Differential spectrophotometry of iodoamino acids and iodinated proteins Aqueous ethylene glycol solutions of iodotyrosines, thyroxine and thyroglobulin

381

Biochimica et Biophysica Acta, 623 (1980) 381--388 © Elsevier/North-Holland Biomedical Press

BBA 38436

DIFFERENTIAL SPECTROPHOTOMETRY OF IODOAMINO ACIDS AND IODINATED PROTEINS AQUEOUS ETHYLENE GLYCOL SOLUTIONS OF IODOTYROSINES, THYROXINE AND THYROGLOBULIN

ISKANDER G. AKHMEDZHANOV, EUGENE E. GUSSAKOVSKY and TULKUN A. BABAEV

Institute of Biochemistry, Uzbek Academy of Sciences, Tashkent, 700125 (U.S.S.R.) (Received August 15th, 1979)

Key words: Iodoamino acid; Iodinated protein; Differential spectrophotometry ; Ethylene glycol

Summary Solvent perturbation difference spectra and corresponding molar extinction coefficients of solutions of monoiodotyrosine, diiodotyrosine and thyroxine with protonated and deprotonated hydroxyls were measured with 20% ethylene glycol as perturbant. Data obtained were applicable to the development of the method of quantitative estimation of the number of iodotyrosine and thyroxine residues accessible to ethylene glycol in iodinated proteins. This method was used for the determination of iOdoamino acids available to ethylene glycol in bovine thyroglobulin (naturally iodinated protein).

Introduction Both naturally and artificially iodinated proteins can contain iodotyrosines such as thyroxine [1--4]. The problem of finding iodoamino acid residues in iodinated proteins accessible to solvents is of great importance for the investigation of their physico-structural properties. The method of solvent perturbation difference spectrophotometry has been sufficiently developed to be applied to quantitative estimation of tyrosine and tryptophan residues accessible to perturbant in proteins [5,6]. Some forms, such as iodotyrosines and thyroxine, are expected to exhibit distinctive solvent perturbation difference spectra under the same conditions as found for unsubs~ituted tyrosines which could be distinguished from the latter because of the closeness of their molar extinction coefficients [7--9].

382 The purpose of the present study is to measure such spectra for monoiodotyrosine, diiodotyrosine and thyroxine solutions with 20% ethylene glycol as perturbant, and to use the results obtained for the direct location of the number of iodoamino acid residues available to perturbant in thyroglobulin, which is a naturally iodinated protein [1]. Materials and Methods In the course of investigations the following preparations were used: thyroxine (Reanal), diiodotyrosine (Chemapol), tyrosine, ethylene glycol and urea (Reakhim). Urea was twice recrystaUized from 70% ethanol. Ethylene glycol was purified by distillation. Monoiodotyrosine was obtained by the iodination of tyrosine at pH 11.5 and at room temperature by an aqueous solution of 0.48 M KI and 0.04 M I2. The formation of monoiodotyrosine was checked by examination of the absorption spectrum [9]. Thyroglobulin was isolated from the bovine thyroid b y gel filtration of salt extracts of thyroid tissues on Sephadex G-200 [10,11], and was a gift f r o m A.A. Nalbandyan (Institute of Biochemistry, Uzbek Academy of Sciences). The concentration of thyroglobulin /A1% was determined spectrophotometrically ~ l c m = 10.0, pH 9.0), taking into account the contribution of light scattering to the measured spectrum. The absorption of iodoamino acids and thyroglobulin solutions was measured with a recording spectrophotometer (EPS-3T, Hitachi) using four silica cells with an optical pathlength of 10 mm [6]. In order to increase the accuracy of differential absorbance measurements, differential transmittance spectra were measured with subsequent recalculation to absorbance with the help of four-figure logarithmic tables. The absolute error of differential transmittance was 0.002, corresponding to an absolute error o f the differential absorbance of 0.001. The spectra were measured at five or six concentrations of iodoamino acids for the purpose of determinating differential molar extinction values. Differential absorbance has a linear dependence on the iodoamino acid concentration. Hence, the results obtained were processed by means of a least-squares analysis for the linear function. The concentration of iodoamino acids in alkaline solution was determined b y spectrophotnmetric examination, using molar extinction coefficients for monoiodotyrosine, diiodotyrosine and thyroxine of 4.10, 6.25 and 6.20 • 103 M-' • cm-', respectively, at maximum absorbance [12]. Results and Discussion Solvent perturbation difference spectra of monoiodotyrosine, diiodotyrosine and thyroxine solutions with 20% ethylene glycol as perturbant [5,6] and pH lower or higher than the pK of dissociation of their hydroxyls are shown in Figs. 1--3. It was impossible to obtain solvent perturbation difference spectra of the thyroxine solution at pH values lower than the pK of hydroxyl because of its poor solubility under these conditions, pK values of the deprotonation of monoiodotyrosine, diiodotyrosine and thyroxine in 20% ethylene glycol are comparable to the values for aqueous solution as spectrophotometrically established. When pH is lower than pK of the deprotonation of hydroxyls, sol-

383

.,Jg.10- 2(M- 1 cm-1) 2C 1.6 12

.,~E.10-2(M-1 cm-1 ) 2C

08

L

A

0.4

I,(~ 1.2

4.(

08

3.6

Q4

j 2

B

32 i

2.8

i

2

24

20

2£

16 1.2

12

o.e

08 0.4

0.4

i

300

340

380

420

A.(nm)

280

i

320

360

400

440 A(nm)

Fig. I . S o l v e n t p e r t u r b a t i o n difference spectra o f 3 - m o n o i o d o t y r o s i n e at PH 4.6 ( A ) and pH 1 0 . 0 (B) due to 20% e t h y l e n e glycol. Curve 1 is a q u e o u s e t h y l e n e g l y c o l s o l u t i o n against a q u e o u s s o l u t i o n . Curve 2 is 8 M u r e a / e t h y l e n e g l y c o l s o l u t i o n against 8 M urea s o l u t i o n . Fig. 2. S o l v e n t p e r t u r b a t i o n difference spectra o f 3 , 5 - d i i o d o t y r o s i n e at pH 3.9 ( A ) and pH 9.1 (B) due t o 20% e t h y l e n e glycol. Curves 1 and 2, see legend to Fig. 1.

vent perturbation difference spectra of monoiodotyrosine and diiodotyrosine are characterized by major and minor maxima; the positions and differential molar extinction coefficient values (Ae) are listed in Table I. Solvent perturbation difference spectra of monoiodotyrosine and diiodotyrosine are similar to that of tyrosine, and Ae values are comparable (cf. Refs. 5 and 6). This similarz/~:. 10- 2 ( f',4-~ cm-~ ) 2.8 2.4 20

2

1.E 1..~ 0.,5 0.,4 280

320

360

400

.~(nm)

Fig. 3. S o l v e n t p e r t u r b a t i o n difference spectra o f t h y r o x i n e at p H 9.6 d u e to 20% e t h y l e n e glycol. Curves 1 and 2, see legend t o Fig. 1.

384 TABLE

I

MOLAR EXTINCTION COEFFICIENTS (Ae) FOR SOLVENT PERTURBATION DIFFERENCE SORPTION OF IODOAMINO ACIDS WITH 20% ETHYLENE GLYCOL AS PERTURBANT

AB-

A e v a l u e s e x p r e s s e d a s M -1 • c m -1 , k m a x v a l u e s i n n m . Iodoamino

acid

pK *

Monoiodotyrosine

8.2

Diiodotyrosine

6.4

pH

4.6 10.0 3.9 9.1

Thyroxine

6.7

9.6

Water

Urea (8 M)

~e

Xma x

~e

kma x

51 ± 5 113 ± 10 136 ± 5

286.5 294.0 317.0

149 ± 9 199 ± 13 201 ± 13

286.5 294.0 318.0

104 206 186 170

289.0 299.0 327.0 314.0-320.0

194 221 368 301

6 7 4 4

289.0 299.0 330.0 319.0

347.0 291.0

286 ± 23 200 ± 16

350.0 291.0

± 5 ± 10 ± 12 ± 11

211 ± 127 ±

8 5

± ± ± ±

* S e e R e f s . 8 a n d 9.

ity is due to the fact that monoiodotyrosine, diiodotyrosine and tyrosine each have the same aromatic system, which determines the properties of electronic transitions. The position of solvent perturbation spectral peaks in the difference spectra of monoiodotyrosine and diiodotyrosine at longer wavelength to that observed with tyrosine seems to reflect the positioning and extent of iodine substitution. A somewhat higher Ae value of monoiodotyrosine and diiodotyrosine as compared to the tyrosine value (Ae = 92.1 M -1 • cm -1 [6]) is probably connected with the electron-withdrawing nature of the iodine atom, thus facilitating a weak perturbance of electronic levels under the influence of ethylene glycol. This fact is supported b y a higher Ae value for diiodotyrosine in comparison with monoiodotyrosine. Solvent perturbation difference spectra of monoiodotyrosine and diiodotyrosine are red-shifted at the deprotonation of the hydroxyls. In this case, the intensity of the minor peak greatly decreases. If the value for m o n o i o d o t y r o sine increases by 20%, then the value for diiodotyrosine decreases by 10%. This difference is possibly due to the difference in symmetry of 7r-electron molecular systems. Solvent perturbation difference spectra of thyroxine at pH > pK have two maxima and a weak shoulder in the wavelength region 330--332 nm. The Ae value of the major maximum is 13% higher than that of diiodotyrosine. This increase can be explained by the influence of iodine atoms in the ~-ring of the iodothyronine structure upon the 7r-electron density distribution in the whole system, the electronic transition of which is designed as L -~ a~ according to Kasha's symbolism [13]. This is similarly due to the increase in Ae when tyrosine changes to monoiodotyrosine and diiodotyrosine. For quantitative analysis of the number of amino acids available to a perturbant in a protein it is necessary to measure the solvent perturbation difference spectra under conditions in which all the aromatic amino acids are accessible to

385 the solvent [5,6]. This is generally achieved b y unfolding the protein molecule in 8 M urea. However, the alteration of the Ae value o f amino acids at the transition in 8 M urea solution must be taken into account. Hence, we have measured solvent perturbation difference spectra of iodoamino acids in such a solvent. The influence of 8 M urea upon the solvent perturbation difference spectra of all iodoamino acids studied mainly manifests itself as a significant increase in Ae values (see Table I and Figs. 1--3). Urea does not cause an additional shift of the monoiodotyrosine absorption spectrum, b u t it shifts the solvent perturbation difference spectra of diiodotyrosine and thyroxine b y 3 nm. This indicates that urea is also an effective perturbant of iodoamino acid absorption spectra. Thus, it is necessary to bear in mind the differences in differential molar extinction coefficients caused b y urea in the quantitative determination o f monoiodotyrosine, diiodotyrosine and thyroxine residues available to a perturbant in iodinated proteins. As tryptophan, tyrosine, monoiodotyrosine, diiodotyrosine and thyroxine are present in thyroglobulin which is the main naturally iodinated protein of the thyroid gland [1], then the solvent perturbation difference spectrum of thyroglobulin must be characterized b y bands of these aromatic amino acids. Solvent perturbation difference spectra of native and denaturated b y 8 M urea thyroglobulin are shown in Fig. 4. These spectra are characterized b y t w o major maxima in the ranges 285--286 and 291--292 nm, as well as b y a wide band in the range 310--360 nm which can be ascribed to tryptophan, tyrosine [5,6] and iodoamino acids, respectively. The presence of these bands in the spectrum of native protein gives qualitative indication of the distribution of residues available to perturbant. The intensity increase when urea is added to the solution demonstrates the presence of aromatic amino acid residues not available for the perturbant in native thyroglobulin molecules. When performing quantitative analysis, one should take into consideration that solvent perturbation difference spectra of monoiodotyrosine, diiodo-

240

z:/~. 10- 2( M-1 cm-1 )

20c 16c 2

12c 8c 40

' 260 ' 280

.~.(nrn)

Fig. 4. S o l v e n t p e r t u r b a t i o n d i f f e r e n c e s p e c t r a o f b o v i n e t h y r o g l o b u l i n at p H 9.0 d u e t o e t h y l e n e glycol. Curves 1 a n d 2, s e e l e g e n d t o Fig. 1.

386

tyrosine and thyroxine depend upon pH values 6--9 because pK values of their hydroxyl deprotonations are in this region. If a solvent perturbation difference molar spectrum of protein is assumed to be a superposition of amino acid residue spectra:

Ae~o=

E

g, Ae[

(1)

then the subscript, i, of linear equation system 1 must be determined b y the pH of the solution. At pH values higher than 9 it is equal to 3 because, in this case, hydroxyls of external residues of iodoamino acid must be deprotonated. These residues have a maximum absorption in the wavelength range above 300 nm (Figs. 1--3) where the differential molar extinction coefficients of tyrosine and tryptophan are small. At pH values below 6--7, hydroxyls of external monoiodotyrosine, diiodotyrosine and thyroxine residues would be protonated and their solvent perturbation difference spectra would be greatly overlapped with tyrosine and t r y p t o p h a n spectra. System 1 would be described with i = 5 as five amino acids are to be determined. In the present study the solvent perturbation difference spectra of thyroglobulin were measured at pH values of approx. 9.0. In order to calculate the portion of iodotyrosine and thyroxine residues available to ethylene glycol, it is sufficient to solve the system of the following linear equations: Ae~ 17-318 = C l ~ e p 7-318 + C2Ae~ 17-318 + C3~e 317-31s3 Ae327-330 _- C1Ae327-330 + C2Ae2327-330 + C3Ae~ 27-330

(2)

Ae~ 47-3s° = C 1Ae34?-3s°+l C2 Ae 347-3s°2 + C3Ae3347-3 s°

where Aelx , Ae} and Ae} are the differential molar extinction coefficients of monoiodotyrosine, diiodotyrosine and thyroxine, respectively; Ci is the molar ratio of the concentration of corresponding iodoamino acid residues available to a perturbant at the protein concentration. If we know the Aeix values we can determine the C/ values b y the measurement of Ae0~ by the use of Gauss' m e t h o d [14] of solving system 2. Substituting the values from Table I into system 2, we obtained recurrent methods for the determination of the value of Ci for the aqueous solution of iodinated protein: C1 = 0.01476Ae0317 -- 0.01529Ae0327 + 0.00474Ae~ 47 C2 = 0.00668Aeo317 -- 0.00190Ae0347 -- 0.85754 Cl

(3)

C3 = 0.00474Aeo347 -- 0.12932 C, -- 0.34202 C2 and for the 8 M urea solution of iodinated protein: C, = 0.00956Aeo31s -- 0.00810Ae~ 3° + 0.00077Ae035° C2 = 0.00450Ae03'8 -- 0.00283Ae03s° -- 0.78957 C1 C3 = 0.00350Aeo3s° -- 0.14490

C 1 --

0.44029 C2

(4)

387 TABLE II QUANTITIES OF IODOAMINO ACID, TYROSINE AND TRYPTOPHAN ETHYLENE GLYCOL IN BOVINE THYROGLOBULIN

RESIDUES AVAILABLE TO

S p e c t r o p h o t o m e t r i c titration data w e r e o b t a i n e d b y the m e t h o d o f E d e l h o c h [ 1 2 ] .

A m i n o acid

S o l v e n t p e r t u r b a t i o n differene spectrophotometry Water

Tyrosine Tryptophan Monoiodotyrosine Ditodotyrosine Thyroxine

76.1 27.9 2.2 5.6 1.0

+ + ± • •

Spectrophotometric titration

Urea (8 M) 6.8 3.9 0.5 1.3 0.4

115.0 50.6 8.1 6.4 2.2

+ ± ± • •

7.1 4.4 1.4 1.3 0.7

110.5 69.0 [15] 9.8 6.9 2.7

The relative error in the determination of Ci values with the help of these methods is equal to the relative errors in the determination of Ae0~ values for the protein solution, which is proportional to the error in differential absorbance measurement. Data concerning the quantitative determination of tyrosine, t r y p t o p h a n and iodoamino acid residues in native bovine thyroglobulin obtained according to systems 3 and 4 are presented in Table II. For purposes o f comparison, the compositions of iodoamino acid and tyrosine residues, obtained b y means of the spectrophotometric titration m e t h o d [12], are also shown. Mean-square deviations, mentioned in Table II, include the error in differential absorbance measurement as well as the divergence of all the studied patterns of bovine thyroglobulin. The relatively large deviation obtained b y us is mainly due to the divergence of data on the patterns as the iodoamino acid composition of thyroglobulin varies according to the physiological state of the thyroid gland [ 1 ]. The above-mentioned values of quantities of tyrosine and iodoamino acid residues correspond to some 'average' protein. The coincidence in the range of mean-square deviations of data relating to the iodoamino acid composition of bovine thyroglobulin, obtained b y means of t w o methods, demonstrates the validity of the use of the recurrent methods 3 and 4 used to find the iodoamino acid residue numbers available to the perturbant. The results given in Table II show that approx. 40 tyrosine residues, 6 m o n o i o d o t y r o s i n e residues and 1 thyroxine residue are not'available to ethylene glycol in native bovine thyroglobulin. Data, mentioned in the present paper, show that solvent perturbation difference s p e c t r o p h o t o m e t r y of iodoamino acids can be successfully applied to the determination of the nunber of 'external' and 'buried' residues of not only tyrosine and tryptophan, b u t also of iodoamino acid residues such as monoiodotyrosine, diiodotyrosine and thyroxine. The application of other perturbants differed from ethyleneglycol in both the range and the mechanism of spectrum perturbation and should make it possible, more conclusively, to investigate external and buried iodoamino acid residues in iodinated proteins. This m e t h o d may be generally useful for the determination of iodoamino acids in iodinated proteins.

388

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