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An analysis of the near ultraviolet circular dichroism of insulin
I45
BIOCHIMICA ET BIOPHYSICA ACTA BBA 35225
AN ANALYSIS OF T H E N E A R U L T R A V I O L E T CIRCULAR D I C H R O I S M OF INSULIN
J. w . S. MORRIS, DANIEL A. MERCOLA AND EDWARD R. ARQUILLA
Department o[ Biophysics and Department of Pathology, Center for the Health Sciences, University of California, Los Angeles, Calif. (U.S.A.) (Received December 28th, 1967) (Revised manuscript received February 27th, 1968)
SUMMARY
I. The near ultraviolet circular dichroism (CD) of insulin has been studied as a function of p H and concentration. The CD spectrum at p H 7 shows a close correspondence with the absorption spectrum, with a peak at 276 m/z, a shoulder at 283 m/z, and short wavelength fine structure. 2. At p H 11.5 the disappearance of CD at 275 m# in time correlates with the increase in absorbance at 293 m#. At low pH and high concentrations the CD follows an ionization curve with a p K of 3.0. At p H 2, the CD is concentration dependent. 3. The above results are shown to be consistent with the known behavior of tyrosine residues A-I 4 and B-26. It is concluded that the near ultraviolet CD of insulin at p H 7 is due to tyrosine and phenylalanine. The C-terminal end of the B-chain is tentatively identified as the monomer-dimer aggregation site.
INTRODUCTION
Circular dichroism (CD) bands of proteins in the near ultraviolet have been associated with optically active side chain chromophores: mainly, aromatic residues and cystine 1. These chromophores absorb in the neighborhood of 270 m/z; thus the particular chromophore associated with a CD band in this region cannot be identified without further elucidation. In the case of ribonuclease, for example, the CD band at 277 m/~ can be ascribed to tyrosine with some certainty, since the well-known spectral shift of tyrosine with increase in p H is accompanied by a shift of the CD band to higher wavelengths 1. Moreover, the CD shift is remarkably similar to that observed in the model compound, N-acetyl-L-tyrosinamide 2. BEYCHOK3,4 has suggested that the near ultraviolet CD of insulin is due to cystine because of the susceptibility of the near ultraviolet CD to disulfide-reducing agents and because the CD exhibits a blue Abbreviation: CD, circular dichroism.
Biochim. Biophys. Acta, 16o (1968) I45-15o
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J, w . s. MORRIS, D. A. MERCOLA, E. R. ARQUILLA
shift rather than a red shift as the pH is raised above IO. This report presents data which suggest that the CD of insulin at neutral and acid pH is due to tyrosine and phenylalanine residues. MATERIALS AND METHODS
The insulin used here was a IO × recrystallized bovine preparation containing 0.74 °/o Zn, with a biological activity of 26 I.U.*. Ribonuclease was purchased from Sigma (Type XII-A). Solutions were prepared with CO2-free H20 and examined immediately at 23 °. CD measurements were made with the prototype of the DurrumJasco ORD/UV 5. The instrument was calibrated daily with camphor sulfonic acid, which also served as a wavelength calibration. All measurements at neutral and acid pH were made on solutions of I ± 0.o5 A 276 mu unit at a constant slit width ofo.8 mm. Under these conditions, successive scans of the same sample virtually superimposed. Spectra and concentrations were obtained with a Cary Model 15 spectrophotometer. Concentration values in the legends and text show ranges and indicate that measurements were made on samples with concentrations within 5 ~o of quoted values. CD results are given as AER: (AL -- AR) × 1 per mole of tyrosine residues per cm. pH measurements were made on a Radiometer 22 pH meter. Difference spectra of 1.25 mg/ml insulin, o.I M borate buffer, p H 8.45, and o.o 5 °/o fl-mercaptoethanol versus 1.25 mg/ml insulin, o.I M borate buffer, pH 8.45, and o.o5 % ethanol were obtained by the use of tandem cells 5. Spectra were recorded with four matched I-cm cuvettes at 15.5 ° and at 25 m#/min. Baselines were recorded with either buffer alone, or with insulin versus insulin treated with ethanol. The two methods gave identical results. RESULTS
Fig. I a shows the near ultraviolet CD spectrum of insulin at p H 7. The main features are a peak at 276 m#, a shoulder at 283 m#, and short wavelength fine structure. These features compare well with the corresponding features of the absorption spectrum (Fig. ib). Since no tryptophan is present 6, only tyrosine and phenylalanine contribute appreciably to the absorption spectrum. The similarity of the short wavelength fine structure of absorbance and CD, and the position of the optical rotatory dispersion fine structure of L-phenylalanine 7 permits the tentative assignment of the insulin CD fine structure to phenylalanine. The CD spectrum of insulin at pH 2, 0.2 mg/ml is shown in Fig. IC. No shoulder or fine structure is noted. However, at p H 2, IO mg/ml, the shoulder and fine structure are still apparent, although the band is reduced in intensity. The disappearance of the shoulder and fine structure at pH 2 is therefore concentration dependent. As a control, the CD of ribonuclease, which has a near ultraviolet CD spectrum very similar to that of insulin (cf. ref. 2), was run in the same cells at p H 7 over a similar 5o-fold concentration range. The magnitude of the main CD band and the presence of the long wavelength shoulder and short wavelength fine structure was unaltered, thus ruling out instrumental and pathlength artifacts. * L o t No. o16666, k i n d l y supplied b y Dr. SCHLICHTKRULL,N o v o l n d u s t r i , A/S, Copenhagen, D e n m a r k .
Biochim. Biophys. Acta, i6o (1968) 145-15o
CIRCULAR DICHROISM OF INSULIN
147 -1.10
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Fig. I. The near ultraviolet CD and a b s o r p t i o n spectra of insulin. Fig. Ia is the CD s p e c t r u m of insulin, i mg/ml, i-cm path, in o.Io M p h o s p h a t e buffer, p H 7.0. Fig. I b is the a b s o r p t i o n spect r u m of the same sample. Fig. ic is the CD s p e c t r u m of insulin, 0.2 mg/ml, 5-cm path, in o.io M KC1-HC1, p H 2.0. Fig. 2. The p H dependence of insulin CD. Conditions: Fig. 2a: concentration IO mg/ml, I - m m p a t h . Fig. 2b: I mg/ml, i-cm path. Fig. 2c: 0.2 mg/ml, 5-cm path. No m e a s u r e m e n t was possible for io mg/ml, p H 7, due to insolubility. Samples at p H 7 were in o. Io M KC1, o.oi M phosp h a t e buffer. All other samples were in o.io M KC1-HC1. Measurements were made at 276 m u for Fig. 2a, 275-276 m?, for Fig. 2b, and varied from 275 m u at p H 7 to 272 m # at p H 2 for Fig. 2c. The d a t a of Fig. 2a have been fitted to an ionization curve with an a p p a r e n t p K of 3.0 (cf. refs. 8, 9).
Fig. 2 shows the pH dependence of CD at three concentrations. At IO mg/ml, the magnitude of the CD band follows an ionization curve with an apparent pK of 3.0 (Fig. 2a), while at I mg/ml and 0.2 mg/ml the behavior is more complicated. Fig. 3 gives the time dependence of the CD of insulin at pH 11. 5. The disappearance of dichroism at 275 m# is correlated with the increase in absorbance at 293 m#, the absorption maximum of the ionized (phenoxy) form of tyrosine. The initial value of absorbance at 293 m# corresponded to the ionization of 3.0 tyrosine
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Fig. 3. The time dependence of insulin CD at p H i 1.5. Conditions: I mg/ml, i - c m path, in o.Io M K C 1 - K O H , p H 11.5 .Solid line: Increase in absorbance at 293 m/~. Points: decrease in circular dichroism at 275 m~. Note the initial lag (cf. ref. 3). Fig. 4. The near ultraviolet difference s p e c t r u m of insulin treated with fl-mercaptoethanol (reference beam) versus insulin treated with ethanol (sample beam) as described in MATERIALS AND METHODS. The s p e c t r u m was recorded IO rain after the solutions were prepared. The spect r u m above 275 m/, was i n d e p e n d e n t of additional increments of fl-mercaptoethanol to the sample b e a m buffer blank.
B i o c h i m . B i o p h y s . Acta, x6o (1968) 145-15o
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MORRRIS, D. A. MERCOLA, E. R. ARQUILL
residues per insulin monomer (cf. ref. 8 and DISCUSSION). As reporteda, 4, the near ultraviolet CD of insulin under these conditions shows no extremum, so the selection of 275 m# is somewhat arbitrary. The reduction of disulfide bonds by/5-mercaptoethanol has a profound effect on the near ultraviolet CD spectral, 4. Therefore, such reduction should also demonstrate a change in the environment of tyrosine residues. Accordingly, a difference spectrum of insulin treated with fl-mercaptoethanol v e r s u s insulin treated with ethanol was recorded (see MATERIALSAND METHODS). The difference spectrum (Fig. 4) with maxima at 278 and 285.5 m# is very similar to insulin tyrosine difference spectra previously describedg, 1°. At the concentration used here, the perturbation of one tyrosine residue per insulin monomer would correspond to o.I A 2 s . m/2 unit (refs. 9, IO). DISCUSSION
It has been shown above that the effect of mercaptoethanol on the near ultraviolet CD does not imply that cystine is the optically active chromophore, since one or more tyrosine residues are also perturbed. Moreover, the failure of the CD band to shift red with tyrosine ionization does not exclude this chromophore from consideration, since there is no a p r i o r i reason to expect that strongly dichroic tyrosine residues in the native molecule would maintain their optical activity when ionized. Since there is no reason to eliminate aromatic amino acids from the CD spectrum at neutral pH, the contribution of tyrosine and phenylalanine to the CD spectrum will be discussed. Difference spectrophotometry studies by SCHERAGA and co-workers% 1° have shown that the B-26 tyrosine residue of insulin is 'anomalous' in that a blue shift is observed when the C-terminal eight amino acids of the B-chain are removed by tryptic digestion. They conclude that this residue is hydrogen bonded to a non-ionizable acceptor, although YANARI AND BOVEY11 have shown that the spectral shift is compatible with the breaking of a hydrophobic interaction. LASKOWSKI, LEACH AND SHERAGA10 also observe a spectral shift when the tryptic digest is acidified, which they take to indicate that an additional tyrosine residue is hydrogen bonded to an ionizable acceptor; presumably, a carboxyl group. Further, it is well knownS,12,13 that three of the four tyrosine residues of insulin ionize normally, with an apparent p K of IO, while the fourth ionizes slowly with a p K of 11. 4. AOYAMA, KURIHARA AND SHIBATA12 have shown that the slowly ionizing tyrosine is located at position A-I4, and that this tyrosine residue is likely the one which shows a spectral shift upon acidification. Thus, at least two of the four tyrosine residues of insulin have been shown to interact, and might be expected to exhibit strong optical activity due to hindered rotation 1. The CD spectrum of insulin at pH 7 (Fig. Ia) shows a close correspondence to the absorption spectrum. The pH dependence of CD at high concentrations (Fig. 2a) is similar to the pH dependence of the tyrosine difference spectrumg, 1°. At p H 2, where tyrosine A-I 4 would not be appreciably hydrogen bonded, the CD spectrum is concentration dependent (Fig. 2). It therefore would seem that the environment of the concentration dependent chromophore is sensitive to aggregation. JEFFREY AND COATES1~ have studied the aggregation of zinc-free insulin at pH 2 and ionic strength o.Io by ultracentrifugation. Their model assumes a m o n o m e r dimer-tetramer-hexamer equilibrium, and predicts that the weight fraction of Biochim. Biophys. Acta, 16o (1968) 145-15 °
CIRCULAR DICHROISM OF INSULIN
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monomers increases from IO % at IO mg/ml to 50 % at I mg/ml, at which point the only species present are monomer and dimer. RUPLEY, RENTHAL AND PRAISSMAN15 have shown by difference spectrophotometry that insulin under the same conditions exhibits a concentration dependent tyrosine blue shift on dilution. Their data correlate well with the model of JEFFREY AND COATES, and they conclude that the tyrosine difference spectrum is due to the monomer-dimer equilibrium, and that one tyrosine residue per monomer is involved. This aggregation-dependent tyrosine blue shift is analogous to the concentration dependence of the magnitude and position of the near ultraviolet CD band at pH 2 (Fig. 2). Further, several observations implicate the B-26 tyrosine as the chromophore of the concentration-dependent CD spectrum. As noted above, at high concentrations the presumed phenylalanyl CD fine structure is insensitive to low pH, and is lost only in the dissociation to monomers. Two of the three phenylalanyl residues of insulin are located in the C-terminal end of the B-chain. Moreover, they are adjacent to tyrosine B-26 e. If the C-terminal B-chain were the monomer-dimer aggregation site, the CD of both tyrosine B-26 and the phenylalanine fine structure would exhibit the observed concentration dependence. It is of interest to note that desoctapeptide insulin le, in which the C-terminal eight amino acids of the B-chain are removed, exists as a monomer in solution, even at pH 717. Preliminary work in this laboratory on other insulin derivatives with Cterminal B-chain alterations indicates that such modifications markedly affect the aggregation state. Because of the non-polar nature of residues B-23 to B-28 (ref. 6), this model implies that the monomer-dimer interaction is predominantly hydrophobic. This model is therefore supported by the data of JEFFREY AND COATES 14, who showed that at pH 2 the free energy of dimerization is independent of ionic strength. On the basis of similar studies in both acid and base, FREDERICQ has concluded that the ... "forces have no electrostatic origin and ... probably originate from Van der Waals attractions between non-polar residues ..."18. Since the CD spectrum of insulin also correlates with the behavior of tyrosine A-I 4 at both low (Fig. 2a) and high (Fig. 3) pH, it is possible to ascribe the near ultraviolet CD of insulin at pH 7 to two or more tyrosine residues and one or more phenylalanyl residues. It has been emphasized3, 4 that the blue-shifted CD spectrum at high pH (Fig. I of ref. 3) cannot be explained by tyrosine residues alone. Again referring to Fig. I of ref. 3, it is possible that at pH 11.52 a disulfide transition is present at about 265 m#. However, the presence of a disulfide transition under this condition is not evidence for its presence in the native molecule at neutral pH. Since tyrosine ionization is far from complete at pH lO.55, it is possible that the spectrum at this pH is a superposition of a disulfide transition at 265 m/z (which is masked or absent at neutral pH), p l u s a tyrosine contribution at 275 m#. The progressive blue shift with increasing pH would only be apparent, caused by a decrease in the tyrosine contribution. In this case, the data of Fig. 3 would imply that the loss of asymmetry or disulfide interchange 3,* of the optically active disulfide bond(s) is dependent on the ionization of tyrosine A-I 4, or vice versa. This interdependence would be similar to the disulfide dependence of tyrosine interactions (Fig. 4). As previously mentioned, the near ultraviolet CD spectrum at pH 2, 0.2 mg/ml is featureless, so it is not possible to identify the responsible chromophore. It is therefore possible that Fig. ic represents a disulfide transition which is masked or absent in Fig. Ia. Biochim. Biophys. Acta, 16o (1968) I45-I5o
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J. w. s. MORRIS, D. A. MERCOLA, E. R. ARQUILLA
ACKNOWLEDGEMENTS
We thank W. F. H. M. MOMMAERTSfor the use of the circular dichroism spectrophotometer and A. N. Glazer for helpful discussions. This study was supported by U.S. Public Health Service Grant No. 5 R o i AM-o6ooi-o6. One of us (D. A. M.) was supported by a Southern California Diabetes Association Predoctoral Fellowship. REFERENCES i 2 3 4 5 6 7 8 9 io II 12 13 14 15 16 17 18
R. T. SIMPSON AND B. L. VALEE, Biochemistry, 5 (1966) 2531. N. S. SIMMONS AND A. N. GLAZER, J. Am. Chem. Soe., 89 (1967) 504 o. S. BEYCHOK, Proc. Natl. Acad. Sci. U.S., 54 (1965) 999S. BEYCHOK, Science, 154 (1966) 1288. T. T. HERSI~OVlTS AND M. LASKOWSKI, JR., J. Biol. Chem., 237 (1962) 2481. A. P. RYLE, F. SANGER, L. F. SMITH AND R. KITAI, Biochem. J., 6o (1955) 541. A. ROSENBERG, J. Biol. Chem., 241 (1966) 5119 . Y. [NADA, J. Biochem. Tokyo, 49 (1961) 217. M. LASKOWSKI, JR., J. M. WIDOW, M. L. McFADDEN AND H. A. SCHERAGA, Biochim. Biophys. Acta, 19 (1956) 581. M. LASKOWSKI, JR., S. J. LEACH AND H. A. SHERAGA, J. Am. Chem. Soc., 82 (196o) 57 I. S. YANARI AND F. A. BOVEY, J. Biol. Chem., 235 (196o) 2818. M. AOYAMA, K. KURIHARA AND K. SHIBATA, Bioehim. Biophys. Acta, lO 7 (I965) 257. E. FREDERICQ, J. Polymer Sci., 12 (1954) 287. P. D. JEFFREY AND J. H. COATES, Biochemistry, 5 (1966) 3820. J. A. RUPLEY, R. D. RENTHAL AND M. PRAISSMAN, Biochim. Biophys. Acta, 14o (1967) 18.5. W. W. BROMER AND R. CHANCE, Biochim. Biophys. Acta, 133 (1967) 219. D. A. MERCOLA, J. W. S. MORRIS, E. R. ARQUILLA AND W. W. BROMER, Federation Proc., 26 (1967) 3212. E. FREDERICQ, Arch. Biochem. Biophys., 6.5 (1956) 218.
Bioehim. Biophys. Acta, 16o (1968) 145-15 o
BIOCHIMICA ET BIOPHYSICA ACTA BBA 35225
AN ANALYSIS OF T H E N E A R U L T R A V I O L E T CIRCULAR D I C H R O I S M OF INSULIN
J. w . S. MORRIS, DANIEL A. MERCOLA AND EDWARD R. ARQUILLA
Department o[ Biophysics and Department of Pathology, Center for the Health Sciences, University of California, Los Angeles, Calif. (U.S.A.) (Received December 28th, 1967) (Revised manuscript received February 27th, 1968)
SUMMARY
I. The near ultraviolet circular dichroism (CD) of insulin has been studied as a function of p H and concentration. The CD spectrum at p H 7 shows a close correspondence with the absorption spectrum, with a peak at 276 m/z, a shoulder at 283 m/z, and short wavelength fine structure. 2. At p H 11.5 the disappearance of CD at 275 m# in time correlates with the increase in absorbance at 293 m#. At low pH and high concentrations the CD follows an ionization curve with a p K of 3.0. At p H 2, the CD is concentration dependent. 3. The above results are shown to be consistent with the known behavior of tyrosine residues A-I 4 and B-26. It is concluded that the near ultraviolet CD of insulin at p H 7 is due to tyrosine and phenylalanine. The C-terminal end of the B-chain is tentatively identified as the monomer-dimer aggregation site.
INTRODUCTION
Circular dichroism (CD) bands of proteins in the near ultraviolet have been associated with optically active side chain chromophores: mainly, aromatic residues and cystine 1. These chromophores absorb in the neighborhood of 270 m/z; thus the particular chromophore associated with a CD band in this region cannot be identified without further elucidation. In the case of ribonuclease, for example, the CD band at 277 m/~ can be ascribed to tyrosine with some certainty, since the well-known spectral shift of tyrosine with increase in p H is accompanied by a shift of the CD band to higher wavelengths 1. Moreover, the CD shift is remarkably similar to that observed in the model compound, N-acetyl-L-tyrosinamide 2. BEYCHOK3,4 has suggested that the near ultraviolet CD of insulin is due to cystine because of the susceptibility of the near ultraviolet CD to disulfide-reducing agents and because the CD exhibits a blue Abbreviation: CD, circular dichroism.
Biochim. Biophys. Acta, 16o (1968) I45-15o
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J, w . s. MORRIS, D. A. MERCOLA, E. R. ARQUILLA
shift rather than a red shift as the pH is raised above IO. This report presents data which suggest that the CD of insulin at neutral and acid pH is due to tyrosine and phenylalanine residues. MATERIALS AND METHODS
The insulin used here was a IO × recrystallized bovine preparation containing 0.74 °/o Zn, with a biological activity of 26 I.U.*. Ribonuclease was purchased from Sigma (Type XII-A). Solutions were prepared with CO2-free H20 and examined immediately at 23 °. CD measurements were made with the prototype of the DurrumJasco ORD/UV 5. The instrument was calibrated daily with camphor sulfonic acid, which also served as a wavelength calibration. All measurements at neutral and acid pH were made on solutions of I ± 0.o5 A 276 mu unit at a constant slit width ofo.8 mm. Under these conditions, successive scans of the same sample virtually superimposed. Spectra and concentrations were obtained with a Cary Model 15 spectrophotometer. Concentration values in the legends and text show ranges and indicate that measurements were made on samples with concentrations within 5 ~o of quoted values. CD results are given as AER: (AL -- AR) × 1 per mole of tyrosine residues per cm. pH measurements were made on a Radiometer 22 pH meter. Difference spectra of 1.25 mg/ml insulin, o.I M borate buffer, p H 8.45, and o.o 5 °/o fl-mercaptoethanol versus 1.25 mg/ml insulin, o.I M borate buffer, pH 8.45, and o.o5 % ethanol were obtained by the use of tandem cells 5. Spectra were recorded with four matched I-cm cuvettes at 15.5 ° and at 25 m#/min. Baselines were recorded with either buffer alone, or with insulin versus insulin treated with ethanol. The two methods gave identical results. RESULTS
Fig. I a shows the near ultraviolet CD spectrum of insulin at p H 7. The main features are a peak at 276 m#, a shoulder at 283 m#, and short wavelength fine structure. These features compare well with the corresponding features of the absorption spectrum (Fig. ib). Since no tryptophan is present 6, only tyrosine and phenylalanine contribute appreciably to the absorption spectrum. The similarity of the short wavelength fine structure of absorbance and CD, and the position of the optical rotatory dispersion fine structure of L-phenylalanine 7 permits the tentative assignment of the insulin CD fine structure to phenylalanine. The CD spectrum of insulin at pH 2, 0.2 mg/ml is shown in Fig. IC. No shoulder or fine structure is noted. However, at p H 2, IO mg/ml, the shoulder and fine structure are still apparent, although the band is reduced in intensity. The disappearance of the shoulder and fine structure at pH 2 is therefore concentration dependent. As a control, the CD of ribonuclease, which has a near ultraviolet CD spectrum very similar to that of insulin (cf. ref. 2), was run in the same cells at p H 7 over a similar 5o-fold concentration range. The magnitude of the main CD band and the presence of the long wavelength shoulder and short wavelength fine structure was unaltered, thus ruling out instrumental and pathlength artifacts. * L o t No. o16666, k i n d l y supplied b y Dr. SCHLICHTKRULL,N o v o l n d u s t r i , A/S, Copenhagen, D e n m a r k .
Biochim. Biophys. Acta, i6o (1968) 145-15o
CIRCULAR DICHROISM OF INSULIN
147 -1.10
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Fig. I. The near ultraviolet CD and a b s o r p t i o n spectra of insulin. Fig. Ia is the CD s p e c t r u m of insulin, i mg/ml, i-cm path, in o.Io M p h o s p h a t e buffer, p H 7.0. Fig. I b is the a b s o r p t i o n spect r u m of the same sample. Fig. ic is the CD s p e c t r u m of insulin, 0.2 mg/ml, 5-cm path, in o.io M KC1-HC1, p H 2.0. Fig. 2. The p H dependence of insulin CD. Conditions: Fig. 2a: concentration IO mg/ml, I - m m p a t h . Fig. 2b: I mg/ml, i-cm path. Fig. 2c: 0.2 mg/ml, 5-cm path. No m e a s u r e m e n t was possible for io mg/ml, p H 7, due to insolubility. Samples at p H 7 were in o. Io M KC1, o.oi M phosp h a t e buffer. All other samples were in o.io M KC1-HC1. Measurements were made at 276 m u for Fig. 2a, 275-276 m?, for Fig. 2b, and varied from 275 m u at p H 7 to 272 m # at p H 2 for Fig. 2c. The d a t a of Fig. 2a have been fitted to an ionization curve with an a p p a r e n t p K of 3.0 (cf. refs. 8, 9).
Fig. 2 shows the pH dependence of CD at three concentrations. At IO mg/ml, the magnitude of the CD band follows an ionization curve with an apparent pK of 3.0 (Fig. 2a), while at I mg/ml and 0.2 mg/ml the behavior is more complicated. Fig. 3 gives the time dependence of the CD of insulin at pH 11. 5. The disappearance of dichroism at 275 m# is correlated with the increase in absorbance at 293 m#, the absorption maximum of the ionized (phenoxy) form of tyrosine. The initial value of absorbance at 293 m# corresponded to the ionization of 3.0 tyrosine
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Fig. 3. The time dependence of insulin CD at p H i 1.5. Conditions: I mg/ml, i - c m path, in o.Io M K C 1 - K O H , p H 11.5 .Solid line: Increase in absorbance at 293 m/~. Points: decrease in circular dichroism at 275 m~. Note the initial lag (cf. ref. 3). Fig. 4. The near ultraviolet difference s p e c t r u m of insulin treated with fl-mercaptoethanol (reference beam) versus insulin treated with ethanol (sample beam) as described in MATERIALS AND METHODS. The s p e c t r u m was recorded IO rain after the solutions were prepared. The spect r u m above 275 m/, was i n d e p e n d e n t of additional increments of fl-mercaptoethanol to the sample b e a m buffer blank.
B i o c h i m . B i o p h y s . Acta, x6o (1968) 145-15o
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residues per insulin monomer (cf. ref. 8 and DISCUSSION). As reporteda, 4, the near ultraviolet CD of insulin under these conditions shows no extremum, so the selection of 275 m# is somewhat arbitrary. The reduction of disulfide bonds by/5-mercaptoethanol has a profound effect on the near ultraviolet CD spectral, 4. Therefore, such reduction should also demonstrate a change in the environment of tyrosine residues. Accordingly, a difference spectrum of insulin treated with fl-mercaptoethanol v e r s u s insulin treated with ethanol was recorded (see MATERIALSAND METHODS). The difference spectrum (Fig. 4) with maxima at 278 and 285.5 m# is very similar to insulin tyrosine difference spectra previously describedg, 1°. At the concentration used here, the perturbation of one tyrosine residue per insulin monomer would correspond to o.I A 2 s . m/2 unit (refs. 9, IO). DISCUSSION
It has been shown above that the effect of mercaptoethanol on the near ultraviolet CD does not imply that cystine is the optically active chromophore, since one or more tyrosine residues are also perturbed. Moreover, the failure of the CD band to shift red with tyrosine ionization does not exclude this chromophore from consideration, since there is no a p r i o r i reason to expect that strongly dichroic tyrosine residues in the native molecule would maintain their optical activity when ionized. Since there is no reason to eliminate aromatic amino acids from the CD spectrum at neutral pH, the contribution of tyrosine and phenylalanine to the CD spectrum will be discussed. Difference spectrophotometry studies by SCHERAGA and co-workers% 1° have shown that the B-26 tyrosine residue of insulin is 'anomalous' in that a blue shift is observed when the C-terminal eight amino acids of the B-chain are removed by tryptic digestion. They conclude that this residue is hydrogen bonded to a non-ionizable acceptor, although YANARI AND BOVEY11 have shown that the spectral shift is compatible with the breaking of a hydrophobic interaction. LASKOWSKI, LEACH AND SHERAGA10 also observe a spectral shift when the tryptic digest is acidified, which they take to indicate that an additional tyrosine residue is hydrogen bonded to an ionizable acceptor; presumably, a carboxyl group. Further, it is well knownS,12,13 that three of the four tyrosine residues of insulin ionize normally, with an apparent p K of IO, while the fourth ionizes slowly with a p K of 11. 4. AOYAMA, KURIHARA AND SHIBATA12 have shown that the slowly ionizing tyrosine is located at position A-I4, and that this tyrosine residue is likely the one which shows a spectral shift upon acidification. Thus, at least two of the four tyrosine residues of insulin have been shown to interact, and might be expected to exhibit strong optical activity due to hindered rotation 1. The CD spectrum of insulin at pH 7 (Fig. Ia) shows a close correspondence to the absorption spectrum. The pH dependence of CD at high concentrations (Fig. 2a) is similar to the pH dependence of the tyrosine difference spectrumg, 1°. At p H 2, where tyrosine A-I 4 would not be appreciably hydrogen bonded, the CD spectrum is concentration dependent (Fig. 2). It therefore would seem that the environment of the concentration dependent chromophore is sensitive to aggregation. JEFFREY AND COATES1~ have studied the aggregation of zinc-free insulin at pH 2 and ionic strength o.Io by ultracentrifugation. Their model assumes a m o n o m e r dimer-tetramer-hexamer equilibrium, and predicts that the weight fraction of Biochim. Biophys. Acta, 16o (1968) 145-15 °
CIRCULAR DICHROISM OF INSULIN
149
monomers increases from IO % at IO mg/ml to 50 % at I mg/ml, at which point the only species present are monomer and dimer. RUPLEY, RENTHAL AND PRAISSMAN15 have shown by difference spectrophotometry that insulin under the same conditions exhibits a concentration dependent tyrosine blue shift on dilution. Their data correlate well with the model of JEFFREY AND COATES, and they conclude that the tyrosine difference spectrum is due to the monomer-dimer equilibrium, and that one tyrosine residue per monomer is involved. This aggregation-dependent tyrosine blue shift is analogous to the concentration dependence of the magnitude and position of the near ultraviolet CD band at pH 2 (Fig. 2). Further, several observations implicate the B-26 tyrosine as the chromophore of the concentration-dependent CD spectrum. As noted above, at high concentrations the presumed phenylalanyl CD fine structure is insensitive to low pH, and is lost only in the dissociation to monomers. Two of the three phenylalanyl residues of insulin are located in the C-terminal end of the B-chain. Moreover, they are adjacent to tyrosine B-26 e. If the C-terminal B-chain were the monomer-dimer aggregation site, the CD of both tyrosine B-26 and the phenylalanine fine structure would exhibit the observed concentration dependence. It is of interest to note that desoctapeptide insulin le, in which the C-terminal eight amino acids of the B-chain are removed, exists as a monomer in solution, even at pH 717. Preliminary work in this laboratory on other insulin derivatives with Cterminal B-chain alterations indicates that such modifications markedly affect the aggregation state. Because of the non-polar nature of residues B-23 to B-28 (ref. 6), this model implies that the monomer-dimer interaction is predominantly hydrophobic. This model is therefore supported by the data of JEFFREY AND COATES 14, who showed that at pH 2 the free energy of dimerization is independent of ionic strength. On the basis of similar studies in both acid and base, FREDERICQ has concluded that the ... "forces have no electrostatic origin and ... probably originate from Van der Waals attractions between non-polar residues ..."18. Since the CD spectrum of insulin also correlates with the behavior of tyrosine A-I 4 at both low (Fig. 2a) and high (Fig. 3) pH, it is possible to ascribe the near ultraviolet CD of insulin at pH 7 to two or more tyrosine residues and one or more phenylalanyl residues. It has been emphasized3, 4 that the blue-shifted CD spectrum at high pH (Fig. I of ref. 3) cannot be explained by tyrosine residues alone. Again referring to Fig. I of ref. 3, it is possible that at pH 11.52 a disulfide transition is present at about 265 m#. However, the presence of a disulfide transition under this condition is not evidence for its presence in the native molecule at neutral pH. Since tyrosine ionization is far from complete at pH lO.55, it is possible that the spectrum at this pH is a superposition of a disulfide transition at 265 m/z (which is masked or absent at neutral pH), p l u s a tyrosine contribution at 275 m#. The progressive blue shift with increasing pH would only be apparent, caused by a decrease in the tyrosine contribution. In this case, the data of Fig. 3 would imply that the loss of asymmetry or disulfide interchange 3,* of the optically active disulfide bond(s) is dependent on the ionization of tyrosine A-I 4, or vice versa. This interdependence would be similar to the disulfide dependence of tyrosine interactions (Fig. 4). As previously mentioned, the near ultraviolet CD spectrum at pH 2, 0.2 mg/ml is featureless, so it is not possible to identify the responsible chromophore. It is therefore possible that Fig. ic represents a disulfide transition which is masked or absent in Fig. Ia. Biochim. Biophys. Acta, 16o (1968) I45-I5o
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ACKNOWLEDGEMENTS
We thank W. F. H. M. MOMMAERTSfor the use of the circular dichroism spectrophotometer and A. N. Glazer for helpful discussions. This study was supported by U.S. Public Health Service Grant No. 5 R o i AM-o6ooi-o6. One of us (D. A. M.) was supported by a Southern California Diabetes Association Predoctoral Fellowship. REFERENCES i 2 3 4 5 6 7 8 9 io II 12 13 14 15 16 17 18
R. T. SIMPSON AND B. L. VALEE, Biochemistry, 5 (1966) 2531. N. S. SIMMONS AND A. N. GLAZER, J. Am. Chem. Soe., 89 (1967) 504 o. S. BEYCHOK, Proc. Natl. Acad. Sci. U.S., 54 (1965) 999S. BEYCHOK, Science, 154 (1966) 1288. T. T. HERSI~OVlTS AND M. LASKOWSKI, JR., J. Biol. Chem., 237 (1962) 2481. A. P. RYLE, F. SANGER, L. F. SMITH AND R. KITAI, Biochem. J., 6o (1955) 541. A. ROSENBERG, J. Biol. Chem., 241 (1966) 5119 . Y. [NADA, J. Biochem. Tokyo, 49 (1961) 217. M. LASKOWSKI, JR., J. M. WIDOW, M. L. McFADDEN AND H. A. SCHERAGA, Biochim. Biophys. Acta, 19 (1956) 581. M. LASKOWSKI, JR., S. J. LEACH AND H. A. SHERAGA, J. Am. Chem. Soc., 82 (196o) 57 I. S. YANARI AND F. A. BOVEY, J. Biol. Chem., 235 (196o) 2818. M. AOYAMA, K. KURIHARA AND K. SHIBATA, Bioehim. Biophys. Acta, lO 7 (I965) 257. E. FREDERICQ, J. Polymer Sci., 12 (1954) 287. P. D. JEFFREY AND J. H. COATES, Biochemistry, 5 (1966) 3820. J. A. RUPLEY, R. D. RENTHAL AND M. PRAISSMAN, Biochim. Biophys. Acta, 14o (1967) 18.5. W. W. BROMER AND R. CHANCE, Biochim. Biophys. Acta, 133 (1967) 219. D. A. MERCOLA, J. W. S. MORRIS, E. R. ARQUILLA AND W. W. BROMER, Federation Proc., 26 (1967) 3212. E. FREDERICQ, Arch. Biochem. Biophys., 6.5 (1956) 218.
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