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Acid- and temperature-induced conformational changes in human chorionic gonadotropin and the mechanism of subunit dissociation
ARCHIVES OF BIOCHEMISTRY Vol. 205, No. 1, November,
AND BIOPHYSICS pp. 104-113, 1980
Acid- and Temperature-Induced Conformational Changes in Human Chorionic Gonadotropin and the Mechanism of Subunit Dissociation HILDA Plasma
Derivatives
FORASTIERI
Laboratory,
American Received
KENNETH
AND
Red Cross February
Blood
C. INGHAM Services,
Bethesda,
Maryland
20014
22, 1980
Human chorionic gonadotropin undergoes a conformational transition in acid which at 4°C is characterized by: (i) a reversible increase in the polarization of tyrosyl fluorescence, P, with a midpoint at pH 5, (ii) a slight decrease in the elution volume on Sephadex G-100 near uv difat pH 3 relative to pH 7, (iii) a slight decrease in s~~,~, (iv) a small positive ference spectrum (AE -2%), and (v) the appearance of a positive CD feature at 235 nm. These observations are compatible with an acid-expanded form of the hormone in which the rotational freedom of one or more tyrosine residues is restricted and/or their proximity to potential quenching groups is altered. The increased value of P following acidification is stable at temperatures below lO”C, but at higher temperatures it decreases with time to an extent which is dependent on the temperature. A substantial portion of this decrease occurs before subunit dissociation can be detected, reflecting the occurrence of a thermal transition with a midpoint near 26°C. A similar transition was observed at neutral pH with a midpoint near 22°C. These results suggest the occurrence of at least two conformationally distinct forms of hCG which may be sequentially encountered prior to subunit dissociation in acid. The kinetics will be either biphasic or strictly first order, depending on the temperature at which the hormone is acidified.
Human ehorionic gonadotropin (hCG)’ is one of several homologous glycoprotein hormones, each comprised of two dissimilar subunits, (Y and p, which are noncovalently linked (l-3). The subunits, which can be dissociated in acid or urea, are biologically inactive (4). Changes in tertiary and/or secondary structure are believed to accompany the dissociation of the subunits (5, 6). However, it is not known whether these changes occur prior to, simultaneously with, or subsequent to, the actual physical separation of the subunits. Spectral studies of the isolated subunits of hCG reveal little evidence for structure of the type which can be disrupted by denaturants or other forms of stress; they appear to
be completely unfolded within the constraints imposed by the disulfide bonds (7). A better understanding of the mechanism of dissociation might provide insight into the structural requirements for biological activity. In previous studies, the rate of acid-induced dissociation was monitored by the loss in the ability to enhance ANS fluorescence. First-order kinetics were reported for hCG (‘7, S), hLH (9), bTSH (lo), and oLH (11). Similar results were obtained when differential absorption was used to monitor the exposure to tyrosyl residues which accompanies the dissociation (7, 10, 11). However, other investigators have observed biphasic kinetics for dissociation of several of these hormones by various methods of detection (12-15). Using polarization of tyrosyl fluorescence, Bolotin and Ingham (ll), also observed biphasic kinetics with oLH under certain conditions. Evidence for a conformational change prior to physical dissociation was provided
’ Abbreviations used: hCG, human chorionicgonadotropin; hLH, human luteinizing hormone; oLH, ovine luteinizing hormone; bTSH, bovine thyroid-stimulating hormone; P, tyrosyl fluorescence polarization; CD spectrum, circular dichroic spectrum; FSH, folliclestimulating hormone. 00039861/80/130104-10$02.00/O Copyright 0 1980 by Academic All rights
of reproduction
in any
Press, Inc. form reserved.
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by the observation of an increase in polarization (P) of tyrosyl fluorescence following acidification of hCG (7). A similar, but smaller increase was observed with oLH (11). The purpose of the present study was to further analyze these acid-induced changes in hCG. This was accomplished by working at lower temperature where the acid form could be reversibly generated and characterized. As a result of working at lower temperature, we discovered a thermal transition which occurs both in acid and at neutral pH and which is manifested by a small change in P and the appearance of biphasic kinetics under certain conditions. MATERIAL
AND METHODS
Highly purified hCG (CR 121, having a biological potency of 12450 IU/mg) was obtained from Dr. R. Canfield of Columbia University via the Center for Population Research, National Institute of Child Health and Human Development, National Institutes of Health. hCG subunits (CR 121) obtained from the same source, had biological potencies of less than 0.5% of the intact hormone. The subunits each exhibited a single band on polyacrylamide disc gel electrophoresis in the presence of 8 M urea, according to Swank and Munkres (16). The hormone exhibited two bands corresponding to those of the subunits. None of the preparations exhibited tryptophan fluorescence, confirming the absence of tryptophan-containing impurities. Protein concentrations were determined from the absorbance at 276 nm using E = 12,000 for hCG, 6700 for hCG-a, and 5400 M-’ cm-’ for hCG$ (7). Differential absorption measurements were made on a Cary 118C spectrophotometer equipped with a thermostatted cell holder, using l.O-cm path length, Teflon stoppered, quartz cuvettes according to the methods of Herkovits (17) and Donovan (18). A Cary Model 61 spectropolarimeter, also equipped with a thermostatted cell holder was used to obtain circular dichroism spectra between 200 and 350 nm at 4°C. Cuvettes having path lengths of 0.5 and 0.05 cm were used for near and far ultraviolet measurements, respectively. Mean residue ellipticities were calculated according
[e]x =
0,b, x 151 1OdC ’
Dl
where Oobs is the observed ellipticity, 151 is the mean residue molecular weight for hCG (including the carbohydrate (19)), d is the path length in
CONFORMATION
105
centimeters and C is the concentration in grams per milliliter. Sedimentation velocity measurements were made at 52,000 rpm in double-sector cells at 4°C using a Spinco Beckman Model E ultracentrifuge, equipped with Schlieren optics and RTIC temperature control unit. Observed sedimentation coefficients were corrected to sedimentation in water at 20°C (szO,,) according to established procedures (20). Samples at neutral pH (0.01 M KPO,) and pH 3 (0.02 M citrate) were analyzed simultaneously in a single experiment, at a hormone concentration of 4 mg/ml. Fluorescence measurements were determined on a Perkin-Elmer MPF4 fluorometer equipped with Polacoat polarizing filters and thermostatted cell holder. Excitation and emission wavelengths of 270 and 310 nm, respectively, were used in order to avoid interference by Raman scattering. Polarization measurements were made as previously described (7, 11) except a uv scrambler was placed between the emission polarizer and the emission monochromator in order to eliminate the need for a grating correction factor. Using vertical excitation, polarization was calculated using Eq. [2]: P = (I,, - Z,)l(Z, + I”),
PI
where Iv and Z H are the observed intensities with the emission polarizer in the vertical and horizontal positions, respectively (21). The observed intensities were corrected for buffer background, which never exceeded 10% of the sample intensity. It was observed that continuous exposure of hCG to excitation at pH 3.0 caused a decrease in P with time which was substantially greater than that observed by periodic monitoring. This effect was eliminated by the use of narrow excitation slits, minimization of exposure time, use of higher hormone concentrations, and stirring of the samples prior to each measurement. The total relative fluorescence intensity for a given polarization measurement was calculated according to Eq. [3] (22): ZT”hl = I, + 21,.
[31
Exclusion chromatography was performed on a 1.0 x 57-cm column of Sephadex G-100 at 4°C using tyrosyl fluorescence to monitor the elution of hormone and/or subunits. Column parameters, as determined using blue dextran (V,) and tyrosine (I’,,,,), were identical at pH 7.0 and pH 3.0. This column does not resolve a mixture of hCG and p subunit. The extent of subunit dissociation of various hCG samples was estimated by nonlinear interpolation between the elution profile for intact hCG and that for fully dissociated hCG usingf = 3Rl (R + 2) wheref is the fraction dissociated and R is the ratio of the peak intensity for hCG-a to that for hCG and/or hCG& This equation takes into account the approximate differences in quantum yield and extinction coefficients of the various species.
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AND INGHAM
RESULTS
A. Acid-Induced
Conformational
Changes
The influence of pH on the polarization (P) of the tyrosyl fluorescence of hCG and its subunits at 4°C is illustrated in Fig. 1. P for the intact hormone exhibited a reversible sigmoidal transition with a midpoint at pH 5.0. By contrast, P for the isolated subunits increased only slightly in acid with little evidence of a transition. The increase in P for the hormone was accompanied by a 20% decrease in fluorescence intensity, whereas the intensity of the (Y and p subunits increased 15 and 34%, respectively. These intensity changes were also reversible. The elution of hCG from a column of Sephadex G-100 at pH 7 and pH 3 at 4°C is shown in Fig. 2A. The hormone was consistently found to elute slightly earlier at the lower pH, with K,, decreasing from -0.13 to 0.10. This suggested that selfassociation might be responsible for the observed increase in P. Sedimentation velocity measurements conducted under conditions identical to those of the chromatography experiments gave values of s20,W of 3.22 at pH 7 and 2.92 at pH 3, eliminating the possibility of self-association. The significance of the small decrease in s20,W is by itself uncertain because of potential primary charge effects. However, when taken together with the chromatographic results, the decrease is compatible with a slight expansion of the hormone in acid. This is analogous to the well-characterized N + F transition in serum albumin (23), whose tryptophan fluorescence also exhibits an increased polarization in acid (24). Further evidence for an acid-induced conformational change in hCG was provided by differential absorption measurements at 4°C (Fig. 3A). The positive difference spectrum at pH 3 vs pH 7, although small ( AE - 2%), exhibits the characteristic features of tyrosyl residues which have experienced a decrease in the polarity of their microenvironment (1’7, 18). There is an apparent shift in the baseline which could reflect small changes in the amount of scattered light. Alternatively there could be
FIG. 1. Effect of pH on the polarization of the tyrosyl fluorescence of hCG and its subunits at 4°C. The protein concentrations were 26 pM in 0.01 M KPO,. Titrations were initiated at pH 7 by adding aliquots of 1 M HCl (0) and reversed with 1 M NaOH (a).
alterations in the extent to which disulfide bonds contribute to the spectrum. Acidification also produced a positive extremum near 235 nm in the near uv CD spectrum (Fig. 3C). A similar feature has been observed in other glycoprotein hormones and has been attributed to tyrosyl and/or disulfide residues (25-30). The far uv circular dichroic spectrum exhibited about a 15% decrease in the intensity of the negative band at 209 nm, suggestive of a slight alteration of secondary structure (Fig. 3B). B. Thermal Transitions Following Acidification
Figure 4 illustrates the time dependence of P following dilution of a chilled sample of hCG into pH 3 buffer which had been equilibrated at the indicated temperature. The initial value of P was independent of temperature between 4 and 37°C. A pattern similar to that in Fig. 4 was also obtained when a sample was first acidified on ice and then quickly brought to the indicated temperature and placed in the fluorometer, previously equilibrated at the same temperature. At PC, P was constant with time even for much longer periods than shown in Fig. 4. Between 16 and 3O”C, P decreased with time, but within 15 min reached an apparent plateau value which was dependent upon tempera-
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20
25
ELUTION VOLUME (ml) FIG. 2. Exclusion chromatography of hCG and its subunits at 4°C on Sephadex G-100 (1.0 x 57 cm). The buffer was 0.02 M citrate, pH 3, except for solid circles (0) panel A, where 0.01 M KPO,, pH 7, was used. Approximately 0.5 mg ofprotein was applied in each determination. (A) Elution ofintact hormone at pH 7 (e) and pH 3 (0); (B) separate elutions of (Y(A) and (0) subunits; (C) hCG dissociated into subunits by incubation in 10 M urea at 40°C for 60 min. Urea was removed from the sample by dialysis into eluting buffer at 4°C prior to application to the column; (D, E) hCG after 40 min incubation at pH 3 and 25” or 3O”C, and 37°C respectively (see Fig. 4); (F-H) hCG after incubation for 210 min at pH 3 and 16”, 25”, and 37°C respectively.
ture. These changes, which could only be reversed by slow cooling, were not accompanied by changes in the intensity of fluorescence. After 40 min, the 25 and 30°C samples were analyzed by exclusion chromatography at 4°C and pH 3.0. As shown in Fig. 2D, there was no indication of dissociation into subunits. At 37”C, the decrease of P with time was continuous and no plateau was evident (Fig. 4). An increase in fluorescence intensity was also observed. Exclusion chromatography of this sample after 40 min revealed two peaks, as shown in Fig. 2E. The first peak elutes in the position corresponding to native hCG (Fig. 2A) and/or hCG-p (Fig. 2B); mixtures of hCG and hCG-P are not significantly resolved on this column. The second peak corresponds to the cx subunit which elutes much later (Fig. 2B),
and indicates that approximately 30% of the hormone was dissociated. This estimate is based on a comparison of the ratio of the two peaks with the same ratio for a sample which was fully dissociated in urea (Fig. 2C, see Methods). Prolonged incubation of similar samples resulted in no detectable dissociation at 16”C, approximately 78% dissociation at 25”C, and complete dissociation at 37°C after 3.5 h, as shown chromatographically in Figs. 2F-H. The final value of P (corresponding to complete dissociation) in this and similar experiments at 37°C was 0.17 ? 0.005 and the total increase in fluorescence intensity was about 30%. Based on polarization measurements alone, the 37°C sample in Fig. 4 would appear to be much more than 30% dissociated after 40 min, since about 60% of the total change in P has occurred by this time.
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,
WAVELENGTH
lnml
FIG. 3. (A) Differential absorption spectrum for hCG at pH 3 vs pH 7 at 4°C (--). The baseline (- - -) was obtained with 54 M hCG in both cuvettes at pH ‘7. The sample solution was then acidified by addition of 2 M HCI and an equal volume of buffer added to the reference solution. (B, C) Circular dichroism of hCG (50 ELM) at 4°C in 0.01 M phosphate buffer at pH 3 (-) and pH 7 (- - -). The path length was 0.05 cm in B and 0.5 cm in C.
This implies that much of the decrease in P during the first 40 min is not due to dissociation and suggests that elevation of the temperature at pH 3 causes the acid form of the hormone to undergo a thermal transition which does not involve, but rather precedes dissociation of subunits. When chilled samples of hCG were diluted into pH 3 buffer at elevated temperatures, the ensuing decrease in P did not obey first-order kinetics. Data obtained at 37 and 42°C are illustrated in Fig. 5A. This type of result would be expected if multiple steps were involved in the dissociation reaction. When the hormone was preincubated for 30 min at pH 3 and 30°C (as in Fig. 4), followed by rapid elevation of the temperature to 37 or 42”C, first-order plots of the decrease in P were linear as shown in Fig. 5B (circles). This suggests that preincubation converted the hormone from a high P, nondissociating form which prevails at low temperature, to a dissociable form characterized by a lower P and which appears with time at higher temperatures. It was subsequently found that if the hormone was first brought to 37 or 42°C at neutral pH and then rapidly acidified, first-order kinetics were also followed and, as shown by the triangles in Fig. 5B, the rates were identical to those obtained after preincubation at pH 3.0 and 30°C. The initial P value for the hormone immediately after acidification at 37 or 42”C, was -0.205, slightly lower than that obtained after preincubation at pH 3 and 30°C.
This value is represented in Fig. 4.
by the dashed line
C. Thermal
at Neutral
Transitions
pH
In the preceding section, kinetic evidence was provided for the occurrence of a thermal transition, not only in acid, but at neutral pH as well. Careful measurements of P for the intact hormone at neutral pH provided additional evidence for such a transition. As shown in Fig. 6, P exhibited a shallow sigmoidal dependence on temperature with a midpoint near 22°C. This was accompanied
:I:.
10
20
30
MINUTES
40
50
FIG. 4. Effect of time on the tyrosyl polarization of hCG at various temperatures. Small aliquots of chilled, concentrated hCG were added to 0.01 M Na-citrate, pH 3, equilibrated at the indicated temperature. Final protein concentration was 22 pM. The dashed line represents the initial value ofP obtained when a sample at 37°C is rapidly acidified and represents the plateau value which would be reached at 37°C if no subunit dissociation occurred (see text).
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CONFORMATION
109
Fig. 6 allowed an estimate of the fractional conversion between the low- and hightemperature forms. The equilibrium data thus obtained are plotted according to the van’t Hoff equation in Fig. 7 (triangles). Although there is considerable uncertainty in the slopes of the dashed lines in Fig. 6, the AH values obtained from the van’t Hoff plots were rather insensitive to arbitrary variations in those slopes. The circles in Fig. ‘7 were obtained by a similar analysis of the plateau values in Fig. 4 using the value of P represented there by the dashed line as the value corresponding to complete conversion at pH 3. Although Fig. 7 clearly indicates that the transition is shifted to higher temperature at pH 3, the uncertainties in the values of AH and
MINUTES
FIG. 5. First-order plots of the time course for the dissociation of 22 M hCG at pH 3. (A) Chilled, concentrated hCG diluted into 0.01 M citrate, pH 3, at 37” (0) and 42°C (0); (B) chilled, concentrated hCG diluted into 0.01 M citrate, pH 3, incubated at 30°C for 30 min (see Fig. 4), and then brought rapidly to 37°C (0) or 42°C (0). The triangles illustrate the results obtained when a 2%pM sample at neutral pH was first equilibrated at the indicated temperature and then rapidly acidified.
by a 50% decrease in fluorescence intensity. By contrast, P for the individual subunits decreased linearly with increasing temperature, with no evidence of a transition. All of these changes were completely and 0.10 ,\ 0 rapidly reversible. Exclusion chromatog10 20 xl 40 50 raphy of the hCG sample revealed no TEMPERATURE (“C) evidence for dissociation. However, at higher temperatures (50-7O”C), hCG exFIG. 6. Effect of temperature on the polarization of hibited a sharp drop in P which was tyrosyl fluorescence of hCG and its subunits, each at found by exclusion chromatography to in- a concentration of 40 pM in 0.01 M phosphate buffer, volve subunit dissociation (data not shown). pH 7. The temperature was increased (0) and decreased (0) at approximately l”C/min with periodic This transition, which was also reversible of P. In order to confirm the subtle provided sufficient time was allowed at each monitoring transition observed for hCG, the experiment was temperature for the state of association to repeated, making multiple measurements of P after reach equilibrium, will be the subject of a equilibration of the intact hormone at each temperaseparate report. ture shown. This accounts for the reduced scatter in Interpolation between the dashed lines of the data for hCG relative to subunits.
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20
0.8
AND
INGHAM DISCUSSION
15
hCG has seven tyrosines (four in (Y and three in p) whose relative contribution to the total fluorescence may vary considerably (‘7). Thus large variations in polarization do not necessarily reflect gross conformational changes but could arise from localized alterations in the environment of one or more tyrosines which contribute disproportionately to the observed intensity. A discussion of the factors which influence P is facilitated by reference to the Perrin equation (21, 31):
h
-=1 p -0.8
I
-1.0
3.3
3.4
3.5
(1rr)xW
FIG. 7. van’t Hoff plots of thermal transition data for hCG at pH 7 (A) and pH 3 (0). The quantityf is the fraction present in the high temperature form as estimated by interpolation between the dashed lines in Fig. 6 or the plateau values in Fig. 4 (see text). The lines were obtained by linear regression and standard deviations of slopes and intercepts are reported in Table I.
AS summarized in Table I do not allow this effect to conclusively be attributed to one or the other of these parameters; a small change in either could account for the observed shift. TABLE
I
CHARACTERISTICSOFTHE THERMAL TRANSITION IN HCG"
PH 7 3
Temperature (“C) 22.5 26.7
AH (kcal/mol) 35 ? 1 37 + 0.6
AS (4 118 ? 4 124 f 2
” The AH and AS values were obtained by linear regressions of the data in Fig. 7. Standard deviations were computed from the variance of the parent distributions assuming all errors occur in the dependent variable (43). Temperature values were taken as the points at which the lines in Fig. 7 intersect the abscissa log (fl1 -f) = 0.
f+ 0
(
$-i 0
(7/p), 1
[41
which relates P for a given tyrosine to its lifetime, T, and the rotational relaxation time, p, of the phenolic group. P, is the maximum polarization observed in the absence of rotation. Thus, events which affect either T or p will influence P. Changes in 7 for a given tyrosine can result from changes in the dielectric properties of its microenvironment and/or changes in the extent of dynamic quenching by neighboring groups on the protein or by other substances present in solution. Changes in p require changes in the rigidity of the microenvironment or in the flexibility of the local polypeptide backbone. Because of the short r for tyrosine fluorescence in proteins (-2, ns (3211, changes in the overall rotational motion of the protein, such as occur during self-association or subunit dissociation, are expected to have relatively little influence on P unless they are accompanied by conformational changes which alter the local rigidity of the tyrosyl environment. Finally, changes in the extent of intertyrosyl energy transfer can also influence P. This phenomenon is very sensitive to donor-acceptor distance and orientation; the critical distance for tyr-tyr transfer is 7-9 A (33, 34). The present observations of the effects of pH and temperature on the conformation and state of subunit association of hCG, as detected primarily through changes in P, can be rationalized in terms of the following scheme:
HUMAN
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GONADOTROPIN
PH where HN represents the native hormone as it exists at neutral pH and temperatures between 30 and 45°C. The subscript A refers to the acid form and the primes to the low-temperature form which prevails below 10°C. Between 10 and 3o”C, the hormone appears to undergo a thermal transition which is rapidly reversible at neutral pH but slow at pH 3.0. The reversible acid transition shown in Fig. 1 corresponds to the equilibrium between HE, and Ha. The shape of the titration curve is consistent with a simple protonation with no evidence for site-site interactions. The midpoint at pH 5.0 suggests that a glutamic or aspartic acid carboxyl group is involved, although abnormal histidines have also been shown to titrate in this region (35, 36). The 20% decrease in fluorescence intensity which accompanies this transition, if it were due entirely to dynamic quenching, could cause a proportional decrease in 7. This could account for a small part of the increase in P without the need for a conformational change. However, protonation of carboxylates is expected to reduce their quenching efficiency, suggesting that the effect on P is indirect, perhaps mediated by a conformational change which decreases the flexibility of one or more fluorescing tyrosines and/or alters their proximity to other potential quenching groups such as disulfide or peptide bonds (37). The effect of acidification on the differential absorption spectrum of hCG at 4”C, although small, reinforces the concept of a conformational change affecting the environment of at least one tyrosine. The fact that the difference spectrum is of opposite sign to that which accompanies dissociation (7, 19) corroborates our assumption that the latter has not occurred, an assumption which was further confirmed by
CONFORMATION
111
exclusion chromatographic analysis. Hence, a distinct form of the hormone occurs in acid which can be reversibly generated and characterized at low temperature. A similar transition also occurs at higher temperatures as evidenced by the instantaneous increase in P following acidification (HN + HA). We suggest that these structural changes which occur reversibly at low temperature are similar to those which occur transiently at higher temperature and which constitute the first step in the mechanism of acid-induced subunit dissociation. The sedimentation velocity and exclusion chromatography results suggest that this first step involves a slight expansion of the hormone. An analogous situation has been well documented for serum albumin, whose structure consists of several globular domains which become separated as the pH is lowered (38). The process occurs in at least two steps, the first corresponding to a loosening and the second to a more dramatic unfolding. Sogami et al. (24) have shown that the first step is accompanied by an increase in polarization of tryptophanyZ fluorescence (analogous to the increase for hCG in Fig. l), while the second step is accompanied by a decrease (analogous to that which accompanies the actual dissociation of the subunit “domains” of hCG). It is not obvious how a “loosening” of the subunit domains causes an increase in P. However, an overall loosening does not preclude a local increase in the rigidity of the environment of a particular tyrosine. Another possibility is that this loosening is accompanied by decreased energy transfer between tyrosines on the same or different subunits. The first and perhaps best evidence for a thermal transition came from the deviations from linearity in the first-order kinetic plots of the polarization data and the elimination of those deviations by preincubating the sample at a temperature above the transition temperature (Figs. 5A and B). In terms of our proposed scheme, acidification of a chilled sample results in the formation of a species, Hi, which is stable at low temperature, and whose conversion to HA following a subsequent increase in temperature is sufficiently slow
112
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to be manifested in the kinetics. On the other hand, acidification above the transition temperature immediately produces HA which undergoes a simple first-order dissociation reaction whose rate depends on pH and temperature. This is the mode of acidification used in earlier studies where first-order kinetics were routinely obtained (g-10). Measurements of P as a function of temperature at neutral pH provided direct evidence for a thermal transition. It is of interest that the slope at either end of the P vs T curve for hCG in Fig. 7, is much smaller than that for the isolated subunits at any temperature. This is compatible with a more restricted amplitude for the temperature-dependent rotational and vibrational motions of the fluorescing tyrosines in the intact hormone relative to the subunits. The increase in P which accompanies acidification could, as mentioned above, be interpreted as a further increase in rigidity, perhaps explaining the slower rate of interconversion between the low- and hightemperature forms at pH 3 vs pH 7. Recall, however, that the thermodynamic parameters governing the equilibria between these forms were not strongly influenced by pH (Table I). Autonomy between pH and temperature-dependent transitions has been demonstrated for human serum albumin (39). The exact origin of the positive feature near 235 nm in the CD spectrum of hCG (Fig. 3) is unclear. A similar feature has been observed in several homologous glycoprotein hormones and/or subunits including bTSH (26), oLH (25,2’7,29), hLH, and FSH (28). It is generally attributed to tyrosyl residues, an interpretation which is supported by the observation that it is most intense in bTSH-P which has a relatively high tyrosine content (26). In addition, the intensity of this band in bTSH decreased significantly following enzymatic removal of two tyrosines from the C terminal region. Pflumm and Beychok observed that a similar feature in the CD spectrum of ribonuclease was strongly affected by tyrosine-specific chemical modifications (40). However, these authors discussed the difficulty of making a definitive assignment of this band because
AND INGHAM
of overlap with neighboring, more intense bands, and provided evidence that disulfide bonds could also be involved. Indeed, a similar but somewhat red-shifted positive band has also been observed with the model compound (Ac-Cys-N-Me)z which lacks tyrosine (41). The intensity of this band, as well as that of the analogous ones in ribonuclease (40, 42) and oLH-a (29), was found to increase with decreasing temperature. Whatever the origin of the 235-nm band, it is clearly accentuated by acidification (Fig. 3), providing additional evidence for a conformational change. A similar effect of acid was reported by Giudice et al. (28), for hLH under conditions where subunit dissociation was not expected. Their spectra, obtained at room temperature, are almost identical to ours at 4°C. However, the 235-nm feature, detectable in our spectrum, even at neutral pH, is absent from published spectra of hCG obtained at room temperature (19, 29), providing additional evidence for a thermal transition in this temperature range. A more definitive assignment of the origin of this band, together with a detailed study of its dependence on pH and temperature, could further clarify the nature of the acid- and temperature-induced transitions in hCG. ACKNOWLEDGMENTS This report constitutes publication No. 467 from the American Red Cross Blood Services Laboratories. The work was also supported by the National Institutes of Health (Grants RR 05737 and AM 19719) and by a Career Development Award to K. C. Ingham (HL 00325). We are indebted to Dr. James C. Osborne for the use of his circular dichrometer and for helpful advice. Thanks also to Mrs. Marcia Robinson for typing the manuscript. REFERENCES 1. CANFIELD, R. E., MORGAN, F. J., KAMMERMAN, S., BELL, J. J., AND AGOSTO, G. M. (19’71) Rec. Progr. Horvn. Res. 27, 121-164. 2. BAHL, 0. P. (1973) in Hormonal Proteins and Peptides (Li, C. H., ed.), Vol. 1, p. 171, Academic Press, New York. 3. MORGAN, F. J., AND CANFIELD, R. E. (1971) Endocrinology 88, 1045-1053. 4. CATS, K. J., DUFAU, M. L., AND TSURUHARA, T. (1973) J. Clin. Endocrinol. Metab. 36, 73430.
HUMAN
CHORIONIC
GONADOTROPIN
5. HOLLADAY, L. A., AND PUETT, D. (1975) Arch. Biochem. Biophys. 171, 708-719. 6. GARNIER, J., SALESSE, R., AND PERNOLLET, J. C. (1974) FEBS Lett. 45, 166-171. 7. INGHAM, K. C., TYLENDA, C., AND EDELHOCH, H. (1976) Arch. Biochem. Biophys. 173, 680690. 8. ALOJ, S. M., EDELHOCH, H., INGHAM, K. C., MORGAN, F. J., CANFIELD, R. E., AND Ross, G. T. (1973) Arch. Biochem. Biophys. 159,497. 9. INGHAM, K. C., ALOJ, S. M., AND EDELHOCH, H. (1973) Arch. Biochem. Biophys. 159, 596605. 10. INGHAM, K. C., ALOJ, S. M., AND EDELHOCH, H. (1974) Arch. Biochem. Biophys. 163, 589599. 11. INGHAM, K. C., AND BOLOTIN, C. (1978) Adz. Biochem. Biophys. 191, 134-145. 12. PERNOLLET, J. C., AND GARNIER, J. (1971) FEBS Lett. 18, 189-192. 13. SALESSE, R., CASTAING, M., PERNOLLET, J. C., AND GARNIER, J. (1975) J. Mol. Biol. 95, 483-496. 14. REICHERT, L. E., AND RAMSEY, R. B. (1975) J. Biol. Chem. 250, 3034-3040. 15. PERNOLLET, J. C., GARNIER, J., PIERCE, J. G., AND SALESSE, R. (1976) Biochim. Biophys. Acta 446, 262-276. 16. SWANK, R. T., AND MUNKRES, K. D. (1971) Anal. Biochem. 39, 462-477. 17. HERSKOVITS, T. T. (1967) in Methods in Enzymology (Kustin, K., ed.), Vol. 16, 748-755, Academic Press, New York. 18. DONOVAN, J. W. (1969) in Physical Principles and Techniques of Protein Chemistry (Leach, S. J., ed.), Part A, pp. 138-170, Academic Press, New York. 19. GARNIER, J., SALESSE, R., AND PERNOLLET, J. C. (1974) FEBS Lett. 45, 166-171. 20. SVEDBERG, T., AND PEDERSON, K. 0. (1940) in The Ultracentrifuge, pp. 273-274, Clarendon Press, Oxford. 21. CHEN, R. F., EDELHOCH, H., AND STEINER, R. F. (1969) in Physical Principles and Techniques of Protein Chemistry (Leach, S. J., ed.), Part A, pp. 171-244, Academic Press, New York. 22. DALE, R., AND EISENGER, J. (1975) in Biochemical Fluorescence: Concepts (Chen, R. F., and Edelhoch, H., ed.), Vol. 1, pp. 115-280, Dekker, New York.
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23. YANG, J. T., AND FOSTER, J. F. (1954) J. Amer. Chem. Sot. 76, 1588-1596. 24. SOGAMI, M., ITOH, K. B., AND NEMOTO, Y. (1975) Biochim. Biophys. Acta 393, 446-459. 25. BEWLEY, T. A., SAIRAM, M. R., AND LI, C. H. (1972) Biochemistry 11, 932-936. 26. CHENG, K. W., GLAZER, A. N., AND PIERCE, J. G. (1973) J. Bid. Chem. 248, 7930-7937. 27. GARNIER, J., PERNOLLET, J. C., TERTRIN-CLARY, C., SALESSE, R., CASTAING, M., BARNAVON, M., DE LA LLOSA, P., AND JUTISZ, M. (1975) Eur. J. Biochem. 53, 243-254. 28. GIUDICE, L. C., PIERCE, J. G., CHENG, K.-W., R. J. (1978) WHITLEY, R., AND RYAN, Biochem. Biophys. Res. Commun. 81,725-733. 29. PUETT, D., AND HOLLADAY, L. A. (1977) Int. J. Peptide Protein Res. 10, 102-105. 30. GIUDICE, L. C., AND PIERCE, J. G. (1979) J. Biol. Chem. 254, 1164-1169. 31. WEBER, G. (1952) Biochem. J. 51, 145-167. 32. LONGWORTH, J. W. (1971) in Excited States of Proteins and Nucleic Acids (Steiner, R. F., and Weinrub, I., eds.), pp. 319-474, Plenum Press, New York. 33. STEINBERG, I. (1971) Annu. Rev. Biochem. 83- 114. 34. EDELHOCH, H., PERLMAN, R. L., AND WILCHEK, M. (1968) Biochemistry 7, 3893-3900. 35. BROWN, F. F., PARSONS, T. F., SIGMAN, D. S., AND PIERCE, J. G. (1979) J. Biol. Chem. 254, 4335-4338. 36. SHINDO, H., COHEN, J. S., AND RUPLEY, J. A. (1977) Biochemistry 16, 3879-3882. 37. COWGILL, R. W. (1976) in Biochemical Fluorescence: Concepts (Chen, R. F., and Edelhoch, H., eds.), Vol. 2, pp. 441-486, Dekker, New York. 38. HARRINGTON, W. R., JOHNSON, P., AND OTTEWILL, R. H. (1956) Biochem. J. 62, 569-582. 39. WALLEVIK, K. (1973) J. Biol. Chem. 248, 2650-2655. 40. PFLUMM, M. N., AND BEYCHOK, S. (1969) J. Biol. Chem. 244, 3973-3981. 41. Takagi, T., OKANO, R., AND MIYAZAWA, T. (1973) Biochim. Biophys. Acta 310, 11-19. 42. SIMONS, E. R., SCHNEIDER, E. G., AND BLOUT, E. R. (1969) J. Biol. Chem. 244, 4023-4026. 43. STRONG, H. D. (1962) Statistical Treatment of Experimental Data pp. 120- 123, McGrawHill, New York.
AND BIOPHYSICS pp. 104-113, 1980
Acid- and Temperature-Induced Conformational Changes in Human Chorionic Gonadotropin and the Mechanism of Subunit Dissociation HILDA Plasma
Derivatives
FORASTIERI
Laboratory,
American Received
KENNETH
AND
Red Cross February
Blood
C. INGHAM Services,
Bethesda,
Maryland
20014
22, 1980
Human chorionic gonadotropin undergoes a conformational transition in acid which at 4°C is characterized by: (i) a reversible increase in the polarization of tyrosyl fluorescence, P, with a midpoint at pH 5, (ii) a slight decrease in the elution volume on Sephadex G-100 near uv difat pH 3 relative to pH 7, (iii) a slight decrease in s~~,~, (iv) a small positive ference spectrum (AE -2%), and (v) the appearance of a positive CD feature at 235 nm. These observations are compatible with an acid-expanded form of the hormone in which the rotational freedom of one or more tyrosine residues is restricted and/or their proximity to potential quenching groups is altered. The increased value of P following acidification is stable at temperatures below lO”C, but at higher temperatures it decreases with time to an extent which is dependent on the temperature. A substantial portion of this decrease occurs before subunit dissociation can be detected, reflecting the occurrence of a thermal transition with a midpoint near 26°C. A similar transition was observed at neutral pH with a midpoint near 22°C. These results suggest the occurrence of at least two conformationally distinct forms of hCG which may be sequentially encountered prior to subunit dissociation in acid. The kinetics will be either biphasic or strictly first order, depending on the temperature at which the hormone is acidified.
Human ehorionic gonadotropin (hCG)’ is one of several homologous glycoprotein hormones, each comprised of two dissimilar subunits, (Y and p, which are noncovalently linked (l-3). The subunits, which can be dissociated in acid or urea, are biologically inactive (4). Changes in tertiary and/or secondary structure are believed to accompany the dissociation of the subunits (5, 6). However, it is not known whether these changes occur prior to, simultaneously with, or subsequent to, the actual physical separation of the subunits. Spectral studies of the isolated subunits of hCG reveal little evidence for structure of the type which can be disrupted by denaturants or other forms of stress; they appear to
be completely unfolded within the constraints imposed by the disulfide bonds (7). A better understanding of the mechanism of dissociation might provide insight into the structural requirements for biological activity. In previous studies, the rate of acid-induced dissociation was monitored by the loss in the ability to enhance ANS fluorescence. First-order kinetics were reported for hCG (‘7, S), hLH (9), bTSH (lo), and oLH (11). Similar results were obtained when differential absorption was used to monitor the exposure to tyrosyl residues which accompanies the dissociation (7, 10, 11). However, other investigators have observed biphasic kinetics for dissociation of several of these hormones by various methods of detection (12-15). Using polarization of tyrosyl fluorescence, Bolotin and Ingham (ll), also observed biphasic kinetics with oLH under certain conditions. Evidence for a conformational change prior to physical dissociation was provided
’ Abbreviations used: hCG, human chorionicgonadotropin; hLH, human luteinizing hormone; oLH, ovine luteinizing hormone; bTSH, bovine thyroid-stimulating hormone; P, tyrosyl fluorescence polarization; CD spectrum, circular dichroic spectrum; FSH, folliclestimulating hormone. 00039861/80/130104-10$02.00/O Copyright 0 1980 by Academic All rights
of reproduction
in any
Press, Inc. form reserved.
104
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GONADOTROPIN
by the observation of an increase in polarization (P) of tyrosyl fluorescence following acidification of hCG (7). A similar, but smaller increase was observed with oLH (11). The purpose of the present study was to further analyze these acid-induced changes in hCG. This was accomplished by working at lower temperature where the acid form could be reversibly generated and characterized. As a result of working at lower temperature, we discovered a thermal transition which occurs both in acid and at neutral pH and which is manifested by a small change in P and the appearance of biphasic kinetics under certain conditions. MATERIAL
AND METHODS
Highly purified hCG (CR 121, having a biological potency of 12450 IU/mg) was obtained from Dr. R. Canfield of Columbia University via the Center for Population Research, National Institute of Child Health and Human Development, National Institutes of Health. hCG subunits (CR 121) obtained from the same source, had biological potencies of less than 0.5% of the intact hormone. The subunits each exhibited a single band on polyacrylamide disc gel electrophoresis in the presence of 8 M urea, according to Swank and Munkres (16). The hormone exhibited two bands corresponding to those of the subunits. None of the preparations exhibited tryptophan fluorescence, confirming the absence of tryptophan-containing impurities. Protein concentrations were determined from the absorbance at 276 nm using E = 12,000 for hCG, 6700 for hCG-a, and 5400 M-’ cm-’ for hCG$ (7). Differential absorption measurements were made on a Cary 118C spectrophotometer equipped with a thermostatted cell holder, using l.O-cm path length, Teflon stoppered, quartz cuvettes according to the methods of Herkovits (17) and Donovan (18). A Cary Model 61 spectropolarimeter, also equipped with a thermostatted cell holder was used to obtain circular dichroism spectra between 200 and 350 nm at 4°C. Cuvettes having path lengths of 0.5 and 0.05 cm were used for near and far ultraviolet measurements, respectively. Mean residue ellipticities were calculated according
[e]x =
0,b, x 151 1OdC ’
Dl
where Oobs is the observed ellipticity, 151 is the mean residue molecular weight for hCG (including the carbohydrate (19)), d is the path length in
CONFORMATION
105
centimeters and C is the concentration in grams per milliliter. Sedimentation velocity measurements were made at 52,000 rpm in double-sector cells at 4°C using a Spinco Beckman Model E ultracentrifuge, equipped with Schlieren optics and RTIC temperature control unit. Observed sedimentation coefficients were corrected to sedimentation in water at 20°C (szO,,) according to established procedures (20). Samples at neutral pH (0.01 M KPO,) and pH 3 (0.02 M citrate) were analyzed simultaneously in a single experiment, at a hormone concentration of 4 mg/ml. Fluorescence measurements were determined on a Perkin-Elmer MPF4 fluorometer equipped with Polacoat polarizing filters and thermostatted cell holder. Excitation and emission wavelengths of 270 and 310 nm, respectively, were used in order to avoid interference by Raman scattering. Polarization measurements were made as previously described (7, 11) except a uv scrambler was placed between the emission polarizer and the emission monochromator in order to eliminate the need for a grating correction factor. Using vertical excitation, polarization was calculated using Eq. [2]: P = (I,, - Z,)l(Z, + I”),
PI
where Iv and Z H are the observed intensities with the emission polarizer in the vertical and horizontal positions, respectively (21). The observed intensities were corrected for buffer background, which never exceeded 10% of the sample intensity. It was observed that continuous exposure of hCG to excitation at pH 3.0 caused a decrease in P with time which was substantially greater than that observed by periodic monitoring. This effect was eliminated by the use of narrow excitation slits, minimization of exposure time, use of higher hormone concentrations, and stirring of the samples prior to each measurement. The total relative fluorescence intensity for a given polarization measurement was calculated according to Eq. [3] (22): ZT”hl = I, + 21,.
[31
Exclusion chromatography was performed on a 1.0 x 57-cm column of Sephadex G-100 at 4°C using tyrosyl fluorescence to monitor the elution of hormone and/or subunits. Column parameters, as determined using blue dextran (V,) and tyrosine (I’,,,,), were identical at pH 7.0 and pH 3.0. This column does not resolve a mixture of hCG and p subunit. The extent of subunit dissociation of various hCG samples was estimated by nonlinear interpolation between the elution profile for intact hCG and that for fully dissociated hCG usingf = 3Rl (R + 2) wheref is the fraction dissociated and R is the ratio of the peak intensity for hCG-a to that for hCG and/or hCG& This equation takes into account the approximate differences in quantum yield and extinction coefficients of the various species.
106
FORASTIERI
AND INGHAM
RESULTS
A. Acid-Induced
Conformational
Changes
The influence of pH on the polarization (P) of the tyrosyl fluorescence of hCG and its subunits at 4°C is illustrated in Fig. 1. P for the intact hormone exhibited a reversible sigmoidal transition with a midpoint at pH 5.0. By contrast, P for the isolated subunits increased only slightly in acid with little evidence of a transition. The increase in P for the hormone was accompanied by a 20% decrease in fluorescence intensity, whereas the intensity of the (Y and p subunits increased 15 and 34%, respectively. These intensity changes were also reversible. The elution of hCG from a column of Sephadex G-100 at pH 7 and pH 3 at 4°C is shown in Fig. 2A. The hormone was consistently found to elute slightly earlier at the lower pH, with K,, decreasing from -0.13 to 0.10. This suggested that selfassociation might be responsible for the observed increase in P. Sedimentation velocity measurements conducted under conditions identical to those of the chromatography experiments gave values of s20,W of 3.22 at pH 7 and 2.92 at pH 3, eliminating the possibility of self-association. The significance of the small decrease in s20,W is by itself uncertain because of potential primary charge effects. However, when taken together with the chromatographic results, the decrease is compatible with a slight expansion of the hormone in acid. This is analogous to the well-characterized N + F transition in serum albumin (23), whose tryptophan fluorescence also exhibits an increased polarization in acid (24). Further evidence for an acid-induced conformational change in hCG was provided by differential absorption measurements at 4°C (Fig. 3A). The positive difference spectrum at pH 3 vs pH 7, although small ( AE - 2%), exhibits the characteristic features of tyrosyl residues which have experienced a decrease in the polarity of their microenvironment (1’7, 18). There is an apparent shift in the baseline which could reflect small changes in the amount of scattered light. Alternatively there could be
FIG. 1. Effect of pH on the polarization of the tyrosyl fluorescence of hCG and its subunits at 4°C. The protein concentrations were 26 pM in 0.01 M KPO,. Titrations were initiated at pH 7 by adding aliquots of 1 M HCl (0) and reversed with 1 M NaOH (a).
alterations in the extent to which disulfide bonds contribute to the spectrum. Acidification also produced a positive extremum near 235 nm in the near uv CD spectrum (Fig. 3C). A similar feature has been observed in other glycoprotein hormones and has been attributed to tyrosyl and/or disulfide residues (25-30). The far uv circular dichroic spectrum exhibited about a 15% decrease in the intensity of the negative band at 209 nm, suggestive of a slight alteration of secondary structure (Fig. 3B). B. Thermal Transitions Following Acidification
Figure 4 illustrates the time dependence of P following dilution of a chilled sample of hCG into pH 3 buffer which had been equilibrated at the indicated temperature. The initial value of P was independent of temperature between 4 and 37°C. A pattern similar to that in Fig. 4 was also obtained when a sample was first acidified on ice and then quickly brought to the indicated temperature and placed in the fluorometer, previously equilibrated at the same temperature. At PC, P was constant with time even for much longer periods than shown in Fig. 4. Between 16 and 3O”C, P decreased with time, but within 15 min reached an apparent plateau value which was dependent upon tempera-
HUMAN
0
CHORIONIC
15
20
GONADOTROPIN
25
10
107
CONFORMATION
15
20
25
ELUTION VOLUME (ml) FIG. 2. Exclusion chromatography of hCG and its subunits at 4°C on Sephadex G-100 (1.0 x 57 cm). The buffer was 0.02 M citrate, pH 3, except for solid circles (0) panel A, where 0.01 M KPO,, pH 7, was used. Approximately 0.5 mg ofprotein was applied in each determination. (A) Elution ofintact hormone at pH 7 (e) and pH 3 (0); (B) separate elutions of (Y(A) and (0) subunits; (C) hCG dissociated into subunits by incubation in 10 M urea at 40°C for 60 min. Urea was removed from the sample by dialysis into eluting buffer at 4°C prior to application to the column; (D, E) hCG after 40 min incubation at pH 3 and 25” or 3O”C, and 37°C respectively (see Fig. 4); (F-H) hCG after incubation for 210 min at pH 3 and 16”, 25”, and 37°C respectively.
ture. These changes, which could only be reversed by slow cooling, were not accompanied by changes in the intensity of fluorescence. After 40 min, the 25 and 30°C samples were analyzed by exclusion chromatography at 4°C and pH 3.0. As shown in Fig. 2D, there was no indication of dissociation into subunits. At 37”C, the decrease of P with time was continuous and no plateau was evident (Fig. 4). An increase in fluorescence intensity was also observed. Exclusion chromatography of this sample after 40 min revealed two peaks, as shown in Fig. 2E. The first peak elutes in the position corresponding to native hCG (Fig. 2A) and/or hCG-p (Fig. 2B); mixtures of hCG and hCG-P are not significantly resolved on this column. The second peak corresponds to the cx subunit which elutes much later (Fig. 2B),
and indicates that approximately 30% of the hormone was dissociated. This estimate is based on a comparison of the ratio of the two peaks with the same ratio for a sample which was fully dissociated in urea (Fig. 2C, see Methods). Prolonged incubation of similar samples resulted in no detectable dissociation at 16”C, approximately 78% dissociation at 25”C, and complete dissociation at 37°C after 3.5 h, as shown chromatographically in Figs. 2F-H. The final value of P (corresponding to complete dissociation) in this and similar experiments at 37°C was 0.17 ? 0.005 and the total increase in fluorescence intensity was about 30%. Based on polarization measurements alone, the 37°C sample in Fig. 4 would appear to be much more than 30% dissociated after 40 min, since about 60% of the total change in P has occurred by this time.
108
FORASTIERI
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,
WAVELENGTH
lnml
FIG. 3. (A) Differential absorption spectrum for hCG at pH 3 vs pH 7 at 4°C (--). The baseline (- - -) was obtained with 54 M hCG in both cuvettes at pH ‘7. The sample solution was then acidified by addition of 2 M HCI and an equal volume of buffer added to the reference solution. (B, C) Circular dichroism of hCG (50 ELM) at 4°C in 0.01 M phosphate buffer at pH 3 (-) and pH 7 (- - -). The path length was 0.05 cm in B and 0.5 cm in C.
This implies that much of the decrease in P during the first 40 min is not due to dissociation and suggests that elevation of the temperature at pH 3 causes the acid form of the hormone to undergo a thermal transition which does not involve, but rather precedes dissociation of subunits. When chilled samples of hCG were diluted into pH 3 buffer at elevated temperatures, the ensuing decrease in P did not obey first-order kinetics. Data obtained at 37 and 42°C are illustrated in Fig. 5A. This type of result would be expected if multiple steps were involved in the dissociation reaction. When the hormone was preincubated for 30 min at pH 3 and 30°C (as in Fig. 4), followed by rapid elevation of the temperature to 37 or 42”C, first-order plots of the decrease in P were linear as shown in Fig. 5B (circles). This suggests that preincubation converted the hormone from a high P, nondissociating form which prevails at low temperature, to a dissociable form characterized by a lower P and which appears with time at higher temperatures. It was subsequently found that if the hormone was first brought to 37 or 42°C at neutral pH and then rapidly acidified, first-order kinetics were also followed and, as shown by the triangles in Fig. 5B, the rates were identical to those obtained after preincubation at pH 3.0 and 30°C. The initial P value for the hormone immediately after acidification at 37 or 42”C, was -0.205, slightly lower than that obtained after preincubation at pH 3 and 30°C.
This value is represented in Fig. 4.
by the dashed line
C. Thermal
at Neutral
Transitions
pH
In the preceding section, kinetic evidence was provided for the occurrence of a thermal transition, not only in acid, but at neutral pH as well. Careful measurements of P for the intact hormone at neutral pH provided additional evidence for such a transition. As shown in Fig. 6, P exhibited a shallow sigmoidal dependence on temperature with a midpoint near 22°C. This was accompanied
:I:.
10
20
30
MINUTES
40
50
FIG. 4. Effect of time on the tyrosyl polarization of hCG at various temperatures. Small aliquots of chilled, concentrated hCG were added to 0.01 M Na-citrate, pH 3, equilibrated at the indicated temperature. Final protein concentration was 22 pM. The dashed line represents the initial value ofP obtained when a sample at 37°C is rapidly acidified and represents the plateau value which would be reached at 37°C if no subunit dissociation occurred (see text).
HUMAN
CHORIONIC
GONADOTROPIN
CONFORMATION
109
Fig. 6 allowed an estimate of the fractional conversion between the low- and hightemperature forms. The equilibrium data thus obtained are plotted according to the van’t Hoff equation in Fig. 7 (triangles). Although there is considerable uncertainty in the slopes of the dashed lines in Fig. 6, the AH values obtained from the van’t Hoff plots were rather insensitive to arbitrary variations in those slopes. The circles in Fig. ‘7 were obtained by a similar analysis of the plateau values in Fig. 4 using the value of P represented there by the dashed line as the value corresponding to complete conversion at pH 3. Although Fig. 7 clearly indicates that the transition is shifted to higher temperature at pH 3, the uncertainties in the values of AH and
MINUTES
FIG. 5. First-order plots of the time course for the dissociation of 22 M hCG at pH 3. (A) Chilled, concentrated hCG diluted into 0.01 M citrate, pH 3, at 37” (0) and 42°C (0); (B) chilled, concentrated hCG diluted into 0.01 M citrate, pH 3, incubated at 30°C for 30 min (see Fig. 4), and then brought rapidly to 37°C (0) or 42°C (0). The triangles illustrate the results obtained when a 2%pM sample at neutral pH was first equilibrated at the indicated temperature and then rapidly acidified.
by a 50% decrease in fluorescence intensity. By contrast, P for the individual subunits decreased linearly with increasing temperature, with no evidence of a transition. All of these changes were completely and 0.10 ,\ 0 rapidly reversible. Exclusion chromatog10 20 xl 40 50 raphy of the hCG sample revealed no TEMPERATURE (“C) evidence for dissociation. However, at higher temperatures (50-7O”C), hCG exFIG. 6. Effect of temperature on the polarization of hibited a sharp drop in P which was tyrosyl fluorescence of hCG and its subunits, each at found by exclusion chromatography to in- a concentration of 40 pM in 0.01 M phosphate buffer, volve subunit dissociation (data not shown). pH 7. The temperature was increased (0) and decreased (0) at approximately l”C/min with periodic This transition, which was also reversible of P. In order to confirm the subtle provided sufficient time was allowed at each monitoring transition observed for hCG, the experiment was temperature for the state of association to repeated, making multiple measurements of P after reach equilibrium, will be the subject of a equilibration of the intact hormone at each temperaseparate report. ture shown. This accounts for the reduced scatter in Interpolation between the dashed lines of the data for hCG relative to subunits.
110
FORASTIERI
20
0.8
AND
INGHAM DISCUSSION
15
hCG has seven tyrosines (four in (Y and three in p) whose relative contribution to the total fluorescence may vary considerably (‘7). Thus large variations in polarization do not necessarily reflect gross conformational changes but could arise from localized alterations in the environment of one or more tyrosines which contribute disproportionately to the observed intensity. A discussion of the factors which influence P is facilitated by reference to the Perrin equation (21, 31):
h
-=1 p -0.8
I
-1.0
3.3
3.4
3.5
(1rr)xW
FIG. 7. van’t Hoff plots of thermal transition data for hCG at pH 7 (A) and pH 3 (0). The quantityf is the fraction present in the high temperature form as estimated by interpolation between the dashed lines in Fig. 6 or the plateau values in Fig. 4 (see text). The lines were obtained by linear regression and standard deviations of slopes and intercepts are reported in Table I.
AS summarized in Table I do not allow this effect to conclusively be attributed to one or the other of these parameters; a small change in either could account for the observed shift. TABLE
I
CHARACTERISTICSOFTHE THERMAL TRANSITION IN HCG"
PH 7 3
Temperature (“C) 22.5 26.7
AH (kcal/mol) 35 ? 1 37 + 0.6
AS (4 118 ? 4 124 f 2
” The AH and AS values were obtained by linear regressions of the data in Fig. 7. Standard deviations were computed from the variance of the parent distributions assuming all errors occur in the dependent variable (43). Temperature values were taken as the points at which the lines in Fig. 7 intersect the abscissa log (fl1 -f) = 0.
f+ 0
(
$-i 0
(7/p), 1
[41
which relates P for a given tyrosine to its lifetime, T, and the rotational relaxation time, p, of the phenolic group. P, is the maximum polarization observed in the absence of rotation. Thus, events which affect either T or p will influence P. Changes in 7 for a given tyrosine can result from changes in the dielectric properties of its microenvironment and/or changes in the extent of dynamic quenching by neighboring groups on the protein or by other substances present in solution. Changes in p require changes in the rigidity of the microenvironment or in the flexibility of the local polypeptide backbone. Because of the short r for tyrosine fluorescence in proteins (-2, ns (3211, changes in the overall rotational motion of the protein, such as occur during self-association or subunit dissociation, are expected to have relatively little influence on P unless they are accompanied by conformational changes which alter the local rigidity of the tyrosyl environment. Finally, changes in the extent of intertyrosyl energy transfer can also influence P. This phenomenon is very sensitive to donor-acceptor distance and orientation; the critical distance for tyr-tyr transfer is 7-9 A (33, 34). The present observations of the effects of pH and temperature on the conformation and state of subunit association of hCG, as detected primarily through changes in P, can be rationalized in terms of the following scheme:
HUMAN
CHORIONIC
GONADOTROPIN
PH where HN represents the native hormone as it exists at neutral pH and temperatures between 30 and 45°C. The subscript A refers to the acid form and the primes to the low-temperature form which prevails below 10°C. Between 10 and 3o”C, the hormone appears to undergo a thermal transition which is rapidly reversible at neutral pH but slow at pH 3.0. The reversible acid transition shown in Fig. 1 corresponds to the equilibrium between HE, and Ha. The shape of the titration curve is consistent with a simple protonation with no evidence for site-site interactions. The midpoint at pH 5.0 suggests that a glutamic or aspartic acid carboxyl group is involved, although abnormal histidines have also been shown to titrate in this region (35, 36). The 20% decrease in fluorescence intensity which accompanies this transition, if it were due entirely to dynamic quenching, could cause a proportional decrease in 7. This could account for a small part of the increase in P without the need for a conformational change. However, protonation of carboxylates is expected to reduce their quenching efficiency, suggesting that the effect on P is indirect, perhaps mediated by a conformational change which decreases the flexibility of one or more fluorescing tyrosines and/or alters their proximity to other potential quenching groups such as disulfide or peptide bonds (37). The effect of acidification on the differential absorption spectrum of hCG at 4”C, although small, reinforces the concept of a conformational change affecting the environment of at least one tyrosine. The fact that the difference spectrum is of opposite sign to that which accompanies dissociation (7, 19) corroborates our assumption that the latter has not occurred, an assumption which was further confirmed by
CONFORMATION
111
exclusion chromatographic analysis. Hence, a distinct form of the hormone occurs in acid which can be reversibly generated and characterized at low temperature. A similar transition also occurs at higher temperatures as evidenced by the instantaneous increase in P following acidification (HN + HA). We suggest that these structural changes which occur reversibly at low temperature are similar to those which occur transiently at higher temperature and which constitute the first step in the mechanism of acid-induced subunit dissociation. The sedimentation velocity and exclusion chromatography results suggest that this first step involves a slight expansion of the hormone. An analogous situation has been well documented for serum albumin, whose structure consists of several globular domains which become separated as the pH is lowered (38). The process occurs in at least two steps, the first corresponding to a loosening and the second to a more dramatic unfolding. Sogami et al. (24) have shown that the first step is accompanied by an increase in polarization of tryptophanyZ fluorescence (analogous to the increase for hCG in Fig. l), while the second step is accompanied by a decrease (analogous to that which accompanies the actual dissociation of the subunit “domains” of hCG). It is not obvious how a “loosening” of the subunit domains causes an increase in P. However, an overall loosening does not preclude a local increase in the rigidity of the environment of a particular tyrosine. Another possibility is that this loosening is accompanied by decreased energy transfer between tyrosines on the same or different subunits. The first and perhaps best evidence for a thermal transition came from the deviations from linearity in the first-order kinetic plots of the polarization data and the elimination of those deviations by preincubating the sample at a temperature above the transition temperature (Figs. 5A and B). In terms of our proposed scheme, acidification of a chilled sample results in the formation of a species, Hi, which is stable at low temperature, and whose conversion to HA following a subsequent increase in temperature is sufficiently slow
112
FORASTIERI
to be manifested in the kinetics. On the other hand, acidification above the transition temperature immediately produces HA which undergoes a simple first-order dissociation reaction whose rate depends on pH and temperature. This is the mode of acidification used in earlier studies where first-order kinetics were routinely obtained (g-10). Measurements of P as a function of temperature at neutral pH provided direct evidence for a thermal transition. It is of interest that the slope at either end of the P vs T curve for hCG in Fig. 7, is much smaller than that for the isolated subunits at any temperature. This is compatible with a more restricted amplitude for the temperature-dependent rotational and vibrational motions of the fluorescing tyrosines in the intact hormone relative to the subunits. The increase in P which accompanies acidification could, as mentioned above, be interpreted as a further increase in rigidity, perhaps explaining the slower rate of interconversion between the low- and hightemperature forms at pH 3 vs pH 7. Recall, however, that the thermodynamic parameters governing the equilibria between these forms were not strongly influenced by pH (Table I). Autonomy between pH and temperature-dependent transitions has been demonstrated for human serum albumin (39). The exact origin of the positive feature near 235 nm in the CD spectrum of hCG (Fig. 3) is unclear. A similar feature has been observed in several homologous glycoprotein hormones and/or subunits including bTSH (26), oLH (25,2’7,29), hLH, and FSH (28). It is generally attributed to tyrosyl residues, an interpretation which is supported by the observation that it is most intense in bTSH-P which has a relatively high tyrosine content (26). In addition, the intensity of this band in bTSH decreased significantly following enzymatic removal of two tyrosines from the C terminal region. Pflumm and Beychok observed that a similar feature in the CD spectrum of ribonuclease was strongly affected by tyrosine-specific chemical modifications (40). However, these authors discussed the difficulty of making a definitive assignment of this band because
AND INGHAM
of overlap with neighboring, more intense bands, and provided evidence that disulfide bonds could also be involved. Indeed, a similar but somewhat red-shifted positive band has also been observed with the model compound (Ac-Cys-N-Me)z which lacks tyrosine (41). The intensity of this band, as well as that of the analogous ones in ribonuclease (40, 42) and oLH-a (29), was found to increase with decreasing temperature. Whatever the origin of the 235-nm band, it is clearly accentuated by acidification (Fig. 3), providing additional evidence for a conformational change. A similar effect of acid was reported by Giudice et al. (28), for hLH under conditions where subunit dissociation was not expected. Their spectra, obtained at room temperature, are almost identical to ours at 4°C. However, the 235-nm feature, detectable in our spectrum, even at neutral pH, is absent from published spectra of hCG obtained at room temperature (19, 29), providing additional evidence for a thermal transition in this temperature range. A more definitive assignment of the origin of this band, together with a detailed study of its dependence on pH and temperature, could further clarify the nature of the acid- and temperature-induced transitions in hCG. ACKNOWLEDGMENTS This report constitutes publication No. 467 from the American Red Cross Blood Services Laboratories. The work was also supported by the National Institutes of Health (Grants RR 05737 and AM 19719) and by a Career Development Award to K. C. Ingham (HL 00325). We are indebted to Dr. James C. Osborne for the use of his circular dichrometer and for helpful advice. Thanks also to Mrs. Marcia Robinson for typing the manuscript. REFERENCES 1. CANFIELD, R. E., MORGAN, F. J., KAMMERMAN, S., BELL, J. J., AND AGOSTO, G. M. (19’71) Rec. Progr. Horvn. Res. 27, 121-164. 2. BAHL, 0. P. (1973) in Hormonal Proteins and Peptides (Li, C. H., ed.), Vol. 1, p. 171, Academic Press, New York. 3. MORGAN, F. J., AND CANFIELD, R. E. (1971) Endocrinology 88, 1045-1053. 4. CATS, K. J., DUFAU, M. L., AND TSURUHARA, T. (1973) J. Clin. Endocrinol. Metab. 36, 73430.
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