THE JOURNAL OF BIOLOCICA~ CHEMISTRY Vol. 235, No. 5, May 1060

Printed in U.S.A.

The Properties of Thyroglobulin

I. THE EFFECTS OF ALKALI

H. EDELHOCH

From the Clinical Endocrinology Branch, National Institute of Arthritis and Metabolic Diseases, National Institutes o;f Health, Bethesda, Maryland

(Received for publication, September 28, 1959)

Several studies have appeared in the past 25 years (l-5) con- cerning the physicochemical properties of thyroglobulin, the pro- tein precursor of the thyroid hormone thyroxine. Most of this work has utilized hog thyroid gland as a source of material. The various investigations are in rather good agreement in ob- taining a molecular weight for thyroglobulin close to -$ million. Moreover, this form of the protein appears to be stable within the pH range -5.0 to 11.3 when neutral salt is present (1).

Lundgren and Williams (2) in 1939 and O’Donnell et al. (5) recently have reported the appearance of a derivative of hog thyroglobulin, which sedimented with a substantially reduced sedimentation coefficient, when the free electrolyte was removed by extensive dialysis. Similar results have been obtained in the present study with calf thyroglobulin at low ionic strengths. However, the interpretation of data obtained on charged macro- molecules, when investigated with insufficient amounts of salt to suppress electrostatic interactions, is usually not free of am- biguities. With thyroglobulin extracted from calf thyroid tis- sue, a new component, which possessed quite similar sedimenta- tion properties to that reported by Lundgren and Williams, was observed to form at higher ionic strengths when the pH was raised above the neutral range. More extensive data, which can be interpreted with greater confidence, have been collected on this form of thyroglobulin. Further conformational changes’ of calf thyroglobulin at still higher pH values are also reported.

EXPERIMENTAL

Materials and Methods

Thyroglobulin was prepared from calf thyroid tissue follow- ing the procedure of Derrien et al. (7) with some minor modi- fications. Normally, about 100 g of tissue (fresh or frozen) was put through an electric meat grinder twice and then ex- tracted with 250 ml of 0.9y0 NaCl for 2 or 3 hours at 5”, with magnetic stirring. Tissue debris was removed by filtration through cotton gauze. The solution was then further clarified by centrifugation for 4 hour at 20,000 X g. Thyroglobulin was precipitated from the supernatant solution by addition of a saturated (3.5 M) potassium phosphate buffer, pH 6.6, to bring the solution to 48 7O saturation (7). The precipitate was separated by centrifugation for 30 minutes at 65,000 x g in

1 We will use the definit,ion suggested by Wolf and Briggs (6) of a conformational change: “. . as any stepwise and reversible change in shape, size, or degree of association that the molecular units of which a protein consists may undergo as a result of change in physical environment.”

the Spinco model L ultracentrifuge. The thyroglobulin was brought into solution again by dissolving the precipitate in a phosphate buffer at 41% saturation. This procedure was re- peated two additional times. The buffer was then removed by dialysis against several changes of distilled water for a period of several days at 5”. The solution was kept frozen until used. This method of purification will be called “phosphate-frac- tionated” or Preparation I.

When examined in the analytical ultracentrifuge, thyroglobu- lin preparations isolated by the above procedure showed small amounts of both faster and slower moving boundaries as shown in Fig. 1A. The concentration of the main component (so,,+, = 19.4) was generally about 85 to 90% of the total protein.

The relative amounts of faster and slower moving boundaries varied somewhat from one preparation to another. O’Donnell et al. (5) also report that their preparations were contaminated with similar types of impurities (cf. Shulman et al. (4)). Since the analytical methods used in earlier reports (l-3) on thyro- globulin did not have the resolving power of recently devel- oped procedures, it is likely that these preparations were simi- larly heterogeneous. Moreover, unless one examines protein concentrations greater than -1 %, small boundaries, represent- ing a few per cent of the total protein, are easily missed. In determining the concentration of components we normally used two solutions, at -1.5 and 0.5%, and sedimented them simul- taneously with the use of a standard and wedge-window cell. The areas of the small peaks were determined from the concen- trated solution, whereas the main peak was analyzed while in the dilute solution.

Since the light scattering molecular weight of this prepara- tion was found to be excessively high compared to the sedi- mentation-diffusion value, it was felt that the impurities could be responsible for the disparity. Moreover, it was observed that the S-25 component was no longer observable when the pH was raised to 9.5 from 6.0. This effect hampered the in- terpretation of the data regarding the relationship between the S-19 and S-12 components. To eliminate these difficulties, the phosphate-fractionated thyroglobulin preparations were purified further. This was effected by a simple differential ultracen- trifugation procedure.

By centrifuging the phosphate-fractionated thyroglobulin preparation (in 0.1 M KNOS) in the No. 40 rotor for 260 min- utes at 40,000 r.p.m. at room temperatures in the model L Spinco ultracentrifuge, most of the thyroglobulin and heavier components were sedimented to the bottom 20 to 30% of the centrifuge tube. By pipetting off the top -70y0 the slower

1326

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FIG. 1. The ultracentrifugal characterization of three different tration = 1.41’$7,‘,. C. Preparation III-R: centrifugally purified thyroglobulin preparations from calf thyroid tissue. A. Prepara- calf thyroid extract. Protein concentration = 1.57%. All solu- tion I-5: phosphate fractionation after method of Derrien. tions were close to pH 6.0 and in 0.10 M KN03. Preparations II Protein concentration = 1.50 and 0.50y0. B. Preparation II-S: and III were performed in the double sector cell and show solvent phosphate fractionated-centrifugally purified. Protein concen- base lines.

sedimenting impurities ( <S-19) were preferentially eliminated. The bottom -30% was then decanted (leaving behind the hard packed pellet) and brought back to the original volume with 0.1 M KNO3. A second centrifugation was then performed for 160 minutes under similar conditions.

The centrifuged solution contained three fairly distinct lay- ers in addition to a small pellet. The bottom one-fifth of the centrifuge tube contained a rather concentrated solution of pro- tein and was easily detected by its color or refraction. The next layer represented thyroglobulin (S-19) essentially free of faster sedimenting protein. The upper boundary of this middle layer was easily detected by observing the light scattered when the celluloid centrifuge tube was placed in a narrow beam of light. Occasionally this boundary was demarcated by strong opalescence. This boundary occurred about two-fifths of the distance from the bottom of the centrifuge tube.

The top layer (-60 %) was discarded again. The next layer (S-19 enriched), about 20% of the total volume, was isolated by careful pipetting. The bottom layer could then be decanted, brought to volume again, and more thyroglobulin obtained by repeating the above separation procedure. Although the yield obtained by differential centrifugation was modest, considerable purification was achieved in a relatively short time without the introduction of new reagents.

No. 40 rotor at 40,000 r.p.m. for 260 minutes. The upper ~60% of solution in the tubes was pipetted off and discarded; the remainder was then brought to 2 to 3% protein with 0.10 M KNOI. This procedure was repeated 3 to 4 times until the solution became free of hemoglobin and appeared yellowish. After the final spin for 160 minutes at 40,000 r.p.m. the mid- dle layer was separated, as described in the preceding paragraph. On ultracentrifugal analysis it showed a more symmetrical thy- roglobulin boundary than had been previously attained (Fig. 1C) which was notably free of faster sedimenting material. The light scattering molecular weight was now in satisfactory accord with the sedimentation-diffusion value (see below for details).

To facilitate discussion of these three types of preparation we will refer to them as follows: Preparation I, phosphate-frac- tionated; Preparation II, phosphate-fractionated, differential centrifuged; Preparation III, differential centrifuged. Unless stated otherwise all the data reported in this communication were obtained on Preparation I.

The sedimentation pattern of the thyroglobulin fraction (mid- dle layer) is shown in Fig. 1B. Careful examination of the schlieren pattern reveals a well developed broad fast shoulder to the large thyroglobulin peak. As seen with the aid of a superimposed solvent base-line (double sector cell) this mate- rial is notably free of slower sedimenting molecules. The light scattering molecular weight proved to be larger than that found from sedimentation and diffusion and in excess of experimental errors (see “Results” for details).

Velocity Sedimentation-Analytical centrifugation was per- formed in the Spinco model E ultracentrifuge which was equipped with a phase plate and temperature controls (RTIC unit). Sedimentation constants were determined by standard procedures. Experimental temperatures ranged between 22 and 25”. All sedimentation constant values were corrected to the standard conditions of water at 20” by the methods suggested by Svedberg and Pedersen (8).

A third type of preparation was secured by means of the differential centrifugation technique directly on the 0.9’$& NaCl- extracted thyroid tissue. The extract was centrifuged in the

The composition of thyroglobulin solutions was determined from the relative areas under the peaks in the schlieren dia- gram. Since solutions of calf thyroglobulin may show more than two overlapping boundaries, the precise determination of individual areas becomes quite difficult. In addition, the com- position, as defined by the schlieren boundary areas, was found to vary somewhat between preparations. Generally this varia- bility did not exceed about 10%. (Preparation II-8 discussed

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1328 Properties of Thyroglobulin. I Vol. 235, No. 5

FIG. 2. ’ central ion = 1.41%.

pH 9.5 ’ plill.b pH 12.0 The influence of pH on the sedimentation behavior of thyroglobulin in 0.10 M KNOa; pH 9.5: Preparation I-5; Protein = 1.74 and 1.04%. pH 11.0: Preparation I-6; concentration = 1.24 and 0.52y0. pH 12.0; Preparation I-5; concentr;

in the text, however, did not conform to the above limit.2) The values reported in Fig. 2 are averages taken from several prep- arations and should be considered provisional until further studies provide more information of the detailed behavior of calf thyroglobulin in alkaline solutions.3 Corrections for the Johnston-Ogston effect (9) were considered to be quite small and were not made. Corrections were made for the radial di- lution in the ultracentrifuge cell, except when more than two boundaries were present.

Di$usion-All solutions used in diffusion experiments were dialyzed against solvents of the same pH and salt concentra- tion for at least 24 hours at 5’. The composition was deter- mined by ultracentrifugation of the solutions used in the di- fusion experiments after completion of the runs.

The diffusion experiments were performed in a Spinco model H instrument. The Rayleigh interference method was used to determine concentration gradients. The procedure (and com- putations) were similar to that reported elsewhere (10). The

2 Preparation I-8 behaved in a manner substantially different from the preceding seven batches prepared by phosphate frac- tionation, in that it showed much greater resistance to alkali in its ability to be broken down into slower sedimenting components (numbers following roman numerals refer to individual batches prepared according to procedure indicated by roman numeral). As seen in Table V, at pH 9.5 only 16% of S-12 was formed, whereas the other preparations showed close to 30%. When Preparation II-8 was adjusted to pH 11.0 its ultracentrifugal composition was approximately that of a pH -9.75 solution, as interpolated from Fig. 1. Finally, a pH 11.3 solution of Preparation II-8 compared favorably with that found with earlier preparations at pH 11.0 (see legend, Fig. 5). Some recent experiments show a difference in behavior of calf thyroglobulin towards conformational changes induced by alkali which appears to depend on whether the thyro- globulin had been dialyzed free of neutral salt (Preparation I) or had been fractionated in 0.1 M KN03 (Preparation III) when pre- pared.

3 This variability in composition in response to alkali may be a reflection of the iodine content of the preparation. If the break- down of thyroglobulin were dependent on either the net charge of the protein or the specific ionization of phenolic hydroxyl groups then. from the known abilitv of iodination of phenols to substan- tially reduce the pK of the hydroxyl group, the degree of iodina- tion of our samples should play a significant role in the variation of both of these functions with pH.

con- ation

experiments were performed at 9.95” and the experimental val- ues were corrected by the Stokes-Einstein relation to those in water at 20”. Fringe positions were read directly from metal- lographic plates by a two-coordinate Mann comparator. Av- erage deviations of AH/AZ values were about 0.9% for Prep- aration I-6 and 0.6% for Preparation II-S. The diffusion constant of 0.5% sucrose was obtained with this instrument and found to be 2.40 X 1O-6 cm2 per second at 1.0”. Av- erage deviations were about 0.2%. This value is in very good agreement with that reported by Gosting and Morris (11).

Viscosity-Viscosity measurements were performed in an Ost- wald viscometer with a flow time of about 100 seconds. The bath temperature was controlled to within 0.01’ of 25.00”. Kinetic energy corrections were considered insignificant. All solutions were clarified by centrifugation at 16,000 X g for ap- proximately $ hour.

Partial Specijic Volume and Refractive Index Increment-The experimental details and results of pycnometric, refractometric, and optical densitometric measurements on a phosphate-frac- tionated (Preparation I) calf thyroglobulin are reported in Table I. A Phoenix differential refractometer was used to obtain the refractometric measurements.

The thyroglobulin preparation was extensively dialyzed against distilled water. All experiments were performed in duplicate and very close agreement was obtained on individ- ual samples. No change in the refractive index increment of calf thyroglobulin was found when the solvent was 0.10 M

KN03. All weights are based on dry weight values obtained by drying to constant weight at 90” under vacuum.

The check on our procedures, using crystalline Armour bovine serum albumin as a standard, showed excellent agreement with literature values as noted in Table I.

Light Scattering-A Brice-Phoenix photometer was used for turbidity measurements. Thyroglobulin solutions were clarified by centrifugation at 30,000 r.p.m. (avg. g = 123,000) for 10 minutes in the swinging bucket rotor of the model L Spinco ultracentrifuge. Solvents were centrifuged in the Servall cen- trifuge for at least t hour at 20,000 X g. Freshly distilled water was used whenever possible.

The instrument was calibrated by a sample of duPont Ludox

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May 1960 H. Edelhoch 1329

by the procedure suggested by Maron and &ou (14). This TABLE I method of calibration gave results essentially identical to those Partial specijic volumes and refractive index increments of calf

provided by the instrument manufacturers by means of their thyroglobulin ;‘ reference” standard (15). The turbidity $ related to the arbitrary instrument values of reduced intensity by r/c = Material /$Fit’$!-/ V* / V (lit.) 1 $ft 1 2 (lit.)

.-

ODt.

8.80 x 10-a RN/c and the molecular weight is obtained by the ~~

standard formula g/100 ml

Preparation I-7 2.24 0.7140.723 (3) 0.1949 8.80 x 10-a

z&o

Preparation I-8 2.40 0.7130.72 (1) 0.1948

M = (T/H&, = 7 . 1.04 x 10-5 Bovine serum 1.732 0.734 0. 7343 (12) 0.1950 0.1954 (13)

The pH values were adjusted, before clarification by cen- albumin

10.6 10.4

6.62

trifugation, to the desired value with dilute base after making solutions 0.10 M in KNO,. The 2.4-cm Phoenix cell was used in all measurements. Small volumes of a -1% protein solu- tion were added stepwise to 15 ml of solvent. The solution was mixed by a small glass-encased magnetic stirrer.

RESULTS

Velocity Sedimentation. A. Effects in Basic Xolutions-In Fig. 2 (and Table II) are shown the relative areas of the schlieren boundaries which are resolved in the analytical ultracentrifuge when the pH of thyroglobulin solutions is increased from pH 5.9 to 12.7 in 0.1 M KN03. At least four new molecular species are formed by the progressive disorganization of thyroglobulin. For convenience we will name each component by the value of its sedimentation constant and “native” thyroglobulin will therefore be called the S-19 component. The s&,~ value of- thyroglobin in 0.10 M KN03 at pH 6.0 was 19.4. The data conform to the equation S = 19.4 (1 - 0.113 C) between 0 and 1 ‘$J protein concentration.

Below pH -9.5 only a single new boundary was evident in the ultracentrifuge. The concentration of this species (S-12) was found to increase gradually between pH 5.9 and 9.5 and comprised a little less than one-third of the total at pH 9.5, as illustrated in Fig. 2A. When a 1.73% solution of thyro- globulin at pH 9.5 was diluted with solvent and examined in the ultracentrifuge, the relative amounts of the S-19 and S-12 components were essentially unaltered between 1.73 and 0.35% protein. The sedimentation coefficients of both components are plotted in Fig. 4 as a function of concentration. The total protein concentration was used to plot the concentration of the fast component whereas the concentration of the slow com- ponent is its actual concentration in solution, i.e. 30% of the total protein concentration (16). The concentration dependence of sedimentation of both components were negative and ap- proximately the same. The extrapolated values of s:o,~ were 19.0 and 12.1. There appears to be a slight decrease in the sedimentation constant of thyroglobulin between pH 6.0 and 9.5.

At pH values slightly above 9.5 the concentration of S-19 drops rapidly and two new sedimenting boundaries appear (see Fig. 3). The faster and more abundant of these moves be- tween the S-19 and S-12 boundaries wih a sedimentation con- stant of about 15, when extrapolated to zero protein concen- tration (see Fig. 5). The S-12 component has an s&,, value of 10.8 at pH 11.0. The slowest moving component, which is present in only quite small amounts below pH -11, sedi- ments with an S value of about 8. The S-12 species increases in concentration up to pH ~11, where it becomes the principal component and then disappears almost completely between pH

* Determined at 20.00 f 0.02” in a 25-ml pycnometer. t Temperature = 23”. The green light filter of the Phoenix

differential refractometer was used for measurements. The table value has been corrected (see Perlmann and Longsworth (13)) by the factor, 1.037 to that of blue light (4360 A); c, in g per ml.

$ Optical density at 280 rnp for a 1.00% solution in a l.OO-cm.2 cuvette.

TABLE II

Effect of pH on ultracentrifugal composition of components derived from thyroglobulin

Protein concentration = 1.2 to 1.4%; KNOI = 0.10 M.

Sedimentation components

PH s-19 S-15 s-12 S-8

% % % %

5.8 >95 <5 8.0 85 15

9.5 70 + 30 + 10.0 31 22 42 5 11.0 8 30 52 10 12.0 23* 72

12.7 75

* This schlieren boundary may include some S-12.

s-3

%

5

25

c% 60-

2 I 40-

PH

FIG. 3. The effect of pH on the variation in composition of thyroglobulin components resolved in the ultracentrifuge. Pro- tein concentration = 1.2 to 1.4oJ,. KNOI = 0.10 M. The numbers indicate the approximate sedimentation coefficients.

11 and 12. The variation in composition with pH of thyro- globulin components is illustrated graphically in Fig. 3.

At pH 12.0 the S-8 unit becomes the predominant species and comprises about 3 of the total protein concentration. At pH 12.7 a smaller moving component (S -3 to 4) is formed.

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1330 Properties of Thyroglobulin. I Vol. 235, No. 5

IO

8 I____

61 I I I I I I I I .O .5 1.0 1.5 2.0

THYROGLOBULIN (g / 100 ml.)

FIG. 4. The concentration dependence of sedimentation of S-19 and S-12 at pH 9.5 in 0.10 M KN03. The abscissae values of S-12 have been reduced to its concentration in solution. The values for S-19 are for the total protein concentration.

I I I I I

6 I I I I

0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6

THYROGLOBULIN (g /IO0 ml.)

FIG. 5. The concentration dependence of sedimentation of S-15 and S-12 in 0.10 M KN03. Open circles were obtained on Prep- aration I-6 at pH 11.0. Closed circles were for Preparation I-8 at, pH 11.3. The centrifuge patterns appeared quite similar (see text for further discussion of pH effects with Preparation I-8). Abscissae values for S-15 are that of the total protein concentra- tion excluding that of S-19. The values for S-12 exclude the con- centrations of S-19 and S-15.

TABLE III Effect of ionic strength on composition of thyroglobulin at pH fO.O*

Sedimentation components Bicarbonate buffer KNOn

s-19 S-15 s-12 S-8

M .?d % % % %

0.0025 0 10 9 67 14 0.0005 0.02 24 17 53 6 0.0025 0.10 31 22 42 5

* Protein concentration % 1.0. Stock solution brought to pH 10.0 with NaOH. The Na+ gegen-ion concentration was therefore -0.005 M.

The S-8 and S-3 components are now the only two boundaries observable in thyroglobulin solutions.

B. Reversibility-When the pH of a 1.5% solution of thy- roglobulin in 0.10 M KNO, was raised from 5.9 to 8.0 by di- lute base and after 5 minutes, returned to pH 5.9 again, the sedimentation pattern was quite similar to the untreated pH 5.9 solution. However, a slight increase in the concentration of the S-12 component was apparent when compared with the control pattern. Similarly, when the pH 5.9 solution was in- creased to pH 11.0 for 5 minutes and then reacidified to pH 8.0, only a small increase in the S-12 component (above the control at pH 8.0) was evident in the schlieren boundaries. In both experiments, therefore, the influence of the hydrogen ion activity on the composition of thyroglobulin products was largely but not completely reversible under the conditions em- ployed.

When a pH 5.9 thyroglobulin solution was brought to pH 12.0 for 5 minutes and then readjusted to pH 8.0, the sedi- mentation pattern neither resembled the pH 8.0, nor pH 12.0 control patterns. Considerably larger amounts of S-12 were observed and the S-19 peak contained a fast shoulder which was not present before the addition of base. The molecular changes that occur at pH 12.0 are therefore either slowly re- versible or partially irreversible. Thyroglobulin solutions ad- justed to pH 12.7 and returned to 6.0 showed no discrete sedimenting boundaries; all the protein, apparently, has been partially or extensively aggregated.

C. Efects of Ionic Strength-Lundgren and Williams (2) ob- served the formation of a new, slower moving boundary in solutions of thyroglobulin when the free electrolyte was elimi- nated by dialysis. They also found that the relative propor- tion of this component increased with pH between pH 6 and 12.

To determine whether calf thyroglobulin exhibited similar properties the variation in its composition has been investigated as a function of ionic strength at several pH values. At neu- trality (pH 6 to 7) the concentration of the S-12 molecule in- creased regularly from a trace at 0.10 M KN03 to about 25% at 0.001 M KNOZ.

At pH 10.0 in 0.10 M KN03 the S-12 unit comprised about 42% of the total protein concentration. The concentration of S-12 increased to about 67% when the ionic strength was re- duced to 0.005. The variation in the other constituents that occurs when the KNOS concentration was reduced is reported in Table III.

At pH 11.0 in 0.20 M KNO,, less than 10% of the S-19 thy- roglobulin species remains, whereas the S-12 becomes the ma- jor component (see Table II). In contrast to the considerable influence of ionic strength on the distribution of sedimenting components observed at lower pH values, the composition changed very little with ionic strength at pH 11.0 between 0.20 and 0.01 M KNO,.

Viscosity-The viscosities of thyroglobulin solutions were de- termined in 0.01 M KN03 at pH 7.0, 9.0, and 11.0 and appear in Fig. 6. At neutrality the intrinsic viscosity was 0.047 dl/g which is only somewhat greater than the Einstein coefficient for nonhydrated spheres (0.019) and almost insignificant when compared to a randomly kinked polymer of similar size. We must conclude, therefore, that thyroglobulin possesses a rather high degree of spherical symmetry and compactness for a mole- cule (or micelle) of molecular weight close to Q million.

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The change in viscosity in going from pH 7.0 to 9.0 is less than from pH 9.0 to 11.0. If one assumes as a model for the shape of the particle(s) in solution that of an unhydrated pro- late ellipsoid of revolution, and computes an average axial ratio from the Simha equation (17), then it is apparent from the results that are shown in Table IV that the pH change from 9.0 to 11.0 introduces about the same change in axial ratio as the pH 7.0 to 9.0 transition. It can be estimated from ultracentrifuge analyses that the relative concentration of the S-12 component has increased from about 15 to 4Ooj, in going from pH 7.0 to 9.0 in 0.01 M KNOS. Further increase to pH 11.0 does not alter the S-12 component greatly (from 40 to 50%) but. does lead to the formation of about 20% of S-15 and 20% of S-8. It would seem, consequently, that the latter two species make a similar contribution to the intrinsic viscosity to that of the S-12 unit.

When the pH of a neutral solution of thyroglobulin was raised t,o 11.0 and then directly returned to pH 7.0, the intrinsic vis- cosity neither dropped to its original value of 0.047 nor re- mained at 0.144, the value reported in Fig. 6 for pH 11.0. In- stead it, returned to an intermediate value of 0.09, which is about. the value observed for the pH 9.0 solution. This re- sult, is approximately in accord with the sedimentation data in which the reversibility in properties was substantial but, not complete.

Di$Gon-In Table V and Fig. 7 are compiled the data of four diffusion experiments on two different thyroglobulin preparations in 0.10 M KN03. The more highly purified frac- tion (Preparation 11-8) had a diffusion coefficient of 2.49 x lo-7 at pH 6.0. When the pH was raised to 9.5, which re- sulted in the formation of 16% of the S-12 component, the dif- fusion component was found to be slightly larger, i.e. 2.56 x 10-7.

With the less purified, phosphate-fractionated thyroglobulin (Preparation I-5) the diffusion coefficient at pH 6.0 was 2.37. The lower value of this fraction probably reflects the greater significance of the impurities sedimenting faster than thyro- globulin (S-19). At pH 9.5 the diffusion constant increased to a value of 2.61. The increase is considerably more than twice that observed with Preparation II and probably results from the breakdown of the S-25 component to smaller frag- ments (since most, of this peak disappears in the sedimenta- tion pattern at pH 9.5) and the formation of a greater pro- portion of S-12 (i.e. 30%) with this preparation.

The concentration dependence of diffusion depends on both the virial coefficient (B) and a frictional factor (k,) related to the concentration dependence of sedimentation (18)) since

It is shown elsewhere in this paper that the virial coefficients are essentially zero at both pH 6.0 and 9.5 (in 0.10 M KN03). Moreover, the concentration dependence of sedimentation of S-19 is equal within experimental errors to that of S-12. Hence, the dependence of diffusion on concentration should be quite similar at pH 6.0 and 9.5. Moreover, O’Donnell et al. (5) have shown that the diffusion coefficient of hog thyroglobulin does not change significantly with concentration from -0.3 to 1% protein. We may, therefore, conclude that the replace- ment of S-19 by S-12 molecules has resulted in an increase in the net rate of diffusion.

I I I I I I I I I I I I .I6 - .I6 -

w w 4 4 0 0 * * 11.0 11.0

.I4 - .I4 -

.I2 - .I2 -

.08 t 7.0 4

.06~ -I

.04 1 I I I I I .O 0.5 I.0 1.5 2.0

THYROGLOBULIN (g /IO0 ml.) FIG. 6. Reduced specific viscosity of thyroglobulin (Prepara-

tion I-5) in 0.010 M KNOB as a function of pH. 28.1”.

Temperature =

TABLE IV

Effect of pH on intrinsic viscosity of thyroglobulin in 0.01 M KNO,

PH 171 a/b*

dug

7.0 0.047 5.5 9.0 0.085 9.0

11.0 0.144 13.0

* a/b = Average axial ratio of prolate ellipsoid of revolution (based on Simha equation) and assuming no hydration.

I 2.7 c

2.6 r Q-m,

3 "

:: 2.5 n

6.0 2.4 *

0 332 04 .06 08 .I0

104/SECONDS FIG. 7. Plot of diffusion data as measured by the Rayleigh inter-

ference fringe method. Filled circles were obtained on Prepara- tion I-6; open circles on Preparation II-S. Experimental details are given in Table V.

Light scattering-Light scattering measurements were made on the three different types of preparations outlined in the Methods section. At neutrality (pH -6.0) in 0.10 pd KN03

all preparations showed no dependence of reduced turbidity on concentration. The weight average molecular weights of these three types of preparations were: (A) Preparation I = 930,000; (B) Preparation II = 820,000; (C) and Preparation III =

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TABLE V

Diffusion coejkients of thyrogEobulin*

Preparation I Preparation 11

PH

6.0 9.5

tinguishable from zero at pH 6.0 and 9.9. However, at pH 11.0, there appears to be a small significant slope factor. In the data shown in Fig. 8, 0.025 M glycine buffer was used in the pH 9.9 and 11.0 solutions to ensure control of the pH.

COllC.Sn- COIlCeIl- (These data were in very good agreement with results obtained

tration S-12t D X 107 tration s-1zt D X 10’ without glycine buffer at the same pH values with this sam- -~

g/l00 ml % g/l00 ml % ple of thyroglobulin.)

0.54 2.37 0.35 2.49 A decrease of 18% in turbidity was found between pH 6.0

0.55 30 2.61 0.33 16 2.56 and 9.9. With this preparation (11-8) the S-12 component con- stituted 20% of the total protein concentration at pH 9.9. Ten per cent of the loss in scatter can be accounted for if the S-12 was formed by dissociation of the S-19. The remaining 8% change probably comes from the dissociation of a small amount of faster sedimenting protein.

* KNO, = 0.10 M. All runs performed at 9.9” and corrected to water at 20’ by Stokes-Einstein formula.

t Sedimentation analyses were performed on samples used in diffusion experiment.

TABLE VI Hydrodynamic properties of thyroglobulin (components) at neutrality

and pH 9.5 in 0.10 M KNO,

PH

6.0

9.5

&lo M x 108 f/h a/b* D;,,, X 10’

19.4 669 1.49 9.0 2.49 a. 12.1 669 2.40 32. 1.55

b. 12.1 335 1.51 9.4 3.11

* Value of axial ratio (a/b) are those for an unhydrated prolate ellipsoid of revolution.

I I I I I I I I

a?

70 - 0 I 1.0 m

I 60

t

Properties of Thyroglobulin. I Vol. 235, No. 5

When the above set of experiments were repeated with Prep- aration III-9 the scattering dropped by only 7% between pH 6.0 and 9.9. An ultracentrifuge pattern showed the formation of 10% of S-12 at pH 9.9. Thus, when the thyroglobulin preparation was essentially devoid of material sedimenting faster than the thyroglobulin peak, the light scattering data are in close agreement with the ultracentrifuge analysis. When the pH was raised to 11.2 the intrinsic scatter dropped to 70% of that observed at pH 6.0.

The further decline in intrinsic scatter observed at pH 11 indicated further molecular disorganization and is compatible with the appearance of slower moving boundaries as reported in Fig. 2 (pH 11.0 and 12.0).

50 I I I I I I I

0 .Ol .02 .03 .04 .05 .06 .07 .00

When a thyroglobulin solution at 1.25% in 0.10 M KNO, was turbidimetrically titrated with dilute base between pH 6.5 and 11.2, the turbidity declined and was in approximate accord with that reported in Fig. 8 at the corresponding pH values. Upon back-titrating with small volumes of dilute acid from pH 11.0 to 8.0, the scattering intensity of the concen- trated thyroglobulin solution retraced the forward curve, al- though it was displaced toward smaller values by about 10% of the total scatter. The addition of dilute acid tended to produce local flocculation in the concentrated solution which disappeared on stirring (magnetic). Nevertheless, it seems un- likely that gross aggregation could account for the smooth re- versal in scatter. When the pH was reduced below pH -8 the solutions tended to become strongly opalescent which pre- cluded further measurements.

FIG. 8. The variation of the reduced intensity of light scatter with pH in 0.10 M KN03. At pH 9.9 and 11.0 the solutions con- tained 0.02 M glycine.

THYROGLOBULIN (g /I00 ml.)

Time E$ects-Practically all measurements reported in this paper were performed at least + hour after any adjustment in pH from the value of concentrated stock solutions. None of the measurements reported above showed any observable time effects, (except the pH-turbidimetric titrations mentioned in the preceding section). Kinetic effects could be observed by light scattering, however, when the pH adjustment was made immediately before observation in the photometer. Most of the decrease in scatter was over in about + hour when the pH was raised from 6.0 to -9.5, whereas somewhat longer times were required when the final pH was 10 and above.

690,000. The progressive decrease in average molecular weight can be correlated with the composition of the different prep- arations as determined by ultracentrifugation (see Fig. 1). The decrease in the average molecular weight follows approximately the decline in the S-25 component and in the material appearing as a fast moving shoulder of the S-19 boundary.

In Fig. 8 is reported the reduced intensities of thyroglobulin (Preparation 11-8) at pH values of 6.0, 9.9, and 11.0 in 0.10 M KN03. The intrinsic scattering and hence the weight av- erage molecular weight of thyroglobulin declined as the pH was increased. It is of interest to note that the slopes of the curves in Fig. 8. i.e. the second virial coefficients. are indis- - I

Denaturation-Some preliminary observations on the rates of denaturation of thyroglobulin in alkaline media are of interest in connection with the sedimentation data and are, therefore, presented. When the pH of a thyroglobulin solution was raised to 11.0 and then rapidly acidified to pH 5.25 with a concen- trated acetate buffer no insoluble protein was found, even after standing at pH 11.0 for 3 hours. However, when the pH was increased to 11.6, a time-dependent formation of insoluble pro-

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May 1960 H. Edelhoch 1333

tein occurred upon the addition of the same acetate buffer (final ~1 = 0.80). About one-half the protein present was pre- cipitated in 5 minutes. At pH 11.9 the rate was much faster and the half-life was only about 1 minute. If we therefore define denatured thyroglobulin as that form which is insoluble at (or near) its isoelectric pH, then we may refer to the S-8 (and slower sedimenting species) as denatured thyroglobulin. The S-15 and S-12 species, therefore, represent modified forms of thyroglobulin which are either soluble at pH 5.3 or in rapid equilibrium with soluble forms at pH 5.3.

DISCUSSlON

Properties of Calf Thyroglobulin at Neutrality-The s$,~ value of thyroglobulin determined in 0.10 M KNOS at pH 6.0 was 19.4, and is in close agreement with the recent value reported by O’Donnell et al. (5), for hog thyroglobulin. The slope, how- ever, is about 50% smaller than theirs.

The value of 2.49 X lo-’ for the diffusion coefficient is slightly smaller than the value of 2.60 X lo-’ reported by both O’Donnell et al. (5) and Derrien et al. (3) for hog thyroglobulin. Whether this difference in diffusion coefficient is related to the impuri- ties present in the hog preparations or to different frictional coefficients for calf and hog thyroglobulin is uncertain at pres- ent. Resolution of this difference will have to await the fur- ther purification of hog thyroglobulin. I f we combine this value with the sedimentation constant (19.4) and the partial specific volume (0.713s) a molecular weight of 669,000 is cal- culated by the Svedberg equation. This value is in the range of recent reports for hog thyroglobulin, of 655 X lo3 by Derrien et al. (3) and 660 X lo3 by O’Donnell et al. (5).

Properties of the S-12 Component-Lundgren and Williams (2) observed the formation of a slower moving boundary in hog thyroglobulin solutions when the free salt was eliminated by dialysis. Since their sedimentation studies showed that the relative area of the slower moving boundary increased with protein concentration between -0.25 and 4% they concluded that the reaction was not a dissociation but an isomerization reaction.

O’Donnell et al. (5) have pointed out that the slower moving boundary would show an increase with increasing protein con- centration due to the Johnston-Ogston effect (9). The failure to observe a decrease may be due to activation energy barriers of the type we have found for calf thyroglobulin which inhibit dissociation on dilution (see final section).

Since the concentration of the S-12 component in calf thy- roglobulin solutions may be enhanced either by reducing the ionic strength at neutral pH or by raising the pH at constant ionic strength (0.10 M), it seems logical to conclude that the component we have called S-12 is similar and probably iden- tical to the “c.u-protein” reported by Lundgren and Williams (2). For reasons presently unknown, the calf protein is more easily dissociated in basic solutions than the hog protein. Hei- delberger and Pedersen (1) observed that at pH 12.0, in .083 M NaCl, hog thyroglobulin is not stable and forms two new sedimenting boundaries with sedimentation coefficients of 12.4 and 9.2. The S-12 particle may also be formed at neutrality in 0.10 M KNOS in much higher yields by treating calf thyro- globulin solutions with relatively small quantities of anionic detergents, such as sodium dodecyl sulfate (19). O’Donnell et al. (5) have reported that a similar enhancement in hog “a- protein” was accomplished by making their solutions 10% in

dioxane. However, to observe their effect they eliminated all free salt.

In Table VI are shown calculated values of the frictional ratio (f/fO) and diffusion coefficient of the S-12 component based on (a) an isomerization process, which necessarily leads to a substantial increase in frictional coefficient, and (b) a dis- sociation into two fragments of equal mass. It is apparent that if the S-12 particle arises from a change in shape only then it would show an increase in frictional ratio, in propor- tion to the sedimentation values of the two forms, i.e. 19.4: 12.1. However, if the S-12 particle is formed by dissociation, it would possess a frictional ratio quite similar to that of the parent molecule. The diffusion coefficient was found to in- crease in both the phosphate- and centrifugally-fractionated thyroglobulin. The increases are in approximate accord with the compositional changes observed in each preparation by sedimentation analysis based on the mechanism fitting Case (b). Since the diffusion coefficient should decrease in Case (a) and increase in Case (b) this experiment gives an unambiguous answer. Viscosity measurements, cannot a priori distinguish between the two cases, unless there were relatively little in- crease or a decrease in viscosity. Then Case (a) could be read- ily excluded. Since an isomerization reaction would require a substantial increase in viscosity (see Table VI), the small increase observed is in harmony with the conclusion drawn from the diffusion data. Differences in the degree of hydration between S-19 and S-12 could account for the small increase observed in intrinsic viscosity.

The reduction in the intrinsic light scatter that occurs be- tween pH 6.0 and 9.9 (or 9.5) can only be viewed as a con- firmation of Case (b). The magnitude of the change is ap- proximately related to the variations in composition, assuming that the molecular weight of S-12 is one-half that of the S-19 component. On this basis, the turbidity is in harmony with the hydrodynamic measurements. Similarly, the increase in scattering that occurs on reducing the pH from 11 to 8 should be viewed as in keeping with the reversibility observed in sedi- mentation patterns under similar pH adjustments.

Properties of Components Formed Above pH B.&-Above pH 9.5 two new molecular species are formed, having sedimentation constants of about 15 and 8. Up to pH -11 the faster sedi- menting component is present at about three times the weight concentration of the slower boundary. Since the S-15 compo- nent has about the same concentration dependence of sedi- mentation as the S-19 and S-12 particles, it is probably globular in form also. On this basis, an approximate value for its mass would be about -2 of the native molecule. Whether this species arises from a 3 to 1 split of the S-19 or by further dissocia- tion of the S-12 into fragments, i.e. halves, with recombina- tion of one of the fragments with the S-12 (on a new site) is not apparent from the data. Since no S-15 material appears until S-8 is seen, dissociation of S-12 into halves and recom- bination to form S-15 seems reasonable.

At pH 12, the S-8 particle becomes the principal component. It has been shown that once this molecule is formed at pH -12 it becomes insoluble when the pH is adjusted rapidly to 5.25 with acetate buffer. We may, therefore, classify S-8 as a denatured (and dissociated) form of thyroglobulin.

Mechanism of Conformational Changes-It seems evident from the ability of ionic strength and pH to produce changes in the conformational properties of thyroglobulin, that the driv-

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1334 Properties of Thyroglobulin. I Vol. 235, No. 5

ing force of the various transformations that occur is largely electrostatic in origin. Since the isoelectric point of thyroglobu- lin is a little below pH 5 (l), an increase in pH will result in a larger negative charge on the protein.

The formation of new sedimentable species following increases in alkalinity occurs at measurable rates and can conveniently be followed by light scattering methods. Although changes in pH produce time-dependent changes in light scattering, varia- tion in protein concentration at a specific pH has little or no effect on the proportions of the various components. The com- position of thyroglobulin solutions, as determined by sedimen- tation analysis, did not vary significantly as the protein con- centration was reduced from -1.7 to 0.35% at pH values of 9.5 and 11.0 (see Fig. 2). Rapid equilibration of components, in accordance with mass action considerations, would probably result in positive slopes in the S versus C plot. The lack of rapid equilibration, however, accounts for the resolution of the individual components in the ultracentrifuge.

The slopes of the turbidity data at pH 9.9 and 11.0 are in accord with the sedimentation experiments in showing that no measurable changes in composition occur with variation in pro- tein concentration. If rearrangement of molecular species oc- curred rapidly with dilution, then a positive slope would be expected in the turbidity function plotted in Fig. 8. Both the sedimentation and turbidity data, therefore, indicate that a significant energy of activation controls the rate of intercon- version of thyroglobulin and its slower sedimenting products. It is of interest to note in this connection that the reduced turbidity of thyroglobulin at pH 6.0 in 0.10 M KNOZ is con- stant from the lowest concentration that has been measured, i.e. 0.0035 to 1.0%. At pH 6.0, therefore, either the equilib- rium constant for dissociation is either very small in 0.10 M KN&, or the activation energy is too high for dissociation to occur at room temperatures.

SUMMARY

The molecular weight of calf thyroglobulin has been deter- mined by sedimentation and diffusion and by light scattering. Both methods give a value close to % million, which is in agree- ment with the molecular weight of hog thyroglobulin.

Calf thyroglobulin has been shown to undergo a series of reversible conformational changes which are governed by the pH and ionic strength of the solution. The first change of this type involves the formation of a particle with sedimentation

properties similar to the a-protein of Lundgren and Williams (2). However, diffusion and light scattering experiments in- dicate that it is formed by dissociation of thyroglobulin into two subunits.

Increase in basicity above pH 9.5 results in the formation of two new sedimenting components which, based on light scattering observations, must be smaller in molecular weight than thyroglobulin.

At room temperatures thryoglobulin is denatured at pH val- ues above -11.4 and the first order velocity constant of de- naturation shows a marked dependence on pH in this region.

Acknowledgment-The author wishes to express his indebted- ness to Mr. Roland E. Lippoldt for providing excellent assist- ance.

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H. EdelhochThe Properties of Thyroglobulin: I. THE EFFECTS OF ALKALI

1960, 235:1326-1334.J. Biol. Chem. 

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