The primary structure of prothymosin a from rat thymus, containing 113 amino acid residues, is reported as follows: AcSer-Asp-Ala-Ala-Val-Asp-Thr-Ser- Glu-Ile-Thr-Thr-Lys-Asp-Leu-Lys-Glu-Lys- Glu-Val-Val-Glu-Glu-Ala-Glu-Asn-Gly-Arg-Asp-Ala- 40 Pro-Ala-Asn-Gly-Asn-Ala-Gln-Asn-Glu-Glu- Gly-Glu-Gln-Glu-Ala-Asp- Glu-Glu-Glu-Glu-Gly-Gly-Gly-Glu-Glu (Asx,Gly,Gly, 70 Glx,Glx,Glx,Glx,Glx,Glx,Glx,Glx,Glx,Glx)Asn-Gly- 80Asp-Glu-Asp-Glu -Glu -Ala -Gl u -Ala-Pro -Th r-Gly -90 100Lys-Arg-Val-Ala-Glu-Asp-Asp-Glu-Asp-Asp-Asp- 110Val-Glu-Thr-Lys-Lys-Gln-Lys-Lys-Thr-Asp-GluAsp-AspOH. The sequence of the first 28 amino acids at the NH2 terminus is identical to that of calf thymosin al. The dicarboxylic amino acids, which account for nearly half of the total residues in prothymosin a, are largely clustered in the central portion of the polypeptide chain. The polypeptide contains no aromatic or sulfur-containing amino acids. A computer analysis of the three-dimensional structure based on the primary sequence suggests that the molecule is composed of at least five a-helical regions interrupted by one short extended chain and three short random coils.A mixture of peptides from calf thymus identified as thymosin fraction 5 (1-3) has been reported to restore immune function in thymectomized mice (1) and to yield positive results in other in vivo and in vitro assays for the induction and maintenance of immune function (ref. 4; for reviews of the earlier literature, see refs. 3, 5, and 6). Thymosin fraction 5 also has shown promise in clinical trials with children with immunodeficiency diseases (7) and with cancer patients (8).Thymosin fraction 5 was shown to be a mixture of peptides ranging in molecular weight from 1000 to 15,000 and in isoelectric point from 4.0 to 7.0 (9). The first component of thymosin fraction 5 to be isolated in pure form and characterized was thymosin a,, an acidic peptide containing 28 amino acid residues (9). This peptide was reported to show many of the biological activities of thymosin fraction 5 (refs. 9-12 (pH 5.5). To this buffer, 10-,/g aliquots of carboxypeptidase Y were added at 0, 4, and 8 hr. At timed intervals 5-10% of the total volume was removed, diluted to 100 ,Al with ice-cold buffer of 67 mM sodium citrate (pH 2.0),

Two peptides related to thymosin a1 have been isolated from preparations of calf thymosin fraction 5. One, lacking four amino acid residues at the COOH terminus, is designated des-(25-28)-thymosin al. The other, named thymosin all, contains seven additional amino acid residues at the COOH terminus. The sequence of this peptide is: AcSer-Asp-Ala-Ala-Val-Asp-ThrSer-Ser-Glu-Ile-Thr-Thr-Lys-Asp-Leu-Lys-Glu-Lys-Lys-Glu-ValVal-Glu-Glu-Ala-Glu-Asn-Gly-Arg-Glu-Ala-Pro-Ala-AsnOH. Thymosin all, in doses of <300 ng per mouse, protects susceptible inbred murine strains against opportunistic infections with Candida albicans. It is m30 times as potent as thymosin fraction 5 and approximately equal in potency to thymosin al.

The major cross-reacting peptide in human plasma detected with a radioimmunoassay (RIA) for thymosin a, was identified as prothymosin a, based on its elution properties in gel-filtration chromatography and its amino acid composition after purification by HPLC. A small quantity (<10%) of the total cross-reacting material was recovered in fractions corresponding to lower molecular weight thymosin al-like peptides. The total quantity of cross-reacting material detected in human blood, expressed as thymosin a, equivalents, was 11-14 pmol/ml (-90% was recovered in the leukocyte fraction, =10% was in the plasma fraction, and 1-2% was in the erythrocyte fraction). The peptide present in leukocytes was also identified as prothymosin a. After correction for the 5-times lower molar reactivity of prothymosin a in the thymosin a, RIA employed in these experiments, we estimate that the content of prothymosin a in human blood is 55-70 pmol/ml (0.6-0.8 pg/ml). The relatively small quantities recovered in the erythrocyte and plasma fractions may be attributed to contamination of the former by leukocytes or to leakage from leukocytes into the plasma.Thymosin a1, an acidic peptide containing 28 amino acid residues (1), was first isolated from a calf thymus preparation called thymosin fraction 5, a biologically active mixture of peptides ranging in molecular weight from 1000 to 15,000 (2). Thymosin a1 has been reported not only to be active in several assay systems for enhancement of immune function, both in vitro and in vivo, but also to be much more potent than the thymosin fraction 5 preparations from which it was isolated (for reviews, see refs. 3-5).Radioimmunoassays (RIAs) for thymosin a1 have been developed in several laboratories (6)(7)(8)25) and employed for the analysis of cross-reacting material in serum or plasma (6, 9-12). Highest levels of immunoreactive peptides were found in cord plasma (6), but detectable quantities (0.6-3.7 ng of thymosin a1 equivalents per ml) were also present in the serum of adolescents (6) or normal adults (12). Of particular interest were reports that the levels of immunoreactive thymosin a,-like material were elevated in serum from patients with acquired immunodeficiency syndrome and also in a group of homosexual men (13,14). Another report, however, suggests that the high levels in serum from patients with acquired immunodeficiency syndrome may be due to the presence of a product of the gag gene of human immunodeficiency virus, which contains an amino acid sequence homologous to the sequence in thymosin a1 that was recognized by the antibody employed (15). This antiserum was also reported to block the replication of human immunodeficiency virus in permissive H9 cells (15).The identity and possible origin of the cross-reacting material in normal blood have not yet been established. A cross-reacting peptide purified from a preparation of Cohn fraction IV-1 by immunoaffinity chromatography was reported to resemble thymosin a1 in size and chromatographic properties (16). However, the i...

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