Simultaneous isolation and determination of prothymosin α, parathymosin α, thymosin β4, and thymosin β10

ANALYTICAL

BIOCHEMISTRY

l&,436-440

(1985)

Simultaneous Isolation and Determination of Prothymosin Parathymosin CX,Thymosin ,&, and Thymosin ,&’

CX,

A. A. HARITOS,* JANET CALDARELLA, AND B. L. HORECKER Roche Institute of Molecular Biology, Roche Research Center, Nutley* New Jersey 07110 Received July 16, 1984 A method was described for the isolation of peptides from rat thymus. Frozen, powdered tissue was suspended in boiling buffer to inactivate endogenous proteinases, the suspension was homogenized, and the peptides were isolated by a two-step procedure including gel filtration and purification by HPLC. The recoveries from rat thymus were, in micrograms per gram of whole tissue, 60-80 for prothymosin a, 50-80 for thymosin &, and 20-30 for thymosin &,. The procedure also yielded smaller quantities of a fourth peptide, designated parathymosin a. The quantities of these peptides in vertebrate tissues can be evaluated by applying radioimmunoassays for prothymosin rx and thymosin & to the boiled tissue extract. @ 1985 Academic

Thymosins are a class of peptides originally isolated from calf thymus (l), particularly from calf thymosin fraction 5 (2) which is a mixture of peptides that has been reported to be effective in the treatment of patients with certain immunodeficiency diseases (3). Thymosin fraction 5 has also been examined for its ability to restore cellular immunity in cancer patients receiving radiotherapy and chemotherapy (4). Thymosin aI, the first peptide to be isolated from calf thymosin fraction 5 (5), has since been shown to be a fragment of a larger native polypeptide, itIr 12,600, designated prothymosin CI (6). The richest source of prothymosin a is the thymus gland, but it has also been isolated from a number of other rat tissues (7). Thymosin &, the second peptide isolated from calf thymosin fraction 5 (S), has been shown to be widely distributed in tissues of vertebrates (9) [for a review see (IO)], and in mammals it is always accompanied by a second related peptide, either thymosin & in ’ Dedicated to Professor Karl Decker on the occasion of his sixtieth birthday. ’ On leave from the Zoological Laboratory, Faculty of Science, University of Athens, Athens 62 I, Greece. 0003-2697185 $3.00 CopyrigJe @ I985 by Academic Press. Inc. All rights of reprcduction in any form reserved.

436

the calf (11) or thymosin PI0 in the tissues of most other vertebrate classes (12). We report here a procedure for the simultaneous isolation of four peptides from rat thymus, including prothymosin a, thymosin fl.,, thymosin &, and a newly identified peptide, similar in size and composition to prothymosin a, named parathymosin a. For three of these peptides, prothymosin a, thymosin &, and thymosin &, the quantities in the tissues can be estimated by radioimmunoassay of the tissue extracts. MATERIALS

AND METHODS

iMaterials. These were from sources as described (6). All solvents used were chromatography grade. Rat thymuses from male Charles River CD rats, 5-6 weeks old, were excised immediately following sacrifice of the animals by decapitation, quickly frozen in liquid N2, and stored at -7OYZ. Methods. Protein and peptide determinations were carried out after alkaline hydrolysis as described by Lai (13). The HPLC separations were carried out with an Altex Ultrasphere ODS C 18 column, using an apparatus equipped with a Waters Associates Model

SIMULTANEOUS

ISOLATION/DETERMINATION

720 system controller and Model 7lOB intelligent sample processor, adapted for fluorescence detection after derivatization with fluorescamine ( 14). Amino acid analyses were carried out on samples hydrolyzed with redistilled 5.7 M HCl at 150°C for I h, using a Glenco MM70 amino acid analyzer adapted for derivatization with d-phthalaldehyde and fluorescence detection as described by Benson and Hare (15).

I

OF THYMOSINS

437 1

I

RESULTS

Isolation of Peptides Extraction from rat thymus. The frozen rat thymuses were pulverized, the powdered material was heated at 100% and the extracts

FRACTION NUMBER FIG. I. Separation on Sephacryl S-200 of peptides in an extract of boiled rat thymus. In the experiment shown, 7 g powdered frozen thymus was boiled in 100 ml 0.1 M Na phosphate buffer, pH 7.0, and the extract was desalted on a SepPak cartridge as described (6). Chromatography was as described in the text at a flow rate of 8,6 ml/h, and 0.86-m] fractions were collected. Aliquots (IO ~1) were lyophylized, hydrolyzed with alkali, and analyzed with fluorescamine (12). Fractions in peaks a and ,!I were pooIed, as shown by the horizontal bars, and combined with corresponding fractions from three similar column separations.

ELUTION TIME hid

FIG. 2. Separation of prothymosin cxand parathymosin a by reverse-phase HPLC. The fractions from peak a (pooled as described in Fig. I) were iyophyhzed, and the residue was dissolved in 900 ~1 of buffer A (I M HCOOH, 0.2 M pyridine). The HPLC experiments (see Materials and Methods) were carried out with 150~~1 ahquots of this solution. Elution was with a gradient of I-propanol in buffer A as shown. Fractions (0.6 ml) were collected every minute, and 5-~1 ahquots were diverted at 6-s intervals for analysis with fluorescamine. In the experiment shown, fractions 49 and SO in peak II, corresponding to prothymosin a, were pooled and combined with similar fractions from five other HPLC experiments. Parathymosin a was purified by rechromatography of fractions 53 and 54 from peak III.

were prepared and desalted on Sep-Pak cartridges (Waters Associates) as previously described (6). Separation on SephacryI S-200. The SepPak eluates from 7 g of rat thymus were lyophylized and the viscous residue was dissolved in 0.6 ml of buffer A (1 M HCOOH, 0.2 M pyridine, pH 2.8). The solution (0.8 ml) was transferred to a column (1.5 X 89 cm) of Sephacryl S200 superfine (Pharmacia) previously equilibrated with buffer A, and the peptides were eluted with the same buffer (Fig. 1). Pur$cation of peptides by HPLC. Peak CY (see Fig. 1), fractionated by HPLC as previously described (6), yielded three major peptides, eluting at approximately 8.5, 13, and 14% n-propanol, respectively (Fig. 2). Rechromatography of the second peak showed it to contain essentially pure prothymosin a

HARITOS,

CALDARELLA,

AND

HORECKER

ELUTION TIME fminl

FIG. 3. HPLC profiles of the purified prothymosin a and thymosins /3,, and pi,,. (A) Protbymosin a (44 pg) from the preparation shown in Fig. 2. (B) Thymosin fl q0xj (20 pg) from the preparation shown in Fig. 4B. (C) Thymosin /3iW0xi (2 I pg) from the preparation shown in 4B. The HPLC analyses were carried out as described in Figs. 2 and 4.

(Fig. 3, Table 1). The third peak in Fig. 2, eluting at 14% n-propanol, contained a previously unidentified peptide, similar in size and amino composition to prothymosin o (unpublished observations). It has tentatively TABLE

I

AMINOACIDCOMP~SITIONOFF'ROTHYMOSIN~ THYMOSINS &AND &,,~oM RATTHYMUV Prothymosin Asx Thr Ser Glx GlY Ala Val Met Ile Leu Phe LYS A% Pro

24.2 6.3 3.5 39.0 10.5 10.1 5.6 1.0 1.1 8.9 2.1 2.3

a

Thymosin 4.0 2.7 3.7 11.0 1.5 2.2 0.1 0.9 1.9 2.0 I.0 9.4 0.2 3.0

& (4) (3) (4) (11) (I) (2) (0) (I) (2) (2) (1) (9) (0) (3)

AND

Thymosin 3.7. 4.5 3.1 9.8 1.4 2.8 1.0 2.6 2.0 1.0 8.3 0.8 2.1

pi0 (4) (5) (3) (9) (I) (3) Kv (I) (3) (2) (I) (8) (0)b (2)

a The values shown were obtained after acid hydrolysis as described under Materials and Methods. The numbers in parentheses are derived from the known sequences of thymosin & (8) and thymosin & (I 1). ’ Arginine was a common contaminant in preparation of thymosin fiiO (1 I) but was not found during the sequence analysis. A variant of thymosin &,, containing an additional arginyl residue, has been isolated from rabbit tissue ( 16).

been named parathymosin a and its structure is under investigation. The peptides in peak /3 (see Fig. l), separated using the same HPLC system, were identified from their elution positions (Fig. 4A) and amino acid compositions (Table 1) as the oxidized and reduced forms of thymosin &, and thymosin /3,,,. This was confirmed by HPLC of another aliquot of peak p carried out after oxidation with HZOZ (17) which yielded only the oxidized forms (Fig. 4B). Rechromatography of the oxidized thymosins pd and pi0 yielded the pure peptides (Fig. 3B, Table 1). The reduced forms of these peptides can be obtained in good yield by HPLC after reduction with fi-mercaptoethanol (17). Quantitative considerations. In the experiment shown, the recoveries of peptides from rat thymus, based on the amino acid analyses of the purified peptides, were, in micrograms/ gram thymus tissue, 82 for prothymosin a, 79 for thymosin &,, and 30 for thymosin B with approximately 25% of each of the 1::; two recovered as the oxidized forms. For parathymosin a, the quantity estimated from the relative peak heights in Fig. 2 was approximately 30 pg/g thymus. The quantities of prothymosin o and thymosins & and fliO in the tissue extracts were evaluated using the radioimmunoassays developed for these peptides (6,18). With these assays we estimated the content of peptides

SIMULTANEOUS

ISOLATION/DETERMINATION

OF THYMOSINS

439

ELLITION TIME (mm)

FIG. 4. Separation of thymosin pd and /3,,, on reverse+phase HPLC. (A) The fractions from peak @ (pooled as described in Fig. 1) were lyophyhzed, and the residue was dissolved in 100 ~1 of buffer A. The HPLC experiments (see Materials and Methods) were carried out with 150-~1 ahquots of this solution. Elution was with a gradient of I-propanol in buffer A as shown. Fractions (0.6 ml) were collected every minute, and 5~1 ahquots were diverted every 6 s for analysis with fluorescamine. In the peaks designated as thymosins &,Xj and /3,e0Xj,respectively, methionine has undergone oxidation to methionine sulfoxide. (B) Another ahquot of peak b (150 ~1) was lyophyhzed, and the residue was dissolved in 100 r.d of buffer A plus SO ~1 of 30% HzOz and incubated for 1 h at room temperature. HPLC was carried out as in (A). In the experiment shown, fractions 47 and 48 corresponding to the oxidized form of thymosin &, and fractions 51 and 52 corresponding to the oxidized form of thymosin j3i0 were pooled and analyzed (see Table I ).

to be approximately 400 pg/g thymus for prothymosin a and approximately 225 pg/g thymus for the sum of thymosin ,& plus thymosin @rO. DISCUSSION

The method described can be employed for the simultaneous isolation of at least four peptides present in thymus extracts, and also for the estimation of their content. The previously described procedures for the isolation of thymosin pd and thymosin fiiO using extraction with guanidinium chloride (17) failed to yield a peptide corresponding to prothymosin cx in the HPLC separations. Whether this can be attributed to a failure to extract prothymosin a with 6 M guanidine hydrochloride or to its loss during desalting of the extract has not been determined. In any event, the present procedure yields comparable recoveries of thymosin & and /3r0 as compared to the recoveries using guanidine hydrochloride extracts. Thymosins ph and &, are highly ubiquitous peptides, and although their functions remain unknown, their wide distribution and con-

servation of structure (9,12) suggest that they must play an important role in the physiology of the cell. Prothymosin a is the native polypeptide from which thymosin aI and other fragments (19) appear to be derived by the action of endogenous proteinases. Thymosin a, has been reported to possess a number of immunoenhancing properties (20), and thymosin a1 (2 1,22), thymosin aI, (19), and prothymosin cz have all been found to protect immunodepressed or susceptible strains of mice against opportunistic infections (unpublished observations). The function of parathymosin cx has not been established. The isolation and analytical procedures described here should aid in further studies on the biological activities of these peptides. REFERENCES I. Goldstein, A. L., Slater, F. D., and White, A. (1966) Proc. Nail. Acad. Sci. USA 56, IO IO- IO 17. 2. Hooper, J. A., McDaniel, M. C., Thurman, G. B., Cohen, G. H., Schulof, R. S., and Goldstein, A. L. (1975) Ann, N. Y. Acad. Sci. 249, 125-144. 3. Wara, D. W., Barrett, D. J., Ammann, A. J., and Cowan, M. J. (1979) Ann. N. Y. Acad. Sci. 332, 128-134.

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4. Chretien, P. B., Lipson, S. D., Makuch, R. W., and Kenady, D. E. (1979) Ann. N. Y. Acad. Sci. 332, 135-147. 5. Goldstein, A. L., Low, T. L. K., McAdoo, M., McClure, J., Thurman, G. B., Rossio, J., Lai, C-Y., Chang, D., Wang, S-S,, Harvey, C., Ramel, A. H., and Meienhofer, J. (1977) Proc. Natl. Acad. Sci. USA 74, 725-729. 6. Haritos, A. A., Goodall, G. J., and Horecker, B. L. (1984) Proc. Natl. Acad. Sci. VSA81, 1008-1011. 7. Haritos, A. A., Tsolas, O., and Horecker, B. L. (1984) Proc. Nat/. Acad. Sci. USA 81, 1391-1393. 8. Low, T. L. K., Hu, S-K, and Goldstein, A. L. (1981) Proc. Natf. Acad. Sci. USA 78, 1162-l 166. 9. Erickson-Viitanen, S., Rqgieti, S., Natahni, P., and Horecker, B. L. (1983) Arch. Biochem. Biophys. 221, 570-576.

10. Horecker, B. L. (1984) in Thymic Hormones and Lymphokines (Goldstein, A. L., ed.), Plenum, New York, in press. 1I. Hannappel, E., Davoust, S., and Horecker, B. L. (1982) Proc. Natl. Acad. Sci. USA 79, 1708-l 7 I I. 12. Erickson-Viitanen, S., Ruggieti, S., Natalini, P., and Horecker, B. L. (1983) Arch. Biochem. Biophys. 225,407-4 13. 13. Lai, C. Y. (1977) in Methods in Enzymology (Hits,

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