Iodination of fibrinogen

Iodination

of Fibrinogen

E. MihBlyP and K. L&i2 From the Medical From the National of Health, Public

Nobel Institute, Stockholm, Sweden and Institute of Arthritis and Metabolic Diseases, National Institutes Health Service, Federal Securily Agency, Bethesda i4, Maryland

Received September 19, 1951

INTRODUCTION

In the second stage of blood coagulation the soluble fibrinogen is transformed by the action of thrombin to the fibrin gel. The thrombin acts catalytically and its role is to modify the fibrinogen molecules which, without further participation of thrombin, polymerize to a three-dimensional network. Very little is known yet about what happens when thrombin alters the fibrinogen molecules and how these molecules are able to form the fibrin clot. The nature of chemical groups involved in the specific function of a protein can’be explored by modifying or removing certain of the characteristic groups of the protein molecule. One such modification is the iodination of the protein molecule. In this paper experiments are presented on the iodination of fibrinogen and fibrin and some of the properties of iodinated fibrinogen are described. The experiments dealing with the action of thrombin upon fibrinogen preparations iodinated to different extents will be published elsewhere. A short report of this work has already been published (1). MATERIALS AND METHODS Fibrinogen Armour bovine fibrinogen dissolved in 0.15 M sodium chloride solution was purified by precipitation at 0.24 ammonium sulfate saturation. The precipitate was dissolved in saline solution and dialyded against a large volume of 0.15 M sodium 1 Present address: Harrison Department of Surgical Research, University of Pennsylvania Medical School, Philadelphia 4, Pa. * Visiting Scientist of the National Institute of Arthritis and Metabolic Diseases. 97

98

E.

chloride solution. Usually these preparations.

about 90% of the protein

MIHtiLYI

AND

K.

LAX1

was clottable

by thrombin

in

Thrombin Hoffmann-IaRoche’s of 1 mg./ml.

commercial

thrombin

Iodination

preparation

was used in solutions

of Fibrinogen

Forty ml. of a fibrinogen solution (10 mg./ml.) was mixed with 40 ml. of borate buffer, pH 8.58 (equal volumes of 0.5 M boric acid and 0.125 M sodium tetraborate), and 100 ml. of 10 M urea solution. The mixture was prepared at O”C., and then 20 ml. of 0.1 N iodine (dissolved in 0.15 M potassium iodide) at the same temperature was added. At given intervals, samples of 20 ml. were withdrawn and the free iodine reduced by adding 1 ml. of 10% sodium thiosulfate solution. Urea and iodide ions were removed by dialysis, then the iodinated fibrinogen was precipitated by adding 0.1 vol. of 1 M acetate buffer, pH 5.3. The precipitate was redissolved in 10 ml. distilled water, which was brought to approximately pH 7.5 by the addition of a few drops of 0.1 N sodium hydroxide.

Iodination

of Fibrin

To 40 ml. of unbuffered fibrinogen solution (10 mg./ml., pH N 6.5) 1 ml. thrombin was added. Clotting occurred in about 2 min., and the gel was left at room temperature for 1 hr. The fibrin gel was dissolved at 0°C. by adding 100 ml. of 10 M urea solution, and iodinated exactly as was the fibrinogen.

Estimation

of Total Protein and Fibrinogen

The total protein concentration nitrogen protein conversion factor content of 16.9% of fibrinogen (2). the percentage of the total protein

Estimation

Concentration

was determined by the Kjeldahl method. A of 5.91 was used on the basis of a nitrogen The purity of the fibrinogen preparations was clottable by thrombin.

of Free and Bound Iodine

The free iodine concentration of samples of 5 ml. was determined by titration with 0.01 N sodium thiosulfate solution. One per cent starch solution was used as indicator, and approximately 0.1 g. of solid potassium iodide was added to each sample. The iodine bound to the protein was determined according to Hunter (3), with slight modification. The alkali fusion of the iodinated fibrinogen was dissolved in water, the solution slightly acidified, and oxidized with bromine water. The excess of bromine was removed by boiling and the cooled solution was acidified with phosphoric acid. Potassium iodide was then added. The iodine liberated was titrated immediately with 0.01 N sodium thiosulfate solution.

Estimation

of Tyrosine and Monoiodotyrosine

The hydrolysis of the iodinated products was performed in 5 N NaOH (4), and the tyrosine and monoiodotyrosine were estimated by the method described by Lugg (5). The optical densities of the colored solutions were determined at two different wavelengths with the Beckman spectrophotometer, and the amounts of

IODINATION

tyrosine and monoiodotyrosine and hlichel (6).

OF

were calculated from the data according to Roche

EXPERIMENTAL

Iodination

99

FIBRINOGEN

AND

RESULTS

of Fibrinogen

and Fibrin

Low temperature and an alkaline reaction were used to promote substitution. The reaction was conducted in 5 M urea solution to make possible the comparative investigation of fibrinogen and fibrin in homogeneous media. Under these conditions the iodine consumption in a fibrinogen-free blank was negligible. Table I shows that the amount of iodine bound in the first 2 min. of the reaction is only a few per cent lower than half of the free iodine which disappears. The reaction in the first few minutes is thus mainly a substitution. Later on the iodine consumption increasingly exceeds the double amount of the bound iodine; thus a considerable amount of iodine is used up for oxidation. TABLE

The Consumption

I

of Iodine

The iodine values are expressed as gram atoms of iodine per lo5 g. of the native fibrinogen. The tyrosine and monoiodotyrosine values are expressed as molecules per lo6 g. of the native fibrinogen. The experiment numbers denote individual experiments, each performed with a different fibrinogen preparation, Total iodine consumed

= I

Iodine bound (sp&r. analysis)

Tyrosine

1

3

4

1

1

4

5

5

_-

____

y;time min.

1 2 10 20 40 60 120

180

.... .

‘&S

102.8 158.6 146.7 186.6 182.7 223.7 222.6 247.5 241.5 284.3 277.4 302.3 294.8

.

..

101.4 154.1 189.6 ..... 251.6 289.7 .....

18.6 45.1 62.6 68.3 76.4 74.1 85.2 90.6

21.1 ....

i,:;

79:5 84.0 86.7 88.9

67.0 76.3 84.1 86.6 88.0 88.0

. . 49.6

.... 46.9

67.1 76.7 86.4 89.5 90.4

67.4 74.7

.. . 14.3 9.3 7.8

i

.

The reactivity of fibrin is somewhat lower in the initial stage of the reaction, but the differences are within the limits of experimental

100

E.

MIHtiLYI

AND

K.

LAKI

error. After 20 min. of iodination these differences disappear completely, the final iodine consumption and the iodine bound maximally being equal in fibrinogen and fibrin. With none of the iodinated fibrin preparations was the clot reconstituted when the urea was removed by dialysis. Solubility and Heat Stability of the Products Iodination for 2 min., where mainly substitution decreased the solubility of fibrinogen. At an ionic iodinated fibrinogen is completely precipitated, protein is precipitated at 2.5. After longer periods

occurred, markedly strength of 0.5 the whereas the native of iodination higher

FIQ. 1. Sedimentation diagram of iodinated fibrinogen dissolved in water. Average speed: 59,780; time after reaching speed: 32 min.

salt concentrations are necessary to cause precipitation and finally the preparations will not precipitate even at half saturation of sodium chloride. The iodinated fibrinogen, even after only 2 min. of treatment, is not coagulable by heat. Figure 1 shows the sedimentation pattern of an iodinated fibrinogen (about 7% of the tyrosines in the diiodotyrosine form) dissolved in water. It can be seen that except for a small amount of heavy component the preparation is homogeneous, indicating that the iodination did not result in a polydisperse material.3 The Development of Yellow Color The fibrinogen solutions which were subjected to the action of iodine for longer than 10 min. gave a visible green color when the excess of 8 We are indebted to Dr. W. R. Carroll, Laboratory of Physical Biology, National Institutes of Health, for the ultracentrifuge experiments.

IODINATION

OF

FIBRINOGEN

101

free iodine was reduced, and turned yellow after 5 to 10 min. The yellow color is not due to free iodine and did not disappear on addition of sodium bisulfite, hydrosulfite, ascorbic acid, or other strong reducing agents. It was firmly bound to the protein and could not be separated from it by boiling for 10 min. in 0.1 N hydrochloric acid, 0.1 N potassium hydroxide, or by dialysis. The extinction coefficients of the yellow solutions at 470 rnp run parallel with the fraction of iodine used for oxidation, indicating that the color develops under the oxidative effect of iodine. The Change in Ultraviolet Spectrum The absorption spectra at pH 9.18 of the fibrinogen preparations changes with the extent of iodination. A new peak appears at 312 rnp and increases with the duration of the iodination. The original maximum at 279 mp increases but little. Finally there is a minimum of absorption in this region. It was shown by Herriott (7) that the absorption maximum is at 305 rnp for monoiodo- and 312 mp for diiodotyrosine, with a minimum for the latter around 280 mp. At this pH the differences between the iodinated and the native protein are more apparent because the diiodotyrosine residues are completely dissociated, their pK being 6.5 (8), whereas the unsubstituted tyrosine residues, with a pK of 11.7 in the native protein (9), are in the undissociated form. The spectral changes reveal the formation of diiodotyrosine groups. The height of the absorption peak at 312 rnp is proportional to the number of iodine atoms bound by the fibrinogen ‘molecule, as is shown in Fig. 2. The determination of the optical density at this wavelength provides a way to estimate the number of bound iodine atoms. Fibrinogen and the equivalent quantity of tyrosine (calculated on the basis of 8.0% tyrosine in fibrinogen) gave the same final density at 312 rnp when iodinated maximally. DISCUSSION

As the result of iodination the following changes were observed on fibrinogen: change in solubility, development of yellow color, and change in the ultraviolet spectrum. The effect of iodine is essentially twofold. Part of iodine oxidizes certain groups of the protein and part of the iodine becomes organically bound. The organically bound iodine is mainly a substitution. The change in solubility would be the result of oxidation or substi-

102

E.

MIHALYI

AND

K.

LAKI

tution. The development of yellow color is undoubtedly due to oxidation. Very likely the tyrosine residues and perhaps tryptophan residues are involved in this reaction. The change in the ultraviolet absorption is undoubtedly mainly due to the iodination of the tyrosine residues. In a number of proteins it has been reported that the substitution occurs exclusively in the tyrosine ring (lo-13), but in other cases (14-20) the iodine content of

5 so5

0

0.2

LOG $ FIG.

against N/ml.

2. The number

the

extinction

I 0.4

AT

I 0.6

I 0.8

312

M,k;

I 1.0

I 1.2

0.1 bjG.

I 1.4

N /

I 1.6

I 1.8

ML.

of gram atoms iodine bound per lo6 g. fibrinogen at 312 rnp of a solution of fibrinogen containing

plotted 0.1 mg.

the iodinated protein was considerably higher than would be accounted for by the iodination of tyrosyl residues [for comprehensive reviews, see Herriott (21) and Olcott and Fraenkel-Conrat (22)]. In the case of fibrinogen the amount of bound iodine exceeds by about 25% the amount needed for the substitutions into the tyrosine ring. Fibrinogen bound maximally 89 g. atoms of iodine per lo6 g. of protein. If all the iodine entered the tyrosine rings, forming diiodotyrosine residues, the above figure would correspond to 8.0% tyrosine content in fibrinogen. This figure is considerably higher than those reported in the literature.

IODINATION

OF FIBRINOGEN

103

By calorimetric (23), photometric estimation (2,24,25), and by microbiological assay (26,27), different authors found in bovine fibrin tyrosine values ranging from 5.5 to 6.5%. Since it was found that the organically bound iodine can be calculated from the optical densities at 312 rnp, the data could point to a higher tyrosine content in fibrinogen or to other groups contributing to the optical densities at 312 rnp. No attempt was made in this work to settle this point. SUMMARY

The iodination of fibrinogen was investigated. Conditions were applied which favor the substitution of iodine. The iodination caused a marked change in the solubility of fibrinogen. The total amount of iodine bound was found to be equal in fibrinogen and fibrin. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27.

LAKI, K., AND MI~LYI, E., Nature 163, 66 (1949). BRAND, E., KASSELL, B., AND SAIDEL, L. J., J. CZin. Invest. 28, 437 (1944). HUNTER, A., J. Biol. Chem. 7, 321 (1909-10). LUGG, J. W. H., Biochem. J. 32, 775 (1938). LUGG, J. W. H., Biochem. J. 31, 1422 (1937). ROCHE, J., AND MICHEL, E., Biochim. et Biophys. Acta 1, 335 (1947). HERRIOTT, R. M., J. Gen. Physiol. 31, 19 (1947-8). DALTON, J. B., KIRK, P. L., AND SCHMIDT, C. L. A., J. Biol. Chem. 88, 589 (1930). TANFORD, CH., J. Am. Chem. Sot. 74,441 (1950). HERRIOTT, R. M., J. Gen. Physiol. 20, 335 (1936-7). NEUBERGER, A., Biochem. J. 28, 1982 (1934). HARINGTON, C. R., AND NEUBERGER, A., Biochem. J. 30, 809 (1936). LI, C. H., J. Biol. Chem. 139, 43 (1941). HUGHES, W. L., JR., AND STRAESSLE, R., J. Am. Chem. Sot. 72,452 (1950). MUNS, J., COONS, A. H., AND SALTER, W. T., J. Biol. Chem. 139, 135 (1941). BLUM, F., AND STRAUSS, E., 2. physiol. Chem. 112, 111 (1920-l). BAUER, H., AND STRAUSS, E., Biochem. 2. 211, 163 (1929). BONOT, A., Bull. sot. chim. biol. 21, 1417 (1939). HAUROWITZ, F., SARAFIAN, K., AND SCHWERIN, P., J. Immunol. 40,391 (1941). SHAHROKH, B. K., J. BioZ. Chem. 161, 659 (1943). HERRIOTT, R. M., Advances in Protein Chem. III, 177-205 (1947). OLCOTT, H. S., AND FRAENKEL-CONRAT, H., Chem. Revs. 41, 182 (1947). FOLIN, O., AND LOONEY, J. M., J. BioZ. Chem. 61, 421 (1922). BRAND, E., AND KASSELL, B., J. Biol. Chem. 131, 489 (1939). BAILEY, K., Advances in Protein Chem. I, 289-317 (1944). GALE, E. F., Biochem. J. 39, 46 (1945). HIER, S. W., GRAHAM, C. E., FREIDES, R., AND KLEIN, D., J. Biol. Chem. 161, 705 (1945).