TB-500 and Thymosin Beta-4: Research Overview

TB-500 is a research name for synthetic thymosin beta-4 (Tβ4), a 43-residue, N-terminally acetylated peptide that binds monomeric actin and is present in nearly every cell. The name is used inconsistently. Some sources apply it to a short acetylated fragment, Ac-LKKTETQ, instead of the full-length peptide, so the molecular weight on the COA is what tells you which one is in the vial. Ours is full-length thymosin β-4. Thymosin β-4 has been studied in cell migration, angiogenesis and cardiac models.

Molecular identity

The TB-500 we supply is full-length thymosin β-4:

Low and Goldstein determined the complete sequence in 1982, working with material purified from thymus preparations. They described a 43-residue peptide with acetylserine at the N-terminus and two predicted helical regions [1]. The single sulfur atom in the formula is the methionine at position 6.

The TB-500 naming problem

Two different molecules circulate under the same name:

Some analytical literature uses the name TB-500 for a synthetic seven-residue fragment of thymosin β-4 (Ac-LKKTETQ). Tidal's TB-500 is full-length thymosin β-4 (CAS 77591-33-4, 4963.44 Da), which is why the molecular weight on its COA matches the full peptide.

Because the full peptide is roughly six times longer than the fragment, mass spectrometry tells them apart easily. A COA for full-length material should report a mass consistent with about 4963 Da, often as a deconvoluted value from multiply charged ions.

The difference matters for interpreting the literature as well. The studies summarized below concern full-length thymosin β-4, and structure–function work shows that different regions of the molecule carry different activities (see below). Findings for the full-length peptide shouldn't be assumed to transfer to the fragment, or the reverse.

Actin biology

Thymosin β-4's best-established function is actin buffering. In 1991, a study showed that "Fx," the peptide holding most of the unpolymerized actin in human platelets, is identical to thymosin β-4. It forms a 1:1 complex with actin monomers and inhibits their polymerization [2]. A later review describes the β-thymosins as the main intracellular G-actin–sequestering peptides, present at high concentrations in almost every cell, that act as actin buffers and supply monomers when filaments are needed [3].

Structure–function work using a DNase I inhibition assay found that oxidizing methionine-6 to the sulfoxide did not abolish actin sequestration. C-terminal fragments lacking the first 12 residues (13–43 and 24–43) did not inhibit actin polymerization, which suggests that residues before position 13 are required for that activity [4].

What the research has examined

Endothelial cells and angiogenesis

In endothelial progenitor cells, thymosin β-4 increased secretion of vascular endothelial growth factor (VEGF). Conditioned medium from these cells increased tube formation by human umbilical vein endothelial cells, and a VEGF-neutralizing antibody abolished the effect. Knocking down Akt or eNOS reduced the VEGF secretion, which implicates the Akt/eNOS pathway [5].

NF-κB, PINCH-1 and ILK

In cell studies, thymosin β-4 interfered with TNF-α-induced NF-κB activation and with nuclear translocation of the RelA/p65 subunit. It also interfered with downstream IL-8 gene transcription. These activities were independent of its G-actin binding. Thymosin β-4 also counteracted the sensitizing effect of its intracellular binding partners PINCH-1 and integrin-linked kinase (ILK) on NF-κB activity [6].

Epicardial progenitors and vessel development

In mice, thymosin β-4 was reported to be essential for coronary vessel development. In adult epicardial explants it stimulated outgrowth and differentiation into fibroblasts, smooth muscle cells and endothelial cells. The study also implicated Ac-SDKP, a tetrapeptide cleavage product from thymosin β-4's N-terminus [7].

Reviews

A 2010 review maps thymosin β-4's activities to short active sites. It identifies the central actin-binding domain at residues 17–23 (LKKTETQ), an N-terminal region that contains Ac-SDKP, and lists ILK/PINCH/Akt activation among the activities not yet assigned to a specific region [8]. A 2001 review of the β-thymosin family summarizes reported extracellular effects, including chemotaxis, angiogenesis and metalloproteinase induction. It notes that the molecular mechanisms of these extracellular effects were not known at the time [3].

Analytical notes

At nearly 5 kDa, thymosin β-4 is large for a synthetic peptide. Expect:

• HPLC: one main peak, with closely eluting deletion or truncation sequences as the typical impurities. The methionine-sulfoxide form may appear as a separate nearby peak.
• Mass spectrometry: electrospray gives a charge-state envelope rather than a single ion. The COA should give the deconvoluted mass and compare it with the theoretical 4963.44 Da.
• Identity: a mass far below 4963 Da would indicate a fragment, such as Ac-LKKTETQ, rather than full-length material.

See reading a certificate of analysis and HPLC purity explained. Batch documents are in the COA registry.

Storage and handling

Store lyophilized thymosin β-4 at −20 °C, away from light and moisture. Because methionine-6 can oxidize, limit how long the powder or solution is exposed to air. After reconstitution, keep it at 2–8 °C and avoid repeated freeze–thaw cycles. Handle it as laboratory material throughout.

Related material

• TB-500 (thymosin β-4)
• BPC-157 + TB-500 blend
• BPC-157 vs TB-500: a side-by-side comparison
• Also a component of the KLOW and GLOW blends. See KLOW vs GLOW

References

1. Low TL, Goldstein AL. Chemical characterization of thymosin beta 4. J Biol Chem. 1982. PubMed
2. Safer D, et al. Thymosin beta 4 and Fx, an actin-sequestering peptide, are indistinguishable. J Biol Chem. 1991. PubMed
3. Huff T, et al. beta-Thymosins, small acidic peptides with multiple functions. Int J Biochem Cell Biol. 2001. PubMed
4. Hannappel E, Wartenberg F. Actin-sequestering ability of thymosin beta 4, thymosin beta 4 fragments, and thymosin beta 4-like peptides as assessed by the DNase I inhibition assay. Biol Chem Hoppe Seyler. 1993. PubMed
5. Zhao Y, et al. Thymosin β4 promotes endothelial progenitor cell angiogenesis via a vascular endothelial growth factor-dependent mechanism. Mol Med Rep. 2018. PubMed
6. Qiu P, et al. Thymosin beta4 inhibits TNF-alpha-induced NF-kappaB activation, IL-8 expression, and the sensitizing effects by its partners PINCH-1 and ILK. FASEB J. 2011. PubMed
7. Smart N, et al. Thymosin beta4 induces adult epicardial progenitor mobilization and neovascularization. Nature. 2007. PubMed
8. Sosne G, et al. Biological activities of thymosin beta4 defined by active sites in short peptide sequences. FASEB J. 2010. PubMed