Humanin: One Free Cysteine and What It Does
Humanin is a 24-residue peptide encoded in mitochondrial DNA rather than in the nuclear genome, and it carries two residues that make it awkward to keep unchanged: a methionine at the N-terminus and a single unpaired cysteine.
Sequence, mass and origin
Humanin is Met-Ala-Pro-Arg-Gly-Phe-Ser-Cys-Leu-Leu-Leu-Leu-Thr-Ser-Glu-Ile-Asp-Leu-Pro-Val-Lys-Arg-Arg-Ala, twenty-four residues with free termini. Its monoisotopic mass is 2685.48 Da and its average mass approximately 2687.3 Da. CAS 330936-69-1.
It is described in the literature as a mitochondria-derived peptide, meaning its open reading frame sits within the mitochondrial genome rather than in nuclear DNA. That places it in the same structural category as MOTS-c, though the two share no sequence similarity and have very different lengths.
The unpaired cysteine
There is exactly one cysteine, at position eight, and there is nothing for it to pair with inside the molecule. A free thiol in an aqueous, aerated solution oxidises, and the usual product is a disulfide-linked dimer of two humanin molecules.
That dimer has almost exactly twice the parent mass, minus two hydrogens, so it appears near 5369 daltons. On a chromatogram it is a distinct later-eluting peak. On a mass spectrum it is unambiguous once the charge envelope is deconvoluted, but in raw form its multiply-charged ions can overlap confusingly with those of the monomer.
The practical consequence is that a purity figure for humanin is time-dependent in a way that a figure for a cysteine-free peptide is not. Dimer content measured on receipt and dimer content measured after a month in solution are different numbers, and both can be correct.
Methionine at position one
The N-terminal methionine adds the second liability. Oxidation to the sulfoxide adds sixteen daltons and shifts retention earlier. Because the residue is terminal and exposed, it oxidises more readily than a buried methionine would.
A sample carrying both a plus-sixteen species and a dimer is showing two independent oxidation processes, not one. They respond to different controls: the methionine to exclusion of oxygen and light, the cysteine to that plus the absence of trace metal catalysts and, in some preparations, a reducing additive.
The S14G variant
A widely studied analog replaces the serine at position fourteen with glycine, written S14G-humanin or HNG. The substitution changes the mass by 30 daltons, since serine at 87.03 gives way to glycine at 57.02.
That 30 dalton difference is the identity check between the two. They are distinct compounds, and a document naming one while reporting the mass of the other has an error in it somewhere. The variant is also sometimes listed simply as HN followed by a letter, which is not informative on its own.
A hydrophobic stretch in the middle
Positions nine through twelve are four consecutive leucines. That is an unusually hydrophobic run for a peptide of this length, and it drives two behaviours: strong retention on reversed-phase columns relative to what the overall composition would suggest, and a tendency to aggregate in aqueous solution at higher concentrations.
Aggregation shows up as a broadened or fronting main peak rather than as a new species, which makes it easy to attribute to a column problem. Diluting the sample and re-injecting distinguishes the two: a concentration-dependent peak shape is the peptide, not the hardware.
Detection and mass spectrometry
One phenylalanine and no tryptophan or tyrosine means 280 nm is not usable. Detection is at 214 nm.
At 2687 daltons the electrospray charge envelope typically runs from two to four protons, with the triply charged ion near m/z 896.8. Deconvolution to a neutral mass is the readable form, and the comparison should be against the average mass rather than the monoisotopic one at this size unless the instrument resolves the isotope cluster.
What confirms a lot
A deconvoluted mass near 2687 Da, a 214 nm chromatogram with the dimer peak either absent or quantified, and a stated figure for oxidised species. For this peptide specifically, the storage form matters more than usual: lyophilised material with the cysteine in its reduced state is a defined thing, and a solution left standing is a mixture whose composition changes.
The other mitochondria-derived peptide
Humanin and MOTS-c share an origin in the mitochondrial genome and nothing else: different lengths, different sequences, different analytical problems. MOTS-c is covered in MOTS-c as a mitochondria-derived peptide and in the mitochondrial peptide research overview.
