European J. Biochem. 5 (1968) 422-428

Effects of Ultraviolet Irradiation (2 5 3 7 A) on the Structure of Human Thyroglobulin

G. VECCHIO, K. DOSE, and G. SALVATORE

Centro di Endocrinologia e di Oncologia Sperimentale del C.N.R., Istituto di Patologia Generale dell'Universit8 di Napoli

(Received April 6, 1968)

The effect of in vitro ultraviolet irradiation at 2537 A on the structure of highly purified human thyroglobulin has been investigated by following the ultracentrifugal pattern and the appearance of free sulfhydryl groups in the irradiated protein.

Thyroglobulin (19 S) dissociates into two 12 S subunits when it is irradiated with doses of a few hundred Einsteins per mole. The quantum yields of dissociation vary between 0.5 x 10-3 and 2 x depending on the experimental conditions. At higher ultraviolet doses, the protein is partially aggregated and denatured.

The extent of dissociation is largely dependent on the ionic strength and the pH of the medium. Doses up to 1,500 Einsteins per mole cause only a partial (3O0iO) splitting of the 19 S molecules, a t neutral pH in 0.1 M KCI, whereas a t pH 10 and low salt (T/2= 0.0042), the same dose produces almost complete dissociation into 12 S subunits.

Ultraviolet light also causes the rupture of some of the disulfide bonds of native thyroglobulin. On irradiation with several hundred Einsteins per mole protein, as many as 21 -SH groups per molecule can be titrated. The quantum yields for -SH production and destruction of cystine residues are both 0.05.

No direct correlation has been found between the extent of dissociation of thyroglobulin and the number of -S-S- bonds cleaved, nor between the reassociation of the 12 S subunits and the reoxidation of -SH groups. It is concluded that two 12 S units present in human thyroglobulin are not linked by interchain disulfide bridges ; the disulfides cleaved by ultraviolet irradiation, however, may play an important role in maintaining the subunit organization of the protein molecule.

The major thyroid protein, thyroglobulin (19 S, mol.wt. = 660,000), consists of two half molecules (12 S, mol.wt. = 330,000) which are in turn made up by two polypeptide chains whose sedimentation coefficient, when in a globular form, approximates 6 S [l]. Most of the 12 S subunits are held together in bovine thyroglobulin by non-covalent bonds whereas the 6 S units are linked by a few interchain disulfide bridges [2]. Most of the disulfide bonds (101 per molecule [ 1,3]) are intrachain rather than inter- chain and no free -SH groups have been detected in thyroglobulin by the use of Ellman's reagent [4]. Very recently Pitt-Rivers and Schwartz [5] have shown the presence in some mammalian species, including human, of a few sulfhydryl groups not titratable with Ellman's reagent, but titratable with other reagents.

Fully reduced and extensively unfolded thyro- globulin has been obtained by treatment with an excess of mercaptoethanol in 8 M urea or 5 M guan-

Unusual Abbreviation. DTNB, 5,5'-dithiobis(Z-nitroben- zoic acid).

____

idine-HC1 [2,6]. Attempts to convert thyroglobulin into globular 6 S species by selectively breaking the interchain disulfide bridges with minimal concen- trations ofreducing agent, have so far been uyuccess- ful [2]. Since ultraviolet irradiation a t 2537 A causes, besides other minor effects, primarily the photolysis of the protein cystine residues [7 - 1 I], in the present work we have investigated the effects of this wave- length on the structure of human thyroglobulin.

The aim of this study was to learn: a) whether any -S-S- linkages are involved in

cross-linking the two 12 S units of human thyro- globulin ;

b) whether ultraviolet irradiation at low levels could yield globular 6 S units by selectively breaking interchain disulfide bridges.

Furthermore, studies on the effects of ultraviolet light on human thyroglobulin may provide some information on the molecular structure of this protein which is less well characterized than bovine thyroglobulin.

A preliminary report of this work has appeared [12].

Vo1.5, Nn.3, 1968 G. VECCHIO, K. DOSE, and G. SALV.4TORE 423

MATERIALS AND METHODS

The thyroid extract from a patient with simple goiter was obtained as previously described [13]. Human thyroglobulin was purified by precipitation of the soluble extract between 1.4 and I .8 M (NH,),- SO,, followed by gel filtration on 7O/, granulated agar gel [13]. The preparation used in the present study was homogeneous in the ultracentrifuge as seen in the bottom pattern of Fig. I. The sedimentation con- stant ( ~ i ~ , ~ ) of this preparation was 18.0. The iodine content of the purified protein was O.Io/, [14]; this low iodine content accounts for the smaller value of the sedimentation constant [15]. The protein was kept as the ammonium sulfate precipitate a t -20". When used, i t was dissolved in and then dialyzed against 0.02 M phosphate + 0.1 M KCI, pH 5.9, which will be referred to as "standard buffer".

Protein concentration was measured spectro- photometrically a t 280 mp using a specific extinction coefficient (&:kl) of 10.0 [13].

Dissociation was achieved by dialysis a t +4" of the protein solution in the standard buffer against either 2 mM phosphate, pH 5.9 ("low ionic strength buffer") or 2 mM bicarbonate-carbonate, pH 10.0 ("low ionic strength-alkaline buffer"). Reversal of dissociation was obtained by dialysis against stand- ard buffer.

Irradiations were carried out with a Hanovia low pressure mercury lamp. The wavelength of irradia- tion was 2537 A ; care was taken to exclude wave- lengths shorter than 2400 A by the use of an appro- priate filter. The intensity of the incident light was 0.125micromoles quanta per cm2 per minute. The protein solutions were irradiated in spectrophoto- metric cells (Suprasyl quartz., 1.0 mm light path) a t a concentration of lolo.

Immediately after irradiation, the protein solu- tion was analyzed by velocity sedimentation in the Spinco model E ultracentrifuge. All the runs were done between 20" and 25". Distances on the ultra- centrifuge plates and tracings of the schlieren patterns were obtained by means of a Nikon com- parator, model 6C. Sedimentation coefficients were calculated by means of standard techniques. The areas under the peaks were estimated by planimetry of the paper tracings. No corrections were made for either Johnston-Ogston or radial dilution effects.

Titration of the -SH groups was carried out as follows: aliquots of 0.3 ml of the protein solutions (control and irradiated) a t a concentration of lo/, were added directly to 0.7 ml 7.15 M guanidine-HCl (recrystallized from methanol a t 40") and 0.05 M phosphate buffer, pH 8.0, in a spectrophotoinetric cell. 20 p1 of a solution of 5,5'-dithiobis (2-nitro- benzoic acid) (DTNB, Pierce Chemical Co., 3.96 mg/ ml) were added last. Absorbance readings were taken

in the same cell. A molar extinction coefficient ( ~ ~ 1 2 ~ ~ ~ ~ ) of 13,600 for the reduced DTNB was used to convert the absorbance at 412mp to -SH concen- tration [4].

The quantum yields were calculated as reported by Risi et al. [lo]. Since the absorbance of ultra- violet irradiated thyroglobulin increases considerably

M

Fig. 1. Ultracentrijugal patterns of native (standard cell , hottorn) and ultraviolet irradiated human thyroglobulin (wedge cell, top). Highly purified 19 S (lo/,,) in 0.1 M KCI + 0.02 &I phosphate buffer, pH 5.9, irradiated with about 500 Einsteins per mole (2537 A). Photograph taken after 16 minutes a t 52,640 rev./min in the An H rotor of the Spinco Model E ultracentrifuge at 20"; bar angle 60"; single sector cells.

Sedimentation is from left to right

with increasing ultraviolet dose, accurate data for the doses applied, that is Einsteins per mole native 19 S thyroglobulin originally present, can be given only for irradiation periods of 15 min and less [lo].

The loss in cystine residues has not been dcter- mined experimentally, but taking into account that in lysozyme and trypsin (which contain tryptophan residues in proportions similar to thyroglobulin) about 1.3 titratable -SH groups are formed per cystine residue photolysed [lo, 161, one may calcu- late the quantum yields for cystine inactivation

424 Ultraviolet Irradiation of Human Thyroglobulin European J. Biochem.

from the quantum yields of -SH production. Quan- tum yields for cystine destruction in proteins may also be estimated from their amino acid composition according to equation ( I ) .

@s-s = fA+ f2@2 + Y (1) where (3s-s is the number of cystine residues de- stroyed per quantum absorbed anywhere in the pro- tein, is the quantum yield for cystinel destruc- tion in free (neutral or acid) solution, Q2 is the quan- tum yield for cystine destruction due to interactions between excited tryptophans and cystines [ll], f l and f i are the fractions of light absorbed by cystine and tryptophan residues, respectively and Y is a function representing the contribution of other fac- tors (mostly excited tyrosine residues) to cystine destruction. and Q2 have been found to be 0.12 and about 0.035, respectively. The mo!ar extinction coefficient of thyroglobulin a t 2537 A is 440,000. Thyroglobulin has 101 cystine residues per molecule [I -31. The molar extinction coefficient of cystine is about 300 a t 2537 A; f l is therefore 101 x 3001 440,000 = 0.07; f 2 is calculated accordingly (85 tryp- tophan residues [17,18]) ( E ; ; S ~ = 3000) yielding 0.58. Y is estimated by multiplying the remainder fraction of light (0.35) by an empirical quantum yield of 0.048 [ll] yielding almost 0.02. The theore- tical value for cystine destruction, according to equation [l], is therefore a little less than 0.05.

Alkylation of the irradiated protein was carried out by adding 100 pl of a solution of iodoacetic acid (recrystallized from petroleum ether and kept a t - 20”) in 0.7 N NaOH to 1.8 ml of lo/o protein solution. Final concentration of iodoacetic acid was 36 mM, corresponding to a molar ratio of iodoacetic acid to protein -SH of 240 (calculated on the basis of 10 -SH’s formed per mole of protein); the pH was kept close to 8.0. The excess iodoacetic acid was removed by dialysis a t pH 5.5.

RESULTS

EFFECTS O F ULTRAVIOLET IRRADIATION ON THE ULTRACENTRIFUGAL COMPOSITION

O F THYROGLOBULIN

High Ionic Strength Buffer ( p H 5.9) The ultracentrifugal pattern of the human 19 S

thyroglobulin in 0.1 M KC1, exposed for 10 minutes to irradiation (accumulated dose of about 500 Ein- steins per mole), is shown in Fig. 1 (top pattern). The appearance of a slow sedimenting boundary, repre- senting about Z O O / , of the total protein, is evident. The sedimentation coefficient (12 S) of the minor component indicates it to be the half molecule (mol.wt. 331,000) of thyroglobulin [l9].

1 In many publications corresponding yields refer to half cystine residues. Those yields, therefore have values double the ones reported here.

The area under the 19 S peak progressively de- creases with higher ultraviolet doses : above 500 Ein- steins per mole an increasing proportion of the native protein is irreversibly aggregated (see Table I ) . The amount of 12s formed, however, does not increase beyond about 30°/, (see Fig. 2) with larger ultraviolet doses. The quantum yield for the decrease of the 19 S peak was calculated to be 0.7 x

Table 1. Dissociation of human thyroglobulin by ultraviolet

Thyroglobulin (0.8O/,, w/v) was dissolved in standard buffer (0.1 M KCI + 0.02 M phosphate, pH 5.9). Light intensity was 0.125 pmoles quanta/cm2/min. Values within brackets are only estimates (see Materials and Methods). Relative proportions were calculated from the area of the schlieren peaks as a percentage of the original non-irradiated 19 S. Protein loss was calculated from the area of the original non-irradiated 19 S minus the areas of the remaining 19 S + formed 12 S (percent). In some instances a few percent of

the protein sedimented faster than 19 S thvroelobulin

light (2,537 A )

Relatire proportions of Protein Of Accumulated dose

exposure 19 s 1” B loss

min Einstrins/nmk - 0 0 100 0

5 300 85 15 0 10 500 73 27 0 15 700 61 21 18

57 22 21 55 18 27

20 (850) 25 (950) 30 (1 j050j 52 21 27

I

M I I I I , I

0 5 10 15 20 25 30 IRRADIATION TIME (min)

Pig. 2. Dissociating effect of ultraviolet irradiation (2537 8 ) on human thyroglobulin. Protein irradiated either in 0.1 M KCl+O.O2 M phosphate buffer, p H 5.9, (high ionic strength, O--O) or in 2 mM phosphate buffer, pH 5.9, (low ionic strength, x----x) with the ultraviolet light a t 2537 d (0.125 pmoles quanta/cm2/min). Ordinate: area under 12 S ultracentrifugal peak as a percentage of original non-irra-

diated 19 S

Low Ionic Strength Buffer (pH 5.9) If the native protein is exposed to a low ionic

strength medium (2 mM phosphate, pH 5.9) about

Vo1.6, No.3, 1968 G. VECCHIO, K. DOSE, and G. SALVATORE 425

20°/, of the 19 S molecules are dissociated into the protein into a slower sedimenting species (approx. 12 S subunits (Fig.2). The cumulative effects of 6 S) which represents an unfolded form of the 12 S irradiation and exposure to low ionic strength are half molecule of thyroglobulin (Fig. 4, lower pattern). not additive but synergistic : about 70°/, of the Ultraviolet irradiation of thyroglobulin in low ionic native protein is dissociated into 12 S subunits after strength-alkaline buffer results in almost complete 10 minutes irradiation (Fig. 3). disappearance of the original 19 S boundary (Fig. 4,

upper pattern).

Fig. 3. Ultracentrijugal pattern of irradiated human thyro- globulin in low ionic strength. Conditions of ultracentrifiiga- tion were as in Fig. 1. Irradiation with about 500 Einsteins per mole (2537 A). Photograph taken after 26minutes a t 52,640 rev./min. Double sector cell. Thyroglobulin in 2 mM

phosphate buffer, pH 5.9, after 10 minutes irradiation

As in high ionic strength buffer a t pH 5.9, the maximum dissociating effect (approx. 700/,) is reached after 10 minutes of irradiation (500 E n - steins per mole) ; longer exposures to ultraviolet light (up to 30 minutes) do not significantly increase the percentage of 12s (Fig.2). The quantum yield for decrease of the 19 S peak was calculated to be about 2 x

Low Ionic Xtrength-Alkaline Buffer ( p H 10.0) Exposure to low ionic strength-alkaline buffer

(2 mM bicarbonate, pH 10.0) dissociates the native

Fig. 4. Ultracentrifugal patterns of native and irradiated human thyroglobulin in low ionic strength and alkalinp p l i . Condi- tions of irradiation and ultracentrifugation as in Fig. 1. Photograph taken after 50 minutes a t 52,64Orev./min. Stand- ard cell (bottom pattern), thyroglobulin in 2 mM bicarb- onate-carbonate buffer, pH 10.0. Wedge cell (top pattern), same preparation after 10 minutes irradiation (500 Ein- steins per mole protein). The slow sedimenting species (ap- prox. 6 S), representing an unfolded form of thyroglobulin half molecules (12 S), is present in larger amounts after

irradiation

SULFHYDRYL GROUPS IN IRRADIATED THYROGLOBTJLIN

It is now generally agreed that one of the most important effects of ultraviolet light on disulfide containing proteins is the disruption of the -S-S- cystine bonds [7-11,16]. Bovine thyroglobulin con- tains a large number of disulfide bonds (101 per molecule, mol.wt. = 660,000) but no free -SH groups titratahle with DTNB [1,2]. It has now been found

426 Ultraviolet Irradiation of Human Thyroglobulin European J. Biochem.

also that human thyroglobulin does not have any free -SH groups titratable with DTNB. However, when human thyroglobulin is irradiated with 300 (or more) Einsteins per mole, a significant number of -SH groups can be titrated (see Table 2 ) . The

Table 2. Formation of -&‘I€ groups in irradiated human thyroglobulin

The intensity of ultraviolet irradiation a t 2537 A was 0.125 pmoles quanta/cm2/min. I n the high ionic strength medium the protein (0.8O/,, w/v) was dissolved in 0.1 M KCI + 0.02 M phosphate, pH 5.9. In the low ionic strength medium the protein (l.OO/,, w/v) was dissolved in 2 mM phosphate,

pH 5.9

-SH groups Irradiation

titllr AccUn~uktted (lose High ionic Low ionic strength strmgtti

min Mristeins/mol(~

0 0 5 300

10 500 15 700 20 (850) 25 (950) 30 (1,050)

No./niole giotcin (niol.mt. - 660.000)

0 0 6.25 7.25

10.40 11.13 18.70 14.24 20.40 17.35 20.40 17.35 2 1 .OO 19.42

phan containing proteins [lo]. On the basis that 1.3 -SH groups are produced per cystine residue destroyed, a quantum yield of about 0.04 can be calculated for cystine destruction. This value agrees with the computed value of a little less than 0.05 (see Materials and Methods).

REASSOCIATION O F THYROGLOBULIN AND REOXIDATION O F -SH GROUPS

Dialysis against standard buffer of the protein previously dissociated in “low ionic strength-alkaline medium”, causes a complete reassociation of the 12 S molecules. Almost no reassociation occurs when the conditions of dissociation are only partly reversed (neutral pH, low ionic strength). Neither dissociation of thyroglobulin nor reassociation of subunits is accompanied by appearance or disappearance of titratable -SH groups when the ultraviolet irradia- tion is omitted (Table 3.)

The relationships between -SH reoxidation and association of the 12 S subunits formed by irradia- tion, were also studied. The sulfhydryl groups were measured after reassociation of thyroglobulin sub- units. The free -SH groups which are formed after

Table 3. Reassociation of human thyroglobulin and reoxidation of --SH groups The protein (0.8,/,, w/v) was irradiated for 10 minutes a t 2537 A (0.125 pmoles quanta/cmz/min)

-SH groups

No. /male protein

0

I~cvcrss lb Expcrirnental ronditinns PH r/r 12 S a

- Dissociation 10 0.0042 26 + dialysis 6 0.0024 25 4 0 + dialysis G 0.124 0 100 0

Dissociation + irradiation 10 0.0042 90 0 13.2 + dialysis 10 0.0042 90 0 G.O + dialysis 6 0.0024 90 0 5.2 + dialvsis G 0.124 62 31 5.8

Area, of the 12 R schlieren peak (formed) as a pcrcentage of the original non-irradiated 19 S. 11 Pcrccntage of dissociated 13 S revertcd to 19 S.

number of -SH groups increases progressively during the first 15 minutes of irradiation (about 700 Ein- steins per mole) and then rapidly levels off with longer irradiation times.

The time-course of -SH appearance parallels that of 12 S subunit formation by ultraviolet irradiation. The number of free -SH groups formed, however, is not significantly different in high and low ionic strength medium, even though the degree of disso- ciation of thyroglobulin is much higher in the low than in the high ionic strength medium. These results suggest that rupture of -S-S- bonds and dissociation of thyroglobulin are, a t least to a first approximation, not directly related.

One -SH group is produced per 20 photons ab- sorbed by thyroglobulin, that is @‘(sH) = 0.05. This value agrees well with those found for other trypto-

irradiation significantly decrease upon dialysis for 16 hours a t f4”. Since such a decrease does not occur in a nitrogen environment (unpublished results), it is most likely due to spontaneous oxida- tion of the -SH groups in the presence of air. It is noteworthy that the same degree of -SH oxidation occurs when the dialysis is carried out against either low ionic strength buffer, which does not permit the reassociation of thyroglobulin subunits, or standard buffer, which produces significant reassociation (see Table 3). Such experiments indicate that both the appearance of - SH groups and their reoxidation are not directly related to the dissociation of thyro- globulin or reassociation of subunits.

This conclusion is further strengthened by the experiments reported in Table 4. Reassociation of thyroglobulin subunits occurs to the same extent

Vo1.5, S0.3, 1968 G. VECCHIO, K. DOSE, and G. SALVATORE 42 7

whether or not the -SH groups in the irradiated and dissociated protein are blocked with iodoacetic acid. Although alkylation of the -SH groups was incomplete, the number of the -SH groups left in the alkylated protein (approx. 3 ) did not decrease after reassociation of about 60°/, of the 12 S units. This indicates that reassociation of the two halves of thyroglobulin is not dependent upon the formation of -S-S- linkages.

Table 4. Reassociation of irradiated thyroglobulin and alkylation of -ASH groups

For dissociation the protein solution (lo/o, w/v) was dialysed against low ionic strength buffer (see Methods) pH 5.9 and irradiated for 10 minutes a t 2537 A (accumulated dose 500 Einsteins per molg protein). Allrylation was by iodo- acetic arid a t pH = 8.0, molar ratio of alkylating agent: -SH in irradiated thyroglobulin = 240: 1. Reassociation was carried out by dialysis of dissociated protein against high ionic strength buffer (see Methods), pH 5.9. The amount of 12 S was calculated from the area of the 12 S schlieren peak (formed) as a percentage of the original non-dissociated 19 S. Reversal was the percentage of dissociated 12 S reverted

to 19 s Treatirient

No./molr "'0 prntcin " 0

- 71 - 10.0 2.8 75

+ + - + 29 59 5.8

+ + + + + 30 60 3.1

- - -

DISCUSSION The results obtained demonstrate that the ultra-

violet irradiation has a considerable effect on the molecular organization of human thyroglobulin. As would be expected, one of the most important effects produced is the cleavage of some of the I01 cystline disulfide bonds which are present in this protein. The quantum yield for cystine destruction is in keeping with the number of free -SH groups experimentally found. Since about 1.3 SH-groups are formed per cystine residue destroyed, the appearance of 20 -SH groups per mole of protein indicates the cleavage of approximately 15 out of the 101 -S-S- linkages present in the native molecule.

A crucial point was to ascertain whether or not the disulfide bridges broken by ultraviolet light were important in holding together the 6 S mononier units present in human thyroglobulin. The results obtained have shown that, under the experimental conditions described here, the ultraviolet light cannot selectively cleave the few interchain disulfides which link the 6 S subunits. The rupture of not less than 15O/, of the total number of disulfides in the pro- tein, in fact, does not yield subunits smaller than 12 S half molecules.

Our results demonstrate that thyroglobulin does dissociate into 12 S subunits after ultraviolet irra- diation at 2537 A. The quantum yield for thyro- globulin dissociation by ultraviolet light (ad) is of the order of but varies with ionic strength and pH. Actually, ultraviolet light alone is not able to induce more than 300/, dissociation of thyroglobulin : high percentages of dissociation are obtained only when the protein is irradiated in a medium which is able by itself to weaken the non-covalent interactions holding the protein subunits together, like a buffer of low ionic strength [20,21]. This means that the quantum yield for dissociation of thyroglobulin is a reciprocal function of the ionic strength, or: 0,~ oc (T/2)-l. If the 19 S dissociation reaction (i.e. 19 S + 2 12 S) is viewed as depending on ionic strength, since :

one has that oc e-APIRT. Consequently, the higher quantum yield ( 0 d ) values a t low ionic strength are explained by the smaller free energy of dissociation, i.e. fewer non-covalent bonds must be destroyed to form 12 S in low than in high ionic strength solu- tions.

The question arises as to the mechanism by which ultraviolet light of 2537 d facilitates the dissociation of 19 S into two halves. The mechanisms to be considered are different whether the 12 S units in human thyroglobulin are linked or not by disulfide bridges. It has been suggested for bovine thyro- globulin [2] that some of the I2 S units in this protein species may be linked by a few disulfides. While this situation may exist also in the case of normal human thyroglobulin, de Combrugghe et al. [24] and Andreoli et al. [25] have independently shown that this is not the case for human thyroglobulin isolated from simple goiter. From the studies presented here (which were performed with the same preparation used by An- dreoli et al. 1251) we have reached, by a different approach, a conclusion which is in agreement with that of the authors mentioned above. In fact :

a) The free -SH groups formed by irradiation of thyroglobulin are not significantly different in high or low ionic strength medium, whereas the extent of dissociation is significantly different in these two media (Table 2 ) .

b) The reoxidation of the free -SH groups formed during the ultraviolet irradiation is independent of the reassociation of thyroglobulin subunits (Table 3) : and conversely, the reassociation of thyroglobulin subunits takes places even if the -SH groups are blocked with an alkylating agent (Table 4) ; in other words, reassociation of thyroglobulin does not require the formation of interchain disulfide linkages.

If the 12s subunits are held together by non- covalent bonds, the most likely possibility is that

428 G. VECCHIO, K. DOSE, and G. SALVATORE: Ultraviolet Irradiation of Human Thyroglobulin Europcan J. Biochcm.

ultraviolet light directly or indirectly affects such bonds.

A mechanism proposed by Augenstine et al. [22], for the inactivation of trypsin by ultraviolet light involves the interaction of an excited disulfide bond “with a neighboring peptide grouping, possibly to extract a proton or hydrogen atom”, thus resulting in the disruption of a hydrogen bond. Other reac- tions, however, may lead to the disruption of hydro- gen and/or other non-covalent bonds. Secondary effects of the ultraviolet light on other aminoacids (tryptophan, tyrosine, phenylalanine, etc. ) cannot be completely ruled out, even though a quantum ab- sorbed by a cystine residue has greater probability of producing modification of the native structure of a protein than one absorbed by other residues of the molecule [23]. Actually, in the present study we have found a very good correlation between radiation dose and reduction of cystine residues provoked by the ultraviolet light. Since there is no evidence that units smaller than half molecules (12 S) are formed by ultraviolet irradiation of human thyroglobulin, it must be concluded that the disulfide bonds broken by the ultraviolet light are intrachain rather than inter chain.

In conclusion, in order to explain the dissociating effect of ultraviolet light on human thyroglobulin, the following mechanism can be envisaged : energy is absorbed by the cystine residues of the protein and the cleavage of some intrachain disulfide bridges alters the quaternary structure of the protein possi- bly affecting some neighboring hydrogen, or other non-covalent bonds which hold together the 12 S units. If the protein is dissolved in a medium of standard ionic strength, a small degree of dissocia- tion results, since other non-covalent interactions keep the 12 S subunits together. In a low ionic strength medium, fewer non-covalent bonds are present, and less quanta are needed to cause the same degree of dissociation.

The authors are deeply indebted to Professor J. Roche (Paris) for advice during the course of this work and to Dr. H. Edelhoch (Bethesda) for revising the manuscript. Mr. A. Montoro provided valuable technical assistance. The work was supported by P. H. S. Grant TW 00236-02 from the United States.

REFERENCES 1. Edelhoch, H., Recent Prop . Hormone Res. 21 (1965) 1. 2. de Crombruaehe. B.. Pitt-Rivers. R.. and Edelhoch. H.. , ,

J . Bid. CYhm.’241 (1966) 2766: ’

3. Edelhoch, H., and Rall, J. E.. in The Thvroid Gland (edited by R. Pitt-Rivers and W. R. Trottkr), Butter- worths, London 1964, Vol. 1, p. 113.

4. Ellman, G. L., Arch. Biochem. Biophys. 82 (1959) 70. 5. Pitt-Rivers, R., and Schwartz, H. L., Biochem. J . 105

6. Edelhoch, H., and de Crombrugghe, B., J . Biol. Chem.

7. Setlow, R., Biochim. Biophys. Acta, 16 (1955) 444. 8. Augenstine, L., Advan. Enzymol. 24 (1962) 359. 9. McLaren, A. D., Photophysiology, 1 (1964) 65.

(1967) 2812.

241 (1966) 4357.

10. Risi, S., Dose, K., Rathinasamy, T. K., and Augen- stine, L., Photochem. Photobiol. 6 (1967) 423.

11. Dose, K., Photochem. Photobiol. 6 (1967) 437. 12. Vecchio, G., Carlomagno, M. S., and Dose, K., Compt.

Rend. Soc. Bid. 161 (1967) 2412. 13. Salvatore, G., Salvatore, M., Cahnmann, H. J., and

Robbins, J., J . Biol. Chem. 239 (1965) 3267. 14. Andreoli, M., Califano, G., and Viscidi, E., Poliu Endo-

crinol. (Pisa) , 19 (1966) 269. 15. de Crombrugghe, B., Edelhoch, H., Beckers, C., and

de Visscher, M., J . Bid. Chem. 242 (1967) 5681. 16. Dose, K., Photochem. Photobiol. (19681, in press. 17. Pierce, J. G., Rawitch, A. B., Brown, D. M., and

Stanley, P. G., Bioehim. Biophys. Acta, 111 (1965) 247.

18. Bismuth. J., Rolland, M., and Lissitzky, S., Acta Endo- crinol. 53 (1966) 297.

19. Aloi. S.. Salvatore. G.. and Roche. J.. J . Biol. Chem. 222 (1967) 3810.’

20. Lundaren, H. P., and Williams. J. W., J . Phws. Chem. 43 T1939) 989.

21. Edelhoch, H., J . Biol. Chem. 235 (1960) 1326. 22. Augenstine, L. G., Ghiron, C. A., Grist, K. L., and

Mason, R., Proc. Nutl. Acad. Sci. U. S. 47 (1961) 1733.

23. Setlow, R., and Doyle, B., Biochinz. Biophys. Acta, 24 (1957) 27.

24. de’crombrugghe, B., Edelhoch, H., Beckers, C., and de

25. Andreoli, M., Sena, L., Edelhoch, H., and Salvatore, G., Visscher, M., personal communication.

unpublished observations.

G. Vecchio and G. Salvatore Istituto di Patologia Generale dell’UniversitA S. Andrea delle Dame 2, 1-80138 Napoli, Italy

K. Dose Max-Planck-Institut fur Biophysik BRD-6000 Frankfurt/Main, Kennedyallee 70, Germany