Optical rotatory dispersion studies of the neutral soluble proteins of embryonic bovine enamel

296

J. ULTRASrRUCTU~ERZSEARCH13, 296--307 (1965

Optical Rotatory Dispersion Studies of the Neutral Soluble Proteins of Embryonic Bovine Enamel 1 LAURENCE C. BONAR, GERALD L. MECHANIC, AND MELVIN J. GLIMCHER

Department of Orthopedic Surgery, the Harvard Medical School, and the Massachusetts General Hospital, Boston, Massachusetts2 Received March 8, 1965 The optical rotatory dispersion characteristics of the neutral-soluble proteins of embryonic bovine enamel, and of a high molecular weight aggregate isolated from the neutral-soluble proteins by Sephadex G-100 filtration have been studied. Both solutions have a high negative optical rotation of [e]~46 = - 140 in 0.1 M NaHCOa, which is increased about 15 % by denaturation with urea. The rotatory behavior is shown to be consistent with the presence of about 8 % c~helix and 6 %/%configuration in 0.1 M NaHCO3 solution, and indicates that the high content of praline in the soluble enamel proteins is distributed randomly in the polypeptide chain, and not segregated in polyproline or praline-pralinepraline segments. X-ray diffraction studies have d e m o n s t r a t e d that a m a j o r constituent of both the organic matrix a n d the neutral soluble proteins of e m b r y o n i c bovine e n a m e l is in the cross-/3 configuration (1, 4). The present report describes studies of the configuration of the n e u t r a l soluble proteins in solution by m e a n s of optical r o t a t o r y dispersion measurements. EXPERIMENTAL

Preparation of proteins. The cold, neutral-soluble enamel proteins (NSEP) from the premolar teeth of bovine embryos ranging in age from 4 months to 6 months were obtained by methods previously described (4, 5). Only the first neutral-soluble extract (3 days) was used in these experiments. The material was freed of salt by dialysis against water, lyophilized, and dried in vacua at 2°C. Dry lyophilized protein was dissolved as a 1% solution in 0.12 M NaHCO3, pH 8.3, and chromatographed at 2°C on a 160 cm × 3 cm column of Sephadex G-100 resin previously equilibrated in 0.12 M NaHCOa. The flow rate was maintained at 41 ml/hour. The first third of the first protein peak emerging was collected and rechromatographed, and i This investigation was supported in part by grants from The National Institutes of Health (AM-06375) and The John A. Hartford Foundation, Inc. 2 Communications should be addressed to the Orthopedic Research Laboratories, Massachusetts General Hospital, Fruit Street, Boston, Massachusetts 02114.

OPTICAL ROTATION OF ENAMEL PROTEINS

297

the first third of this rechromatographed peak was used in these studies. Ultracentrifugation of this fraction at pH 8.3 revealed that it consisted in most instances of a single, high molecular weight aggregate (HMWA), although in some cases it contained also a very small amount of lower molecular weight material? Chemical analyses. The amino acid composition of the proteins was determined on aliquots hydrolyzed in triply distilled, constant boiling 6 N HC1 at 105°C for 24 hours, utilizing a commercial model 2 of the automatic amino acid analyzer described by Piez and Morris (12). The nitrogen content of protein solutions and of known weights of dry salt-free proteins were determined by Kjeldahl analysis. Optical rotation. The optical rotatory dispersion measurements of the protein solutions were made with a Rudolph photoelectric polarimeter equipped with a quartz prism monochromator. General Electric A H 6 and Hanovia SH mercury arc lamps, and an Osram XBO 450 xenon arc lamp were used as light sources. Measurements were made from the 579 m# mercury llne down to the 365 m# line in all cases, and down to the 302 m# line where the transparency of the solution permitted. Monochromator slit widths were kept below 0.1 mm at wavelengths above 365 m~ and below 0.2 mm at 365 m# and below. Two decimeter pathlength polarimeter tubes, 3 mm in inside diameter, with fused silica windows, equipped with a jacket for temperature control were used, except where high absorption at lower wavelengths required the use of similar tubes of 1.0 or 0.5 dm pathlengths. The polarimeter tubes were maintained between 4°C and 6°C, except for the measurements made at acid pH, where temperatures of 2-3°C were necessary to prevent precipitation of the protein. The optical rotation of relatively concentrated protein solutions (approximately 1%) and aliquots diluted tenfold were measured with a Bendix Type 143 A automatic polarimeter, modified by the addition of a thermostatted cell compartment long enough to accommodate standard 1 dm polarimeter tubes. A tungsten filament lamp and 546 m# interference filter with a band-pass half-width of 30 m/~ was used as a light source. The concentration solutions were measured in 0.5 dm tubes, the diluted solutions in 1 dm tubes. Preliminary experiments had demonstrated that this instrument was capable of a precision of +0.0015°C at 576 m/~ when used under the conditions employed here. Measurements were made at 6°C. Procedures. Protein solutions for polarimetric studies were prepared by dissolving the salt-free, lyophilized proteins in 0.1 M NaHCO3, pH 8.15, at 2°C. The solutions were clarified by ultracentrifugation at 20,000 rpm for 1 hour or by filtration through fine sintered-glass filters. Aliquots were removed for concentration determinations after clarification. The protein concentration was in some instances determined by dissolving a known weight of lyophilized protein and weighing the undissolved material after ultracentrifugation (which constituted only a few per cent of the total weight). Corrections also were made for the moisture content of the protein. In some cases, the first third of the peak recovered from the Sephadex G-100 resin column was used directly without prior dialysis or lyophilization. In such instances the solutions were diluted to reduce the concentration of NaHCO3 from 0.12 M t o 0.10 M. To investigate the effect of concentration on the specific rotation, 1% protein solutions at pH 8.15 were prepared in the following solutions: (a) 0.1 M NaHCO~; (b) 0.02 M Tris; or (c) 0.02 Tris and 0.08 M KC1. Similar solutions containing 4 M urea were also prepared. An aliquot of each of the six solutions was then diluted tenfold. 1 Mechanic, G. L., Katz, E. P., and Glimcher, M. J., in preparation. 2 Phoenix Precision Instrument Company, Philadelphia, Pennsylvania. 20-- 651825 J. Ultrastructure Research

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L.C. BONAR, G. L. MECHANICAND M. J. GLIMCHER

The concentration of protein in urea was determined by pipetting a solution of known protein concentration into a preweighed portion of urea (reagent grade, recrystallized from a 1 : 1 (v/v) ethanol-water solution and stored dry in brown bottles). From the known density of the urea solution (6) and the weights of protein solution and of urea, the volume, and hence the dilution, resulting from the addition of urea can be determined. At 4°C, addition of solid urea to a concentration of 4 M results in a dilution to 0.811 of the initial protein concentration. The refractive index of 4 M urea was determined by use of the Sellmeier equation (18) to interpolate between published values of n for 8 M urea at 265 m/~ (17) and 589 m~ (15), assuming n to vary linearly with the concentration of urea. Solutions of H M W A in dilute acid were prepared by titrating the protein in 0.1 M NaHCO a to pH 4.68 with 0.3 N HC1. RESULTS The optical r o t a t o r y dispersion curves of the neutral-soluble enamel proteins (NSEP) and of the high molecular weight aggregate ( H M W A ) isolated f r o m the N S E P by Sephadex G-100 filtration, are both linear when plotted either as (l/e) vs. 22 or )2 ~ vs. 2 ~ and thus follow a single-term D r u d e equation. The values of [~]54G and the r o t a t o r y parameters calculated by the weighted least squares method of Schellman (I4) are listed in Table I. There is no significant difference between the parameters for N S E P and H M W A . The addition of urea to a final concentration of 4 M results in an increase in the levorotation of both preparations of approximately 16 % (Table I). There is no further increase in the specific rotations when the urea concentration is raised to 8 M. The values of [~]~46 in urea (Table I) have been corrected for the refractive index difference between water and 4 M urea (15). There is no detectable change in the specific optical rotation of the N S E P with protein concentration (1.0-0.1%) at p H 8.15, either in 0.1 M N a H C O a , in 0.02 M Tris buffer and 0.08 M KC1, or in 0.02 M Tris buffer alone. Similarly, no difference in the specific optical rotation is noted with dilution of the protein solutions in 4 M urea. The sensitivity of the instrument is such that a change of more than 3 % would be readily detectable. Acidification of the neutral or slightly alkaline protein solutions at temperatures above 5°C resulted in the precipitation of some of the protein, and even at temperatures below 5°C, scattering of the polarized light by colloidal particles precluded optical rotation measurements at wavelengths below 406 m/~. The values of [c~]546 and the dispersion parameters of the H M W A at p H 4.68, determined over this limited wavelength range, are given in Table I. These data demonstrate that there is no change in the optical r o t a t o r y properties of the protein with acidification. The amino acid compositions of b o t h the N S E P and the H M W A are substantially the same, b o t h being characterized by their high contents of proline, glutamic acid, histidine, leucine, and isoleucine (Table II).

OPTICAL ROTATION OF ENAMEL PROTEINS

299

TABLE I O P T I C A L R O T A T O R Y DISPERSION OF ENAMEL PROTEINS a Solvent t 0. I M NaHCOs p H 8.15

0.1 M N a H C O ~ + 4 M Urea pH 8.15

0.1 M NaCI p H 4,68

Sample

2C

k

[~]5~6t~

2C

k

[cq~a6b

[c~]s4.c

2C

k

[~]~4~b

NSEP HMWA

215 214

36.3 34.6

- 144 - 138

212 213

41.9 " 40.5

- 166 - 160

- 162 - 156

-212

-35.4

-- 140

a Measurements at pH 8.15 made at 6°C, those at pH 4.68 at 2°C. b Calculated from the dispersion constants. c Corrected for the refractive index differences between water and 4 M urea. TABLE II THE A M I N O A C I D COMPOSITION OF THE N E U T R A L SOLUBLE PROTEINS OF EMBRYONIC BOVINE ENAMEL ( N S E P ) AND THE H I G H M O L E C U L A R W E I G H T A G G R E G A T E ( H M W A ) SEPHADEX G E L F I L T R A T I O N Amino Acid

NSEP*

Cystine (half) a 3-Hydroxyproline 4-Hydroxyproline Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine

1.6 Trace Trace 32 29 48 159 258 51 23

HMWA

2.0 -Trace 33 27 38 170 252 50 23

Amino Acid

Valine Methionine b Isoleucine Leucine Tyrosine Phenylalanine Hydroxylysine Lysine Histidine Arginine

NSEW

38 48 35 100 42 23 4 14 79 15

ISOLATED BY

HMWA

46 51 40 96 42 22 1 13 80 15

Recovered as cysteic acid. b Recovered as methionine sulfoxide. e Data from Glimcher et aL (5). DISCUSSION B e c a u s e of t h e p h y s i c a l h e t e r o g e n e i t y of t h e n e u t r a l - s o l u b l e p r o t e i n s , a n d t h e f a c t t h a t t h e h i g h m o l e c u l a r w e i g h t species i s o l a t e d b y S e p h a d e x

G - 1 0 0 f i l t r a t i o n is a

noncovalently bonded aggregate,1 conclusions concerning the structure of these proteins i n s o l u t i o n b a s e d o n r e s u l t s of o p t i c a l r o t a t i o n s t u d i e s m u s t b e m a d e w i t h c a u tion.

However,

despite their marked

have demonstrated isolated

by

physical heterogeneity, unpublished

that the chemical compositions

Sephadex

gel-filtration and

studies

of t h e v a s t m a j o r i t y of s p e c i e s

by ion exchange

Mechanic, G. L., Katz, E. P,, Glimcher, M. J., in preparation.

chromatography

have

300

L . C . BONAR, G. L. MECHANIC AND M. J. GLIMCHER

many striking similar characteristics, principally their relatively high content of proline (250 or more residues per 1000 total amino acid residues). With the exception of collagen and the synthetic polypyrrolidine polypeptides (8), the negative optical rotation of the enamel proteins in nondenaturing solvents is higher than most undenatured proteins (18). Unlike collagen or polypyrrolidine peptides, but similar to proteins having s-helical or/3 configurations, denaturation in urea is accompanied by an increase in the levorotation (18). The latter results clearly indicate that the enamel proteins do not exist in a completely random configuration in solution, but possess some ordered structure. Furthermore, this configuration does not appear to depend on any tertiary structure produced in the proteins by aggregation, since physical chemical studies1 have shown that dissociation of the higher molecular weight aggregates occurs on dilution of the protein concentration and on acidification. Two different methods of analyzing optical rotatory dispersion data for evidence of regular structure have been used (18). The method most commonly employed is based on the empirical use of the Moffitt equation (11). The optical rotatory dispersion of a solution of polypeptide or protein molecules in s-helical, /3, or random configurations can be described by equations 1 a or l b (•8): [@

or

n~+2 100 [a~2oz +f~( a~2~ , b~2~ \ ~ [ a~o2~ b~o2t ]] 3 M R W [22- 4o2 k~.2- 2° + (2~-- 2o~) +J¢ k22 - ~ o + (25 ~ 2 1 1 [~]_

~ + 2 100 [(ao+fza~÷f~ao)2o (fr~b~+f~b~°)4~] 3 MRW L 2~-2o~ ~- (43-4~) 2 "

(la) (lb)

In this expression, n is the refractive index; M R W is the mean residue weight of the polypeptide; a~, a~, and a~o are constants descriptive of the 42 dependence of the rotatory dispersion of the polypeptide in its random, helical and/3 forms, respectively; b~ and b~o are similar constants describing the 44 dependence, and f~ and f, are the fractions of the polypeptide in the s-helical and/3 configuration, respectively. These equations apply to polypeptides which, in the random form, are described by a single-term Drude equation (4c~4o), so that no b~ constant is required. 40 is assumed to be about 212 m# for most systems. The values of a~ and a~ have been observed to vary considerably with the amino acid composition and with the nature of the solvent, while b~ is almost independent of these influences (18). In practice, b~6s which is the sum of fEb~ +f,b0~, is determined from the dispersion measurement. The values of b~ and bPoare determined for the given polypeptide when converted as fully as possible to the helix or fi-form, or, where this is not possible, values determined for other polypeptides which do exist in the appropriate forms are 1 Katz, E. P., Mechanic, G. L., Glimcher, M. J., in preparation.

O P T I C A L R O T A T I O N OF E N A M E L P R O T E I N S

301

used. Unless the c~-helix or/3 configuration is known to be absent or the amount of either configuration present can be determined independently, however, no unique determination of frr and ffl is possible. Because of the sensitivity of a~o, a~, and a~ to changes in solvent composition, the value of agb~, the sum of ~ +fnaoH+f~Jo, is seldom used for structural considerations. Equations 1 a and l b can be generalized to include the rotatory contributions of configurations differing from random, ~helix, and/3 configurations, providing the dispersion of such contributions can be described by the sum of the 22 and 2 ~ terms with the appropriate value of 2o. In principle a0~, a~, and a0~ should be determined for a given polyeptide under identical conditions, but this is usually not possible. In practice a~ois usually estimated from the rotatory dispersion of the material in the denatured state, where f~ =f~ =0, although it is clear that the use of different solvents to effect denaturation may result in a somewhat erroneous value of this parameter. Values of a0g of + 600 for Pinna nobilis tropomyosin, a protein with a very high ~helical content, a n d + 6 5 0 a n d + 9 5 0 for poly-L-glutamic acid and poly-L-lysine, respectively, in an aqueous environment, have been reported (18). b~ values for these structures were in the - 6 0 0 to - 6 5 0 range. Because it has been difficult to obtain soluble polypeptides or proteins in the/3 configuration free of contamination with components in other configurations, corresponding parameters for the/3 configuration have not been investigated as thoroughly as those of the c~-helix. Values of the a0~ of +840, +234, and +600, and values of b~0 of +420, +250, and +190 have been reported for low molecular weight poly-y-benzyl-L-glutamate (19), bovine serum albumin (9), and poly-benzyl-L-serine (2), respectively. For both H M W A and NSEP, a~b~ and b~~ were calculated from a plot of 3

n2 + 2

M R W 22-22[~] 20~ 100 2~ vs. ) 2 _ 202

as described by Urnes and Doty (18), using a value of 212 m# for 20. For HMWA, values for a3b~ varied from - 6 0 0 to - 6 9 0 in 0.1 M NaHCO3, and from - 7 1 0 to - 780 in 4 M urea in a number of different preparations. Values for bSb~ ranging from - 9 to +12 in 0.1 M N a H C Q , and from - 1 to +31 in 4 M u r e a were found. Addition of urea resulted in an increase ranging from +5 to +29. The values of these parameters for NSEP fell within the above ranges. The turbidity of H M W A solutions was observed to increase on standing. Solutions prepared by dissolving lyophilized salt-free H M W A in 0.1 M NaHCOa were also turbid. Because of the known tendency of H M W A to aggregate, it was thought desirable to measure the optical rotatory dispersion of H M W A immediately after fractionation. Accordingly measurements were begun on one sample within 2 hours after elution from a Sephadex G-100 column, and completed within 5 hours after

302

L.C.

BONAR,

G. L. MECHANIC

A N D M. J. G L I M C H E R

-I00

-200

.
----0.1M

NaHCO 3

--0.1M

NaHCO 3 +

4M

Urea

-500

-400

-500

~+

C4

-600

c -700

-800

-900

-

tt

~------~

i

!

z

i.

:[

I

I

I

I

I

I

I

I

I

0.1

0.2

0.5

0.4

0.5

0.6

0.7

0.8

0.9

1.O

~2_~o ~

FIG. 1. Moffitt plot for the high molecular weight aggregate of the neutral-soluble embryonic enamel proteins, measured within several hours after elution from a column of Sephadex G-100 resin. The results in 0.1 M Nat-ICOs and in 4 M urea are shown. A volume of 208.2 m# for Zo was used in the calculations. elution. U n d e r these conditions, it was possible to determine the optical rotation d o w n to 302.5 m # without any clarification of the solution. There was no indication of change in the optical rotation values during the course of the measurements. Because of the increased wavelength range over which measurements were made, and the greater precision of the measurements due to the reduced a m o u n t of scattering depolarization, the measurements obtained under these conditions are probably somewhat more precise than measurements on slightly more turbid solutions. The values of the rotatory dispersion parameters obtained under these conditions are indicative of the H M W A in its freshly prepared condition. Using 20=212.0 m/~, a~bs and b3b~ were f o u n d to be - 6 9 0 and + 2 in 0.1 M N a H C Q , and - 7 8 5 and +27 in 4 M urea. The disruption of an a-helix would result in a positive change in b~bs, while disruption of a/3 structure would cause a negative change in b8bs. Either change would lead to a negative change in a~bS. The observed decrease in a °b~0, accompanied by a positive change in b~b~ u p o n addition of urea suggests that a portion of the proteins

OPTICAL ROTATION OF ENAMEL PROTEINS

303

exists in 0.1 M NaHCO3 in the ~-helix form, and that this helical portion is disrupted by the addition of urea. The positive value of b~bS in 4 M urea suggests the presence in this solvent of a portion of the protein in the/3 configuration. However, the presence of any regular configuration in 4 M urea seems unlikely, particularly since increasing the urea concentration to 8 M produces no further change in optical rotation. The values obtained for b8bS are strongly dependent upon the choice of 20. In the present investigation, as in most optical rotatory dispersion studies, the dispersion data are not sufficiently precise to permit an independent assignment of 20; values of 20 ranging from 200 to 225 m# were found to yield equally good straight lines when plotted as described above. The value of 212 m# is usually chosen for 2 o because of its applicability in studies of helical synthetic polypeptides, and because the values of b~b~ calculated using this value can be compared with the results of other studies (18). If it is assumed that the neutral-soluble enamel proteins are completely denatured in 4 M urea, i.e., that all structural regularity capable of contributing to the optical activity has been destroyed, a value of 20 can be cbosen so that b ~ b s ~ 0 in urea. For freshly prepared HMWA, 2o=208.2 m# yields values of a8b~ and b~b~ of - 7 2 3 and - 2 5 , respectively, in 0.1 M N a H C Q , and - 7 0 0 and 0 in 4 M urea. These values are consistent with the presence in 0.1 M N a H C Q solution of about 5 % of the protein in the ~-helix configuration, which is destroyed on addition of urea. Figure 1 shows the plot of 3 M R W 2 2 - 2~ 2~ n 2 + 2 100 2~ [cq vs. 23_2~ for freshly prepared H M W A with 2 o =208.2 m#. Another method commonly employed to assess the configurational implications of optical rotation measurements is based on the change in [~] at a given wavelength which would result from the destruction of an c~-helix or/3 configuration (18). This method avoids dependence on the small and variable value of b~b~, but ignores the difference in dispersion of the a-helix compared to the/3 configuration, and, since it includes changes in the a o terms in equation 1 as well as the b o terms, probably reflects configuration-independent changes due to alteration of the solvent in addition to those due to configuration changes. From the data of Schellman and Schellman (16) and Urnes and Doty (18), the change in [~]5~6 resulting from the destruction of an a-helix can be estimated as - 110. The corresponding value of A [~]54Gfor the disordering of a/3 configuration can be calculated using equation 1 to be - 180, using a~b~= -775, the value found for freshly prepared H M W A in 4 M urea, and the values of Jo and b~o reported by Wada et al. (19). The change in [0~]5~G observed on denaturation of the soluble dental enamel proteins is about - 2 0 , corrected for the

304

L . C . BONAR, G. L. MECHANIC AND M. J. GLIMCHER

change in refractive index of the solvent (15). This observed change would be produced by the denaturation of about 20 % of a-helix configuration, 11% of/3 configuration, or some combination of the two. The relatively large amount of configurational change indicated by the magnitude of the change in [a]54G,compared with the 5 % loss of a-helix predicted by the change in b~bs, suggests that, in fact, the b~bS contribution of the a-helix is partially compensated by a smaller amount of fl configuration. Thus, taking b~ = - 6 0 0 and b0¢ = + 420, a solution containing about 8 % 0~-helix and 6 %/3 configuration would yield b~b~= - 2 5 , and A[c~]5~a on denaturation of - 2 0 , in agreement with the values found for freshly prepared HMWA. The observed differences in the rotatory parameter of different preparations might be the result of slightly different proportions of ~helical and/3 configurations. Because of its unusual amino acid composition, and in particular its high imino acid content, it is conceivable that the neutral-soluble enamel proteins exist in 0.1 M NaHCO3 solution in a configuration distinct from:both the a-helix and/3 configuration. Significant amounts of such a structure could exist if the values of a 0 and b0 for the structure were such that a relatively small change in a~bS, b~b~, and [e]~46 occur on denaturation. The high proline content of NSEP and HMWA, which is apparently randomly distributed in the protein (see below) indicates that an a-helix could exist only in a low-proline component or region of the total protein. Studies of the amino acid composition of various components which can be separated from NSEP reveal that all but very minor constituents have a characteristically high content of proline. R is possible however, that small proline-free segments of polypeptide chains exist, which can form an a-helix under appropriate conditions. X-ray diffraction patterns of decalcified embryonic bovine enamel matrices, such as those used as the source of the material studied here, show no evidence of an o~-helical pattern (4) nor do patterns obtained from fibers prepared from NSEP (1). However, 10% or less of c~-helical configuration might be undetected by X-ray diffraction methods. While cross-/~ X-ray diffraction patterns are obtained from the protein precipitated from solutions of NSEP (1), the amount of/3 material necessary to give the observed diffraction patterns--probably of the order of 50 % of the total--is much higher than the 11% or less indicated by the optical activity studies. Thus extensive molecular rearrangement of the protein into a/3 form probably occurs on preparation of the solid specimen for X-ray diffraction studies, and the observation that the dried, reconstituted protein is in a cross-/3 configuration does not necessarily imply that it exists in this form in solution. The contribution which a regular, asymmetric structure in a polypeptide chain makes to the optical activity of the polypeptide may be estimated by calculating [e]oon~,

OPTICAL ROTATION OF ENAMEL PROTEINS

305

as described by Josse and Harrington (10) and Harrington and von Hippel (8). This parameter is the amount by which the observed rotation exceeds the rotation which the constituent amino acids would have in a completely random polypeptide chain; it incorporates a~o, a~, bg, boa, and any similar configuration-dependent terms in equation 1. It is assumed that glycine makes no rotational contribution and that the specific rotatory contribution of the remaining residues, with the exception of the pyrrolidines, is typical of proteins in denaturing solvents: about - 1 3 0 at 546 m/~. The intrinsic residue rotation of proline is about - 2 5 0 at 589 m# (3, 7, 20) and that of hydroxyproline, - 2 0 0 at 589 m# (8); these values correspond to - 3 0 0 for proline and - 2 4 0 for hydroxyproline at 546 m/~. Using these values, Lr~l°°n~gJ54~for gelatins derived from mammalian collagens can be calculated-as - 4 0 (8, 10). In gelatin, a substantial portion of the pyrrolidine content is in the pro-hypro sequence. Since adjacent pyrrolidine residues are sterically prevented from attaining a random configuration with respect to one another, the presence of this pro-hypro sequence probably accounts for the large [~]°°~g for gelatin. From the results of Yaron and Berger (20), r~loo,~t j546 for proline polymers with an average degree of polymerization of 2 can be calculated to be approximately - 100. r~lconfig For NSEP and H M W A in urea, by contrast, L ~546 was calculated to be 0 to + 10. If it is assumed that the protein is completely denatured in 4 M urea, so that there are no compensating positive contributions to [~]oo~n~, the lack of a negative value for this parameter indicates that no significant amount of the proline present is segregated into poly-proline or even proline-proline-proline segments. That is, the proline is randomly distributed in the polypeptide chains. The alternate explanation, that the proline is segregated but with approximately equal numbers of proline residues in the polyproline I and polyproline II configurations, which would result in a net configurational contribution close to zero, is considered unlikely, particularly since the polyproline I configuration appears to be unstable in an aqueous environment (8). The results of the optical rotatory dispersion investigation of NSEP and H M W A clearly indicates the existence of small amounts of a regular configuration in these proteins which is destroyed by the addition of urea. The values of the significant rotatory parameters are consistent with the presence of about 8.4 % s-helix and 6 % /3 configuration in freshly prepared HMWA. The results also indicate that the large amount of proline present is randomly distributed in the polypeptide chain. There appears to be no significant difference in the rotatory properties of NSEP and HMWA. The values of a0, b 0, and [~]5~ for NSEP are in the range recorded for H M W A in both 0.1 M NaHCO3 and in 4 M urea. This suggests that components of NSEP which are eluted from Sephadex G-100 after HMWA, and which presumably are lower molecular weight aggregates, have substantially the same configuration

306

L. C. BONAR, G. L. MECHANICAND M. J. GLIMCHER

as the H M W A . With the exception of a few minor constituents, the amino acid compositions of all components show m a n y similar features. It would therefore appear that most of the components present in the physically heterogeneous NSEP, although aggregates of varying molecular weights, are similar in composition and configuration. In the solid state, the embryonic enamel proteins have been shown to be in the cross-/3 configuration (1, 4). The optical rotation results presented here indicate that the large amount of proline in the protein is not segregated from the remainder of the residues, and thus is presumably in a configuration giving the cross-/3 diffraction pattern. It has usually been assumed that the Pauling-Corey pleated sheet configurations, which are thought to account for the/3 class diffraction patterns, cannot accommodate significant amounts of proline. However, Ramachandran (13)has described a modification of the Pauling-Corey pleated sheet which can accommodate proline residues without distortion. The structure has alternate residues rotated approximately 180 degrees about the chain axis from the Pauling-Corey structure, to produce a pleated sheet with all the C = O ' s extending on one side of the axis, and all of the N - H ' s extending in the opposite direction. Preliminary structure-building experiments conducted in this laboratory with molecular models have indicated several other possible structures which can accommodate large amounts of proline and which are consistent with the observed cross-/3 diffraction pattern. These latter structures have not yet been critically examined for concurrence with accepted values of interatomic distances and bond angles, and will be described in a later publication. It is clear from the amino acid composition and the results reported here, however, that any proposed structure must be capable of accommodating at least one residue in four of proline. The authors wish to acknowledge the technical assistance of Miss Kathy Rutstein and Miss Diane Brickley. REFERENCES

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