute of Allergy and Infectious Diseases, of the National Institutes of Health, U. S. Public Health Service. (2) Presented in part before the Division of Biological Chemistry at the 131st meeting of the American Chemical Society, Miami, Florida, April, 1957. (3) (a) Research Fellow of the National Heart Institute, U. S. Public Health Service, 1952-1956. (b) On Sabbatical leave of ahsence, 1956-1957, from the Division of Protein Chemistry of the Wool Research Laboratories, C.S.I.R.O., Melbourne, Australia. (4) A. P. Ryle, F. Sanger, L. F . Smith and R. Kitai, Biockem. J . , 60, 541 (1955). (5) C. Tanford and J. Epstein, THISJOURNAL, 76, 2163, 2170 (1954). (6) For a study of the thermodynamics of the ionization of the single lysyl amino residue in insulin see L. Gruen, hl. Laakowski, Tr., and € I . A. Scheraga, ibid , 81, 3891 (1950).
(8) G . W. Schwert and Y.Takenaka, ibid., 16, 570 (1955). (9) D. B. Wetlaufer, J. T. Edsall and B. R. Hollingworth, J . B i d . Ckem., 233, 1421 (1958). (10) J. W. Donovan, M. Laskowski, Jr., and H . A. Scheraga, Biochim. Biophrs. Acta, 29, 455 (1958). (11) M. Laskowski, Jr., J. M. Widom, M. L. McFadden and H . A. Scheraga, ibid., 19, 581 (1956). (12) H . A. Scheraga, i b i d . , 28, 196 (1957). (13) C. C. Bigelow and M. Ottesen, ibid., 82, 574 (1959). (14) D. Shugar, Biockem. J . , 62, 142 (1952). (15) W. F. Harrington and J. A. Schellman, C o m p t . rend. frau. Lab. Carlsberg., Ser. Ckim., 80, 21 (1956). (16) M. Sela and C. B. Antinsen, Biochim. Biopkys. Acta, 24, 229 (1957). (17) C. H. Chervenka.. ibid... 26.. 222 (1957): . , . 31. 85 (1959). . . (18) A. N . Glazer, H. A. McKenzie and K. G. Wake, Nature, 180, 1286 (10.57). (19) H. I'raenkel-Conrat, E ' e d r r n l i o 7 ~P r o c . . 1 6 . 810 (1957).
572
31. LASKOWSKI, JR.,
s. J . LEACH A N D H.x. SCHERAGA
been clear to what extent vicinal and other similar effects could be excluded. The distinctions between these possibilities will be examined later, The absorption spectrum of tyrosine in the 270290 mb region undergoes an enhancement in intensity and a shift toward longer wave lengths if the tyrosyl group becomes hydrogen bonded as a donor. X difference spectrum between the hydrogen -bonded and the non -hydrogen-bonded f orms shows two major peaks11f12 in the vicinity of 2SO and 2S7 mp. The tyrosyl hydrogen bonds in a protein can be broken in several different ways, e.g,., by acidification so that the acceptor group gains a proton, by treatment with urea or by enzymatic hydrolysis of peptide bonds leading to the liberation of one or more peptide fragments containing eithcr the acceptor group or the tyrosyl donor. Variation of the pH, in the acidification iiiethod, yields a titration curve for the acceptor group in which the pH is correlated with the extent of hydrogen bond breakage. I n this way the pK and identity of the acceptor group may be determined. l l , 1 2 , 2 ? It is important to note that in the technique of difference spectrophotometry the rupture of tyrosyl hydrogen bonds is detected under conditions (pH 2-8) where the tyrosyl hydroxyl group does not ionize. This is in contrast t o the method of Crammer and Keubergerz3in which the protein is treated with alkali to rupture the hydrogen bond, this occurring in the pH region of tyrosyl ionization. In the present work the diFference spectra produced by acidification and by enzytnatic hydrolysis of insulin have been investigated in more detail. I n the latter case data have been obtained for the rate of appearance of the difference spectrum, and this has been correlated with the rate of tryptic hydrolysis of the B22-B23 arg-g!y bond of the phenylalanyl-(R) chain. Digestion of insulin with carboxypeptidase indicates that the spectral changes are not produced by hydrolysis of the U29-I330 lys-ala bond. As regards thc spectral shift produced by acidification, evidence on the nature and location of the acceptor groups has been adduced by studying the spectral shifts at varying PI-I, ionic strength and concentration. In addition, by studying the pH-dependcnce of the spectral changes both before and after tryptic digestion, it has been possible t o show that there is more than one tyrosyl residue involved in hydrogen bonding. Xttenipts have been made t o interpret the data in terms of current hypotheses about the configuration of insulin in solution. In view of its importance to the discussion of the data obtained here, the amino acid sequence de( 2 0 ) 1s. J. Williams and J. 12. Poster, THISJ O U R N A L , 81,865 (1959). (21) 0. 0. Blumenfeld and G. E. Perlmann, J . Gen. Physiol., 42, 308 (19.jU) ( 2 2 ) J t shoi!ld, however, he pointed out t h a t more complex interpretations of this pK dependence are also possible. Ionization of a group in a protein may cause some cooperative structural changes leading t o breakage u f the hydrogen bond even though t h e ionizing g r o u p was not t h e acceptor.*Q While such a possibility should he twrrie in mind, it is not possible a t present t o prove it or rule i t out. A s will he e r n ~ ~ h a s i z e tin i the Discussion section. tyrosyl hydrogen bontiinji will be assumed t o be the origin of the spectral changes re1,urted here. (23) J . L. Crammer and A . Seuherger, Biochem. J . , S T , 302 (1943).
VOl. S'
duced by Sanger and co-workers4 is reproduced in Fig. 1, where the tyrosyl residues and free COOH groups are given special prominence.
Experimental Materials.-Crystalline zinc insulin was obtained from the Eli Lilly Co. (Batch No. 535,664). Zinc-free insulin was prepared, according to the directions of Dr. 0. K. Belirens, by dissolving 1 g. of zinc insulin in 60 rnl. of 0.025 N HC1 and dialyzing a t 0" for 5 days in a rocking dialyzer with frequent changes of 0.025 N €1'21. The zinc-free insulin was recovered by lyophilization and found to contain less than 0.0470 zinc compared t o 0.57% (1 atom of zinc per 12,000 mol. wt. dimer) for the dry crystalline starting material. The trypsin used was a Worthington twice-crystallized, salt-free product (Batch No. TRGOSF). The soybean trypsin inhibitor (STI) and carboxypeptidase were also Worthington preparations (Batch Nos. SI5308 and C0557D F P , the latter being DFP treated). The synthetic substrate p-toluenesulfonyl-L-arginine methyl ester hydrochloride (T.lMe) was obtained from the PI. hl. Chemical Co. Ltd., of Santa Monica, California. Ammonium acetate buffers and other salt solutions were prepared from analytical grade reagents. Ionic strengths were maintained at 0.10 except where otherwise stated. The standard KOH solutions were COz-freez4and HC1 solutions were diluted as required from glass-distilled, constantboiling HCI. De-ionized water was used throughout. Dioxane was purified by refluxing over solid S a O H for 12 hours, followed by fractionation over fresh solid NaOH. Insulin Methyl Ester.-Methylated insulin was prepared by Dr. M. L. McFadden according to the procedure of Mommaerts and Neurath . 2 5 Zinc insulin was suspended in 0.1 N HC1 in anhydrous methanol (100 mg. per 10 ml.) a t 25' for 24 hr. with occasional shaking. Complete solutinn was nnt effected in this time, hlethanol was removed by dialysis against 0.025 N HCI. The lyophilized insulin inethyl ester hydrochloride was soluble to the extent of a t least O . l ~in O 0.075 JI KCl below p H 5 . 3 . Zinc Analysis of Insulin.-Zinc analyses were carried out by Dr. L. Gruen using the Versene titration method developed by Flaschka.*B The sample was adjusted t o p H 8 t o 10 with an ammonia-ammonium chloride buffer and titrated with a standard solution of 0.01 d l Versene using a 0.1-ml. ultramicroburet and Erioehrome Black T indicator. The Versene was standardized agninst a standard solution of Zn++ prepared by dissolving the weighed metal in a small amount of triply glass-distilled HCl and diluting to volume with zinc-free water. The presence of insulin did not interfere with the zinc titration, as was verified by adding known amounts of zinc to solutirms of zinc-free insulin and obscrving that the amounts of zinc found in ashed and unashcd samples were the same xithin the experilnentd error. Spectrophotometry.-Spectrophotometric measurements were made manually with the Beckman model DU spectropliotometcr using a photomultiplier attachment at full sensitivity. The concentration of the insulin solutions varied from 0.1 to 0.574, and matched silica cells of 0.5 and 1 cm. light path, respectively, were used. Many of the results, however, have been converted to a standard basis of 0.570 insulin for a 1 cm. cell. Difference spectra were obtained by using reference solutions of identical protein eoncentration to the solution under examination.2' As will be discussed below, a difference spectrum arises from changes in the environment of the tyrosyl residues, produced either by pH changes or as a result of enzymatic digestion. I n either case, the optical density di.tferences ( A D ) are relatively small, arising as they do from shifts of only 1-2 mfi in the absorption spectra. It is therefore necessary to use comparatively concentrated insulin solutions of up to 0.5% which, even in 0.5 cm. cells, would have very high optical (24) I. h l . Kolthoff, Z . ~ n a lChe7n., 61, 48 (1922). ( 2 ; ) W. F. €I. M. Mommaerts a n d €1. Neurath, J . B d . Cketn., 186, 909 (1030). (26) II. I'laschka, A f i k u o Chemisi A n a l y s t , 4 2 , 84 (I!( (27) A similar u s e of the rnrthod of difference spectra f l J r the itccw rate determination of copper was made by I
