THOMAS A. BEWLEY Hormone
CHOH HA0 LI
Received July 25, 1974 Porcine pituitary lactogenic hormone has been examined by exclusion chromatography, analytical ultracentrifugation, free-boundary electrophoresis, solubility behavior, circular dichroism, and fluorescence spectra. The results have been compared with previous studies of ovine pituitary lactogenic hormone. These two hormones were found to resemble one another very closely by all criteria studied. While the homology between these lactogenic hormones and growth hormones is strongly reflected in the sharing of certain physicochemical properties, a number of distinct structural differences between these two closely related evolutionary lines are now becoming apparent.
The purification and partial characterization of porcine prolactin’ was first described in 1964 by Eppstein (1, 2). A complete amino acid sequence for the molecule was reported in 1973 by Li (3). A comparison (3) of the primary structure of PP2 with that of OP (4) indicated that 162 of the 198 residue positions were occupied by identical amino acids. According to the scheme described by Bewley and Li (5), the 36 nonidentical residues may be characterized as: 14 highly acceptable, 8 acceptable, and 14 unacceptable replacements. Thus, the homology between these two proteins is nearly as extensive as that between HGH and HCS, and somewhat greater than that between HGH and SGH (6). The present investigation was undertaken to physico’ Paper XXXVI in the Studies on Pituitary Lactogenie Hormone series. The terms “prolactin” and “lactogenic hormone” are equivalent, as are the terms “somatotropin” and “growth hormone.” Paper XXXV appeared in Arch. Biochem. Biophys. 161, 313-318 (1974). z Abbreviations: PP, porcine pituitary lactogenic hormone; OP, ovine pituitary lactogenic hormone; HGH, human pituitary growth hormone; HCS, human chorionic somatomammotropin; SGH, ovine pituitary growth hormone; BGH, bovine pituitary growth hormone; CD, circular dichroism. 80 Copyright 0 1975by Academic Press, Inc. All rights of reproduction in any form reserved.
chemically characterize the porcine hormone and to compare the physical properties of PP with those previously reported from studies of OP. The combined properties of these lactogenic hormones may then be compared with similar properties of growth hormones in order to gain further insight into structure-function relationships in these proteins. EXPERIMENTAL Highly purified PP was prepared from fresh porcine pituitary glands by a modification of procedures previously described for the isolation of OP (4,7). For the studies contained herein, the monomer form of the protein was obtained from lyophilized material by exclusion chromatography on Sephadex G-100 in either 0.1 M Tris-HCl buffer (pH 8.2) or 0.01 M ammonium acetate buffer (pH 4.0). This monomer was always used directly without subsequent lyophilization. In some instances, this required either concentration of the solution in an Amicon Diaflo apparatus using a PM-10 membrane, or changing buffers by exhaustive dialysis in the cold, or both. In no case was the protein subjected to pH changes which would require passing through the isoelectric point. Ultrapure guanidine hydrochloride was obtained from Heico, Inc., Delaware Water Gap, PA. All other chemicals were of reagent grade and used without further purification. The concentration of PP in all solutions was determined spectrophotometrically using the relation
PORCINE previously for OP (4). 4m l,,“rn = 9.09 determined The validity of assuming an identical value for the absorbance of PP is supported both by the results of spectrophotometric titration and the fact that solutions of OP and PP which show equal absorbance also give equal fringe counts in low-speed synthetic boundary experiments in the ultracentrifuge. Absorption spectra were scanned from 360 to 245 nm and corrected for light scattering as described by Beaven and Holiday (8). Sedimentation behavior. The sedimentation behavior of PP was studied in the Spinco Model E analytical ultracentrifuge equipped with both schlieren and Rayleigh optics. Rotor temperature was maintained at 2O.O”C by the RTIC unit on the centrifuge in all cases. Sedimentation velocity was measured by the moving boundary method at 52,640 rpm in an AN-D rotor. Sedimentation equilibrium was carried out by the high-speed meniscus depletion method described by Yphantis (9) on a solution of the protein (0.25 mg/ml) in 0.1 M KCl, 0.01 M NH,HCO, buffer (pH 10.35) at 35,580 rpm. The attainment of equilibrium was tested by reading successive interference plates. The molecular weight was calculated from the relationship:
A value for the partial specific volume of the protein (V = 0.732 cmg,‘g) was computed from the amino acid composition according to the primary structure of the hormone (3). Stokes radius. The Stokes radius (a) of PP was measured at rmrn temperature by exclusion chromatography on Sephadex G-100 in 0.1 M Tris-HCl buffer (pH 8.2), following the approach outlined by Laurent and Killander (10). The chromatographic column was calibrated with two proteins of known Stokes radii: bovine serum
where K., = (V,-V,)/V*V,,). V, and V, are the elution volume of the protein and the total volume of the gel bed, respectively. The values of L and R are column constants determined by the calibration proteins. From a combination of the Stokes radius, sedimentation coefficient (s.,,,) and the partial specific volume of the protein, the molecular weight was also calculated from the relationship: M _ 6*Nwlo,,a (l-tip)
where N is Avagadro’s number, and I) and p were taken to be the viscosity and density of water at 2O”C, respectively. Free-boundary electrophoresis. Free-boundary electrophoresis of the protein was carried out in a Spinco Model H electrophoresis-diffusion apparatus. All experiments were performed in sodium acetate or sodium phosphate buffers (I = 0.05) at 1.7”C. Protein solutions were prepared by concentrating a sample of the monomer which had been previously obtained by exclusion chromatography in either an acidic or basic buffer to 2-3 mg/ml. This concentrate was then dialyzed in the cold against 200 vol of the appropriate acetate or phosphate buffer. The conductivity of the buffer was determined on an LKB Conductivity Bridge, Model 3216 B, also after dialysis against the protein solution. All experiments were conducted at an applied current of 8 mA. In several experiments on either side of the isoelectric point, when the boundary had migrated to a position almost out of view, the field potential was reversed and the experiment continued until the protein boundary had returned approximately to its original position. The mobility (p) was calculated from the relationship: p = u/E, where u is the velocity of the boundary in cm/set and E is the field potential gradient in volts/cm. E was computed from: E = Z/KA, where I is the applied current, K the specific conductivity of the buffer, and A the cross-sectional area of the electrophoresis cell. mobility is expressed The in the units cmz.sec-‘.volt-‘. Circular dichroism spectra. Circular dichroism spectra of PP in various solvents were taken on a Gary Model 60 Spectropolarimeter equipped with a Model 6002 Circular Dichroism attachment according to procedures previously described (11). The content of a-helix was estimated as described by Bewley et al. (12). The mean residue weight of PP was calculated to be 115. Difference absorption spectra and spectrophotometric titration. Difference absorption spectra of PP in various solvents, relative to the protein in 0.1 M Tris-HCl buffer (pH 8.2) were obtained on a Beckman DK-2A spectrophotometer by procedures described elsewhere (11). Spectrophotometric titration of the tyrosine residues in PP were performed by the difference-spectra technique as described by Bewley et al. (12). Briefly, a sample (~1 mg/ml) of the protein at pH 8.2-8.3 is placed in the reference beam of the spectrophotometer. A second aliquot is titrated with 10 N KOH to a higher pH and placed in the sample beam. A spectrum is taken from 360 to x250 nm. The sample aliquot is again titrated to a still higher pH and another spectrum is taken. After reaching pH 13, the titration is reversed, using constant-boiling HCl as the titrant. A sample of N-acetyl-L-tyrosinamide was also titrated as a model in order to determine the tyrosine residues AE H, 196“In for peptide-linked
under identical experimental conditions. This value was found to be 2380 per mole, with a PK. of 10.0 in good agreement with previously published values (8, 13). Fluorescence emission spectra. The fluorescence emission spectra of both PP and OP in 0.1 M Tris-HCI buffer (pH 8.2), and PP in 0.1 M sodium acetate buffer (pH 4.0) were measured on a Hitachi-Perkin-Elmer spectrofluorimeter, Model MPF-2A. The instrument was equipped with a temperature-controlled cell holder and maintained at 27.O”C. The excitation monochromator was set at 292 nm. All spectra were corrected for solvent blanks. Samples of known concentration were quantitatively diluted before taking the fluorescence spectra to provide solutions with maximum optical densities less than 0.1. Relative quantum yields were estimated from the areas under the emission spectra after correcting for solvent blanks and differences in protein concentration. Solubility. The solubility experiments of both PP and OP in ammonium sulfate solutions were carried out in a cold morn at 2°C as previously described (14). (NH,),SO, solutions of various concentrations had a pH value of 6.60. RESULTS
Exclusion chromatography. When highly purified, lyophilized PP is submitted to exclusion chromatography on Sephadex G-100 in 0.1 M Tris-HCl buffer (pH 8.2), the elution pattern shows a small amount of aggregated material ( <5%) appearing at the void volumn of the column, with the major component appearing as a single, essentially symmetrical, peak. The trailing edge of this peak is usually a little steeper than the leading edge. However, this slight departure from symmetry is not significantly improved by cutting samples from either side of the peak and submitting them to rechromatography. The rechromatographed peak does not show any aggregate indicating that high molecularweight forms of PP are not in rapid equilibrium with the monomer. The elution position of the peak is very reproducible, exhibiting a VJV,, ratio of 1.86 with a standard error of f 0.03. The K,, for PP was found to be 0.414 and appeared to be independent of protein concentration. Exactly the same values for VJV, and K,, were found for OP on the same column. A V,lV, ratio of 1.85 has been previously reported for GP under these same conditions (15). Sedimentation behavior. The values for
sedimentation coefficient of PP at a number of protein concentrations in Tris buffer (pH 8.2) are shown in Table I. The regression line through these points, obtained by the least-squares method, gives a value for the sedimentation coefficient at infinite dilution (s2,,+.) of 21.8 S with a standard error of f 0.06. The slope of this line was not significantly different from zero at the 99% confidence level according to the standard F test. This indicates there is no apparent concentration dependence of ‘s’ in this solvent between 2 and 8 mg/ml. In all cases, the schlieren patterns appeared as single, symmetrical peaks. The same value, s20,W= 2.18 has been reported for OP in 0.1 M NaHCO, buffer at protein concentrations of 3-5 mg/ml (16). The behavior of PP in a high-speed sedimentation equilibrium experiment at pH 10.35 is shown in Fig. 1. From the innermost radius (r) at which the fringe displacement (Ay) could be measured accurately, out to the fluorocarbon meniscus, the plot of log,, (Ay) vs r2 appears linear. The slope of the least-squares regression line was found to be 0.744 with a standard error of 0.015 cme2. From this slope and the value of V = 0.732 cm3 x g-l, a weight average molecular weight for the anhydrous protein was computed to be 22,400 * 500. This value is in good agreement with the molecular weight of 22,870 calculated from the amino acid sequence (3). Lowspeed sedimentation equilibrium experiments in Tris-HCl buffer (pH 8.2) invariaTABLE
SEDIMENTATION COEFFICIENT OF PORCINE PROLACTIN Protein concentration (mg/ml) 7.75 6.10 5.20 3.55 1.85 O.Ob
2.20 2.19 2.16 2.18 2.19 2.18 f 0.06
“Measured in 0.1 M Tris-HCl buffer, pH 8.2, at 2O.O”C. *Value at infinite dilution extrapolated from the least-squares regression line * standard error of the extrapolated intercept.
T Ls 0 $
r2 (cm21 FIG. 1. Sedimentation equilibrium of porcine prolactin at 2O.O”C in 0.1 M KCl, 0.01 M NH,HCO, buffer (pH 10.35) performed according to the meniscus depletion method. The log,, of the vertical fringe displacement (Ay) as a function of the square of the distance from the axis of rotation (r) was obtained from an interference photograph taken after 330 min at 35,580 rpm. Protein concentration at the bottom meniscus was 1.90 mg/ml.
bly showed small amounts of higher molecular-weight components accumulating with time as the run was prolonged (12-72 h). Under these conditions, it was not possible to reach a stable condition of equilibrium, Stokes radius. A Stokes radius of 24.9 + 0.5 A was calculated for PP from its elution pattern on a calibrated Sephadex G-100 column, in Tris-HCl buffer (pH8.2) and at 27°C. On the same column, a Stokes radius of 24.9 A was also found for the ovine hormone. From a combination of the Stokes radius, sedimentation coefficient, and partial specific volume, a molecular weight of 23,000 was computed for both PP and OP. Free-boundary electrophoresis. Although both the ascending and descending boundaries were photographed, all the following results and observations refer to the descending boundary. Under all conditions
studied, only a single, symmetrical moving boundary appeared throughout the run, with no evidence of electrophoretic heterogeneity. Even in those instances in which the field was reversed and the protein allowed to return to its starting position, no sharpening of the boundary was observed, which further indicates the electrophoretic homogeneity of the preparation. Figure 2 shows a typical plate taken after 4200 set in acetate buffer of pH 3.65 and 0.05 ionic strength. The electrophoretic mobilities of PP in various acetate and phosphate buffers are shown in Table II. Extrapolation of these data indicate an isoelectric pH of 5.85 for the hormone. This value may be compared with an isoionic pH of 6.90 calculated from the amino acid composition (3), and an isoelectric pH of 5.73, previously reported for OP under similar conditions (17). Solubility. The solubility of PP and OP in ammonium sulfate solutions as a function of ionic strength is shown in Fig. 3. The linear relationship follows Cohn’s equation (18, 19) for the solubility of proteins in a salt solution: log S = B - K,‘P, where S represents the solubility of protein in g/liter; @,the intercept constant at p = 0; K,‘, the salting-out constant; ~1, the ionic strength. Table III presents the values of /3 and K,’ for porcine and ovine prolactins. Circular dichroism spectra below 250 nm. Circular dichroism spectra of PP in
the region dominated by amide bond absorption are shown in Fig. 4A. At pH 8.2, the protein shows the two negative bands characteristic of a-helical polypeptides (20, 21) with negative maxima at 223 and 209 nm. At pH 4.0, there is a decrease in the negative ellipticity of both bands, and a definite blue-shift in the 223-nm band to 221 nm. This shift is accompanied by a reversal in the relative intensities of the two bands. This same phenomenon has been reported for OP (13). The two proteins show very similar spectra under these conditions with about 50% of either polypeptide chain exhibiting a-helical structure in both solvents. It is also clear from
BEWLEY AND LI
FIG. 2. Free boundary electrophoresis of porcine prolactin (2 mg/ml) in acetate buffer, I = 0.05, pH = 3.65 at 1.7”C. Schlieren photograph of the descending boundary taken after 4200 set under a potential gradient of 5.66 V/cm. TABLE II ELECTROPHORETIC MOBILITY OF PORCINE PROLACTIN ACETATE AND PHOSPHATE BUFFERS’
Acetate Acetate Acetate Phosphate Phosphate Phosphate
3.65 3.90 4.50 6.60 7.40 7.50
jl x lo5 (cmz.u~‘.sec-‘) +6.4 +6.5 +5.1 -3.6 -4.1 -4.3
“I = 0.05, Temp = 1.7%. The sign p indicates whether the protein is cationic or anionic at the pH of measurement.
Fig. 4A that 6 M guanidine hydrochloride (pH 8.2) causes extensive denaturation of the protein, with a loss of most of the secondary structure. Similar results have been found for OP in 5 M guanidine hydrochloride (13) and 10 M urea (22) at pH 8.0-8.2. Although not shown in Fig. 4A, the spectrum of PP taken 1 h after titration to pH 11.0 was found to be indistinguishable from the spectrum taken at pH 8.2. Because of excessive optical absorption from the solvent, it is not possible to obtain
reliable CD spectra in 50% acetic acid below x240 nm. Circular dichroism spectra above 250 nm. CD spectra of PP in the region of side-chain absorption are presented in Fig. 4B. At pH 8.2, the protein shows a weak, asymmetric positive band with a maximum near 295 nm, a negative maximum at 289 nm, with rapidly increasing negative ellipticity below 287.5 nm. In this latter region, a broad, negative shoulder appears around 280 nm. Two very weak negative maxima are found at 269 and 261 nm. At pH 4.0, both the positive band and the negative band at 289 nm are no longer visible. This spectrum is best described as a broad negative envelope between 320 and 257 nm, containing several barely discernible negative bands near 282-283, 277, 269, and 261-262 nm. Below 257 nm, the spectrum becomes rapidly more negative as it enters the region dominated by the amide bond absorption. Essentially the same type of spectra are shown in 50% acetic acid and 6 M guanidine hydrochloride. In these latter two solvents, the negative envelopes between 320 and 257 nm are not as intense and show somewhat less complex fine
I 40 IONIC
shift spectrum is generated by 50% acetic acid with well-defined blue-shift maxima at 291 and 286 nm, and shoulders near 298 and 280 nm. The spectrum generated by 6 M guanidine hydrochloride shows blue-shift peaks at 290-291 and 285-286 nm, a shoulder near 280 nm, with an additional weak shoulder between 295 and 300 nm. The spectra generated by the pH 4.0 acetate buffer and 50% acetic acid are quite comparable in both pattern and intensity to spectra previously reported for OP (23). A spectrum of OP in 6 M guanidine hydrochloride is not presently available for comparison. Spectrophotometric titration. The spectrophotometric titration of PP in 0.1 M KC1 was performed by the difference spectra technique as outlined in the Experimental
FIG. 3. Solubility of porcine and ovine prolactin in (N.H,),SO, solutions at 2%; ionic strength in moles per liter. TABLE VALUES
OF (3 AND
FOR PORCINE AND
0.356 zt 0.014 0.369 * 0.019
’ Mean + standard
structure than the spectrum at pH 4.0. The spectrum in 6 M guanidine hydrochloride appears to contain weak, negative finestructure bands near 283-284,267-268, and 260 nm. These spectra exhibit substantial similarities,
FIG. 4. Circular dichroism spectra in the region (A) dominated by the amide bond absorption and(B) sidechain absorption for PP in 0.1 M Tris-HCI buffer, pH 8.2 (-•-); 0.1 M ammonium acetate buffer, pH 4.0 (- - -); 50% acetic acid (* l l l ); and 6 M guanidine hydrochloride, 0.1 M Tris-HCl buffer, pH 8.2 (-•-). For the native protein (Tris buffer) in (A) and all cases in (B), points actually read from the CD spectra are shown as large filled circles (0).
r is 0
\ ‘..‘\ .. ‘: \ ‘4,
5 \y,+i z
....‘.‘/ .. ,/
0 b ;
,/’ -20 :
FIG. 5. Difference absorption spectra of porcine prolactin in 0.1 M acetate buffer, pH 4.0 (---); 50% acetic acid (- - -); and 6 M guanidine hydrochloride, 0.1 M Tris-HCl buffer, pH 8.2 (0 0 0). The reference contained porcine prolactin in 0.1 M Tris-HCl buffer, pH 8.2. Protein concentrations were 1.28 mg/ml in all cases.
Section. The family of curves generated during the titration all showed absorption maxima at 295 nm. Plotting the change in absorption at 295 nm versus pH produces the ionization curve shown in Fig. 6. Only six of the seven tyrosine residues in PP can be titrated at pH 13.1. The pK, of these six groups estimated from the midpoint of the ionization curve, was found to be 11.15. These results are quite comparable to those previously reported for OP in which only six of the seven tyrosyls could be titrated in 0.15 M KCl, with a pK, of 11.2-11.3 (24). One striking difference does appear, however. The titration of OP is reported to be irreversible above pH 11.8 (24). Figure 6 clearly shows that the titration of PP is completely reversible from pH 13.1. No time dependence was found in the reversibility of the PP titration although a more prolonged exposure to extremely alkaline conditions would be expected to produce some irreversibility. Even relatively shortterm exposure of OP to pH > 12 is reported to bring about irreversible denaturation (24). This point has been verified in repeated titrations of PP. Fluorescence emission spectra. The fluorescence emission spectra of both PP and OP have been measured at 27°C in Tris-HCl buffer (pH 8.2)) as well as PP in acetate buffer (pH 4.0). Excitation was effected at 292 nm in order to limit the emission predominantly to tryptophan. In addition, this wavelength was found to be the excitation maximum of both proteins
in the Tris-HCl buffer. The wavelengths of maximum emission were found to be 337.9 & 1.0 nm for PP and 336.5 & 1.0 nm for OP at pH 8.2. The relative quantum yield of OP to PP was found to be 1.03 k 0.09. At pH 4.0, the emission maximum of PP shifts to 340.0 nm with an increase in quantum yield to 1.42 times that found at pH 8.2. A very similar increase in quantum yield has been reported to accompany acidification of OP solutions (22). Table IV summarizes known physicochemical properties of prolactin isolated from porcine and ovine pituitary glands. DISCUSSION
It is now generally accepted that the conformation of a protein results from secondary intramolecular forces produced by interaction between the solvent and the primary structure (25, 26). Numerous examples have shown that when two or more proteins contain sufficient similarity in their primary structures, they will also exhibit similarities in secondary and tertiary structure (27-30). In many cases these similarities are further reflected in an overlapping of biological function. The pituitary and placental somatotropins have been found to obey these principles (6, 11, 15, 31-33), and increasing evidence suggests that the pituitary and placental gonadotropins, including pituitary thyrotropin, do also (34-41). In addition, ovine prolactin has been shown to be related in sequence, conformation, and biological activity to various pituitary and placental somatotropins (6, 13, 22, 23). Accordingly, the extensive homology reported between the primary structures of PP and OP (3) should produce additional similarities in those physical properties which are sensitive to any inter- and/or intramolecular interactions that are dictated by the conformations of the molecules. The results of the present investigation are in full accord with this view. The elution behavior of PP on exlcusion chromatography at pH 8.2 shows it to be a homogeneous preparation by this criteria. Using a calibrated Sephad$x column, equivalent Stokes radii of 24.9 A have been calculated for both hormones.
. ,.I.,*? s
0s 92 z
FIG. 6. Spectrophotometric
titration of tyrosyl residues in porcine prolactin in 0.1 M KCI: forward titration (0) and reverse titration (0). This ionization curve was constructed from a family of difference absorption spectra as described in the text. TABLE
This is of some importance as it indicates that subsequent optical measurements made on fresh solutions of the protein, represent the properties of essentially Prolactin Properties pure PP monomer. The szO,.,+. value for PP Porcine Ovine was found to be the same as previously reported for OP monomer (16). When this Molecular weight value is combined with the Stokes radius Sedimentation equilib,2,400 23,300 and V, the computed value for the molecrium ular weight of PP (23,000) is in excellent 22,550 ‘2,870 Amino acid sequence. 2.18 2.18 Sedimentation coefficient, agreement with that obtained by ultracens.0.w trifuge data, indicating good consistency Chromatography” on between the sedimentation and exclusion Sephadex G-100 chromatographic behavior of the hormone. 1.86 1.85 VeIV, equilibrium High-speed sedimentation 0.414 0.414 K.” (Fig. 1) at pH 10.35 indicates homogeneity Stokes radius, A 24.9 24.9 of the preparation with a weight average Isoelectric point, p1 5.85 5.73 molecular weight (22,400) which is in exa-Helix,” % 55 55 cellent agreement with the value obtained 336.5 Maximum fluorescence 337.9 from the primary structure (3). Low-speed emission,“, * nm sedimentation equilibrium at pH 8.2 Titratable tyrogl residues’ showed the presence of a slow aggregation 6 6 number reaction. The effect of this reaction be11.15 11.2-11.3 P% comes apparent only after 12-24 h of sediSolubility in (NH,),SO, mentation, but is sufficient to prevent the solutions, pH 6.60 establishment of a stable equilibrium con0.35f 0.369 B dition throughout a period of 72 h. Appar0.202 0.187 K’ ently, the sedimentation velocity experia 0.1 M Tris-HCl buffer, pH 8.2. ments at pH 8.2 were completed before b Excitation at 292 nm. this time-dependent aggregation could c Both PP and OP contain a total of seven tyrosyl produce a noticeable effect. Increasing residues. aggregation of purified OP monomer, occurring as the solvent pH approaches Sedimentation velocity experiments at either side of the isoelectric point, has been pH 8.2 again indicate PP is a homogeneous previously described (16, 42). These earlier studies on OP also show that aggregation is protein which exhibits no concentration dependence in sedimentation coefficient not appreciable above pH 9. Free-boundary electrophoresis (Fig. 2) between 2 and 8 mg/ml protein (Table 1). PHYSICOCHEMICAL.
PROPERTIES OF PORCINE PROLACTIN
indicates this preparation is also electrophoretically homogeneous. The fact that the isoelectric point for PP is slightly more basic than that of OP is in accord with the amino acid composition of the two hormones. Although both contain the same total number of carboxyl groups, PP has two more arginine and one more histidine residue than OP (3, 4). As shown in Fig. 3, the solubility behavior of both PP and OP in ammonium sulfate solutions obeys Cohn’s equation (18, 19). It is evident that OP is more soluble than the porcine hormone; however, the values of p for both hormones are nearly identical (Table III). This is to be expected (18), as the isoelectric points of PP and OP are very close to each other. The far ultraviolet CD (Fig. 4A) of PP was found to be very similar to that previously reported for OP (13). At pH 8.2, both proteins show hyperchromic and bathochromic shifts in the spectral region usually associated with the n + a* amide bond transition. These shifts disappear, presenting a more “normal” spectrum, when either protein is adjusted to pH 3.6-4.0. The structural origin of these characteristic shifts in OP has been discussed previously (13), but their exact nature is still uncertain. No such shifts have been reported in the spectra of various growth hormones (11, 13, 15, 31-33), even though two of these hormones (HGH and HCS) also display lactogenic activity (43-46). CD spectra of PP in the region of sidechain absorption (Fig. 4B) exhibit qualitative similarities but small quantitative differences to OP spectra. A weak negative band near 290 nm, whose presence could only be inferred previously in the OP spectrum (13), is clearly evident in the PP spectrum at pH 8.2. This same band is also seen in the spectrum of BGH (33), SGH (33), and HCS (15, 31), and has been assigned to a lo, indole transition in these molecules. While this band is more intense in PP than OP, the reverse is true of the asymmetric positive dichroism seen above 292 nm in both hormones. This dichroism has been assigned to overlapping IL, and IL, indole transitions, and is also seen in spectra of HGH (13, 32) but not in spectra
of BGH, SGH, or HCS. These results suggest there may be very slight differences between the immediate environments of one or both of the tryptophan residues in OP and PP. However, the fact that these two proteins exhibit nearly identical difference absorption and fluorescence spectra strongly argues that any differences in the tertiary structures around their tryptophan residues must be relatively small. Both the CD and difference absorption spectra (Fig. 5) in pH 4.0 buffer and 50% acetic acid indicate that at least one tryptophan residue is buried within the hydrophobic interior of these molecules in the pH range 4.0-8.2. This residue becomes exposed to the external solvent in 50% acetic acid. In OP it has been shown that the buried residue is tryptophan-90 (23). The second tryptophan, in OP at position 149, was found to be exposed under all conditions studied (23). A similar structural transition involving the homologous tryptophan-86 residues, buried in HGH and HCS, has also been reported (11). Although it may be presumed that PP will closely resemble OP in this regard, it remains to be proved by direct chemical modification studies. It might appear from the difference absorption spectra that 6 M guanidine hydrochloride causes less denaturation of the protein than 50% acetic acid. This is not the case, however, as is clearly shown by the CD spectra. The intensity of the denaturation blue-shift peaks are proportional to the net difference in refractive index between the hydrophobic interior of the protein and the external solvent (47, 48). The vDzo value for 50% acetic acid is only 1.3524 (49) while that for 6 M guanidine hydrochloride is 1.4278 (50). Therefore, even though the tryptophan residues are completely exposed in the 6 M guanidine hydrochloride solvent, they have been subjected to a smaller net change in refractive index, leading to less intense blue-shifts. There appears to be one nontitratable tyrosine residue in both PP and OP (24) with the six ionizing residues in each showing similar and abnormally high pK, values. However, while the titration of OP is reported (24) to be irreversible from pH >
COMPARATIVE PROPERTIESOF LACTOCENIC AND GROWTH HORMONES A. Some properties which are shared by lactogenic and growth hormones 1. Approximately 50% homology in primary structures, especially near the COOH-terminal half. 2. Monomer molecular weights near 22,000. 3. High content of a-helix (4555%). 4. Unusual st.ability of a-helix to extremes of pH. 5. A tryptophan residue occurring slightly NH,-terminal to the center of the primary structure which is not exposed to the external solvent in the native conformation. B. Some properties which distinguish lactogenic from growth hormone 1. Two of the three disulfide bonds in lactogenic hormones are homologous with similar bonds in the growth hormones. The third disulfide in the lactogenic hormones forms a small loop near the NH,-terminus and has no counterpart in the growth hormones. 2. The lactogenic hormones contain two tryptophan residues rather than the single tryptophan found in the growth hormones. One of these, occurring in the COOH-terminal half of the lactogenic sequence, is nonhomologous with any residue in the growth hormones. This residue is exposed to the external solvent in native lactogenic hormones. 3. The far ultraviolet CD of lactogenic hormones at pH 8.2 exhibits both hyperchromic and bathochromic shifts in the band usually assigned to the n + ** transition, appearing in these proteins at 222-223 nm and in growth hormones at 220-221 nm. In lactogenic hormones, these unusual spectral features are “normalized” by acidification below pH 4.0 without causing significant changes in the estimated a-helix content.
11.8, the titration of OP is completely reversible from pH 13 (Fig. 6). In a previous publication (51) we have proposed a scheme for the evolution of growth and lactogenic hormones. This scheme was based entirely on an analysis of sequence homologies. Two distinct and separate lines of evolution are seen to diverge from a common ancestral polypeptide. These have been designated as the lactogenic and the growth hormones lines. The names given to these two evolutionary lines are actually somewhat arbitrary, but they do reflect what has been historically recognized as the predominant activity of most of the members of each line. It is important to note that in the future, the placement of a protein within either line should be based on sequence similarities rather than on the type of biological activity displayed. With the addition of the primary structure and some of the chemical and physical properties of PP to the lactogenic hormones, we can designate certain properties as characteristic of each line. Other properties seem to be shared by both lines, but are not generally shared with most other proteins. Some of the properties which can be placed into these two categories are
summarized in Table V. Undoubtedly, as more information is obtained from studies of additional lactogenic and growth hormones, modifications of these tables will be required. Human pituitary prolactin will be of special interest in this regard. ACKNOWLEDGMENTS The authors thank Dr. P. Tarli for the solubility experiments. We also thank P. Geffen and C. M. Behrens for their excellent technical assistance. This work was supported in part by U. S. Public Health Service Grant AM-6097. REFERENCES 1. EPPSTEIN, S. H. (1964) Nature (London) 202, 899-900. 2. EPPSTEIN, S. H. (1965) Encerpta Med. Found. Znt. congr. Ser. 112, 340-349. 3. LI, C. H. (1973) J. Znt. Res. Commun. 1,19. 4. LI, C. H., DIXON, J. S., Lo, T. B., SCHMIDT, K. D., AND PANKOV, Y. A. (1970) Arch. Biochem. Biophys. 141, 705-737. 5. BEWLEY, T. A., AND LI, C. H. (1970) Science 168, 1361-1362. 6. BEWLEY, T. A., DIXON, J. S., AND LI, C. H. (1972) Znt. J. Peptide Protein Res. 4, 281-287. 7. LI, C. H., SIMPSON, M. E., AND EVANS, H. M. (1942) J. Biol. Chem. 146.627-631. 8. BEAVEN, G. H., AND HOLIDAY, E. R. (1952) Aduan. Protein Chem. 7, 319-386. 9. YPHANTIS, D. A. (1964) Biochemistry 3, 297-317.
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