Thymosin beta-4 is a 43-residue, N-terminally acetylated peptide and the most abundant member of the beta-thymosin family in mammalian cells. Its defined biochemical role is to bind monomeric actin one molecule to one molecule and hold it out of the polymer pool, which makes it a standard reagent in cytoskeletal work. It is also the parent molecule of the fragment supplied as TB-500, and the relationship between the two is set out below.
Key facts
| Type | Endogenous beta-thymosin, N-terminally acetylated |
| Amino acid count | 43 |
| Actin-binding motif | LKKTETQ, residues 17 to 23 |
| Molecular formula | C212H350N56O78S |
| Molecular weight | 4963 g/mol (free peptide) |
| CAS number | 77591-33-4 (acetate salt) |
| Synonyms | T-beta-4, TB4, Fx peptide, prothymosin beta-4 |
| Supplied form | Lyophilised powder in a sealed vial |
Structure and chemistry
The sequence begins Ac-Ser-Asp-Lys-Pro-Asp-Met-Ala-Glu-Ile-Glu-Lys and runs to a C-terminal glutamate-serine pair, with acetylation of the N-terminal serine present on the native molecule and reproduced in synthetic material. The composition is unusually charged for its length: fourteen acidic residues and nine lysines give a low isoelectric point and a strongly negative net charge at neutral pH, which is why the peptide is freely water soluble and shows little tendency to adsorb to polypropylene compared with more hydrophobic sequences of similar size.
In free solution the peptide is intrinsically disordered, with no stable secondary structure detectable by circular dichroism. Order appears on binding. Nuclear magnetic resonance and crystallographic work on beta-thymosin complexes shows an N-terminal amphipathic helix that docks on the barbed-end face of actin, a central extended segment carrying the LKKTETQ motif that runs across the actin surface, and a C-terminal helix that contacts the pointed-end face. The molecule therefore behaves as a coupled folding and binding system, which matters when comparing the intact peptide with short fragments that carry only part of the interface.
Two residues govern handling. Methionine at position 6 oxidises to the sulfoxide on exposure to air or peroxide, adding sixteen mass units and producing a resolvable earlier-eluting peak on RP-HPLC. The N-terminal acetyl group removes the free alpha-amine, so Edman sequencing fails on intact material and identity is confirmed by mass spectrometry, with the acetylated parent ion at roughly 4963 Da distinguishing correctly synthesised peptide from a non-acetylated impurity 42 units lighter.
Mechanism of action
The characterised molecular function is actin sequestration. Thymosin beta-4 binds globular actin in a one to one complex with a dissociation constant in the low micromolar range and, because intracellular concentrations of the peptide are high, a large fraction of the unpolymerised actin pool is held in this complex. The bound actin cannot add to either end of a filament, so the peptide sets the size of the free monomer reservoir and thereby the rate at which new filaments can elongate. Removing or adding the peptide in a pyrene-actin polymerisation assay shifts the lag phase and the plateau in a way that is quantifiable and reproducible, which is the basis for its use as a calibration reagent.
Distinct from sequestration, the peptide is a substrate for tissue transglutaminase, which cross-links it to extracellular matrix components, and it is cleaved by prolyl oligopeptidase at the N-terminal Ser-Asp-Lys-Pro tetrapeptide to release Ac-SDKP. That tetrapeptide is a separate signalling entity with its own literature and its own assays, so experiments on the intact peptide in serum-containing medium are generating both species at once unless a peptidase inhibitor is present. Reported effects on endothelial migration, cell survival signalling and gene expression in culture are attributed variously to the intact peptide, to Ac-SDKP and to the LKKTETQ motif, and the assays that separate these are the ones worth reading.
Research applications
- Pyrene-actin polymerisation and depolymerisation assays, with the peptide used to set the free monomer concentration.
- Fluorescence anisotropy and isothermal titration calorimetry measurements of actin binding affinity and stoichiometry.
- Circular dichroism studies of coupled folding and binding, comparing free peptide with the actin complex.
- Structure-activity comparison between the intact 43-residue peptide, the LKKTETQ heptapeptide and the fragment supplied as TB-500.
- Prolyl oligopeptidase and transglutaminase substrate assays, following release of Ac-SDKP and cross-link formation by LC-MS.
- Cell migration and wound-closure assays in endothelial and fibroblast monolayers, run against fragment and vehicle controls.
Compounds studied on this axis sit in NuVion’s Tissue Repair Signalling category.
Handling in the laboratory
Peptides of this class are supplied lyophilised and reconstituted with bacteriostatic water added slowly down the wall of the vial, then swirled until dissolved. The high net negative charge means dissolution is straightforward in water or neutral buffer without organic co-solvent. The reconstitution calculator converts vial content and diluent volume into a stock concentration. Stocks intended for polymerisation assays are prepared in the same buffer as the actin to avoid a salt or pH step at the point of mixing.
Unopened vials are kept sealed, dry, protected from light and refrigerated as stated on the product documentation. Reconstituted solution is refrigerated and used within the period given on that documentation, or aliquoted and frozen to avoid repeated freeze-thaw cycles. Because methionine 6 is oxidation sensitive, headspace air is minimised and reducing agents are avoided only where the assay permits, since they interfere with some detection chemistries. Purity is determined by RP-HPLC and identity by mass spectrometry, where the sulfoxide sits as a distinct earlier peak.
Testing and supply from NuVion
The compound NuVion supplies on this axis is TB-500, the synthetic fragment carrying the LKKTETQ actin-binding motif at 889 g/mol. It is manufactured at a GMP-audited facility and independently tested by Janoshik Analytical, with purity determined by RP-HPLC and identity confirmed by mass spectrometry. Most batches are tested and the Certificate of Analysis for a tested batch is published on the product page and in the COA library. It is supplied lyophilised in sealed vials and dispatched from within Australia. The intact 43-residue protein is not part of the current range.
Related compounds
The closest compound in the range is TB-500, the synthetic fragment built around the LKKTETQ actin-binding motif. Other compounds used alongside it in cytoskeletal and matrix work include BPC-157 and the copper tripeptide GHK-Cu.
Frequently asked questions
Is TB-500 the same as thymosin beta-4?
No. Thymosin beta-4 is the full 43-residue acetylated peptide. TB-500 is a short synthetic fragment built around the LKKTETQ actin-binding motif, so it carries part of the binding interface and not the flanking helices. The two have different molecular weights and behave differently in binding assays.
Why does the molecular weight vary between suppliers?
Most synthetic material is supplied as an acetate salt, and catalogue figures sometimes quote the salt mass or a trifluoroacetate mass instead of the free peptide at roughly 4963 g/mol. Counter-ion content also varies by batch, which is why peptide content is reported separately from chromatographic purity.
What does the LKKTETQ motif do?
It is the central actin-binding segment, residues 17 to 23, which runs across the surface of monomeric actin in the bound complex. It is the region shared with TB-500 and the reason short fragments retain some actin-related activity in vitro.
Is thymosin beta-4 a therapeutic good in Australia?
No. Compounds of this class are laboratory chemicals for in vitro research. Thymosin beta-4 is not included in the Australian Register of Therapeutic Goods, has not been assessed by the Therapeutic Goods Administration, and is not for human or veterinary use.
Research use only. The compound described on this page is discussed as a laboratory chemical used in in vitro research. It is not included in the Australian Register of Therapeutic Goods and has not been assessed by the Therapeutic Goods Administration for quality, safety or efficacy. It is not for human or veterinary use, and nothing on this page is a representation about therapeutic use.
