Histidine Structure: The Imidazole Side Chain, Its pKa and Why It Binds Copper

Histidine structure comes down to one feature: an imidazole ring hung off the amino-acid backbone by a single CH₂ group. That ring carries two nitrogens, can swap a proton between them, gains or loses a positive charge within about one pH unit of neutral, and holds metal ions such as copper(II), nickel(II) and zinc(II). For anyone working with synthetic peptides, those four properties decide how a histidine-containing sequence behaves in a buffer, on a purification column and in a mass spectrum.

This guide covers the formula and masses, the two ring nitrogens and their tautomers, measured pKa values, His-tag purification, how the tripeptide GHK holds copper(II), and what histidine means when you check a peptide’s documentation.

Histidine structure in numbers

Histidine is one of the 20 standard amino acids, written His or H (see our amino acid codes chart). In ribosomally made peptides it is the L-form, the (2S) configuration; the mirror-image D-form appears in synthetic sequences, as our guide to D-amino acids explains.

The molecular formula of free histidine is C₆H₉N₃O₂. The Merck Index lists a molar mass of 155.15 g/mol, and the monoisotopic mass, which a high-resolution mass spectrometer measures, is 155.06947 Da. Built into a chain, histidine loses one water to the peptide bond, so the residue contributes C₆H₇N₃O, or 137.0589 Da monoisotopic. That is the value used to add up a sequence; our guide to peptide molecular weight walks through the arithmetic.

The imidazole side chain and its two nitrogens

Imidazole is a flat five-membered ring containing three carbons and two nitrogens, with the nitrogens separated by one carbon. In histidine the ring carries a hydrogen on one nitrogen and a lone pair of electrons on the other. The lone-pair nitrogen is the one that accepts a proton when the pH drops and the one that donates electrons to a metal ion.

Naming the nitrogens: pros, tele, δ1 and ε2

The IUPAC Gold Book records that biochemists numbered the nitrogen next to the side chain 1 while organic chemists numbered it 3, so “N1” could mean either atom. The adopted solution names them by position: pros (“near”, symbol π) for the nitrogen adjacent to the side-chain attachment, and tele (“far”, symbol τ) for the other one. In protein-structure notation the near nitrogen is Nδ1 and the far one Nε2, so Nπ = Nδ1 and Nτ = Nε2.

Histidine tautomers

The neutral ring holds one N-H proton, which can sit on either nitrogen. The two forms are tautomers: the Nδ1-H form (proton on the near nitrogen) and the Nε2-H form (proton on the far one). They interconvert, and which one dominates depends on the residue’s surroundings. A 2003 study in the Journal of the American Chemical Society showed that the ¹³C chemical shift of the ring’s Cδ2 carbon separates them, with values above 122 ppm pointing mainly to the Nδ1-H form and values below 122 ppm mainly to the Nε2-H form. Tautomers matter because only the nitrogen without a hydrogen can bind a metal.

When the ring takes up a second proton, both nitrogens carry hydrogens, the ring is positively charged (imidazolium), and the tautomer distinction disappears.

Histidine pKa: why the charge flips near neutral

A pKa is the pH at which a group is half protonated. Free histidine has three: the Merck Index lists 1.82 (carboxyl), 6.00 (imidazole) and 9.17 (amino), giving a calculated isoelectric point of about 7.6.

Inside a chain the side-chain value shifts. Thurlkill and colleagues titrated blocked alanine pentapeptides, a model for an unfolded chain, in 0.1 M KCl at 25 °C and found 6.54 ± 0.04 for histidine. A survey of folded proteins found an average of 6.6 with a standard deviation of 1.0 across 131 histidines, so individual values spread over several pH units.

Sources: Merck Index (free amino acid); Thurlkill et al. 2006 (model peptide); Grimsley et al. 2009 (folded proteins).

What a pKa near 6 means in numbers

The Henderson–Hasselbalch relationship turns a pKa into the fraction protonated at any pH: fraction = 1 / (1 + 10^{(pH − pKa)}). Using the model-peptide value of 6.54, a histidine side chain is about 97% protonated at pH 5, 78% at pH 6, 26% at pH 7 and 12% at pH 7.4. Of the common ionizable side chains, histidine’s is the closest to neutral: in the same model-peptide study aspartate (3.67) and glutamate (4.25) sit well below it, and cysteine (8.55), tyrosine (9.84) and lysine (10.40) well above.

Because the unprotonated ring can act as a general base and the protonated ring as a general acid, histidine sits in the catalytic site of serine proteases and many other enzymes.

Why histidine binds copper, nickel and zinc

A neutral imidazole nitrogen with a free lone pair is a good electron-pair donor, and transition-metal ions are good acceptors. Sundberg and Martin’s 1974 Chemical Reviews survey covers histidine and other imidazoles with transition-metal ions, and in metalloproteins histidine’s imidazole is a frequent metal ligand.

Only the deprotonated nitrogen can bind a metal, so protons and metal ions compete for the same lone pair; lowering the pH far enough releases the metal, which makes the binding reversible.

His-tags and IMAC

Porath and colleagues introduced metal chelate affinity chromatography in Nature in 1975, using immobilized metal ions to separate proteins. In 1988 Hochuli and colleagues paired a new metal chelate adsorbent with a genetically added histidine tail, an approach that led to the nickel-nitrilotriacetic acid (Ni-NTA) resins in wide use today. In one manufacturer’s description, NTA is a tetradentate chelator that holds Ni²⁺ through four coordination sites, leaving the remaining sites for the imidazole rings of a six-histidine (6xHis) tag. Bound protein is released by free imidazole, which competes for the nickel, or by low pH: the same manufacturer’s denaturing elution buffer is pH 4.0, where histidine is almost fully protonated.

Worked example: how GHK holds copper(II)

GHK is the tripeptide glycyl-L-histidyl-L-lysine, Gly-His-Lys in sequence notation, a simple example of histidine binding copper. In 1984 Perkins and colleagues reported a crystal structure of a copper complex of GHK at 1.1 Å resolution in Inorganica Chimica Acta. A 2011 study by Hureau and colleagues combined X-ray crystallography with EPR, HYSCORE, absorption and NMR spectroscopy to compare Cu(II)-GHK with Cu(II)-DAHK, another copper-binding peptide found in blood plasma.

According to the abstract, in the solid state Cu(II)-GHK forms a dimer, while in solution it is a monomer in which copper is held by nitrogen donors from the glycine N-terminal amine, a deprotonated backbone amide nitrogen and the histidine imidazole. A fourth equatorial position is labile; in the crystal it is taken by a carboxylate oxygen.

The difference is where histidine sits. In GHK it is residue 2; in DAHK it is residue 3, giving the classic amino-terminal Cu(II)- and Ni(II)-binding (ATCUN) arrangement: a free N-terminal amine, two backbone amide nitrogens and a histidine together form a square-planar complex, as in the simplest example, Gly-Gly-His.

AHK-Cu differs from GHK-Cu only by replacing glycine with alanine. With histidine still at position 2, similar binding is a reasonable expectation, not a published structure we could find. Our GHK-Cu vs AHK-Cu comparison covers what the research does separate, and the GHK-Cu research guide covers the peptide’s wider literature. For the size vocabulary, see dipeptides, tripeptides and oligopeptides.

Histidine in other research sequences

Histidine also sits at or near the N-terminus of several other sequences on our shelf:

• GHRP-6 is His-D-Trp-Ala-Trp-D-Phe-Lys-NH₂. Histidine is residue 1, so this hexapeptide has a free N-terminal amine directly beside an imidazole ring. Our GHRP-6, GHRP-2 and hexarelin comparison covers the family.
• VIP (vasoactive intestinal peptide) is HSDAVFTDNYTRLRKQMAVKKYLNSILN-NH₂, a 28-residue member of the secretin/glucagon family, which also starts with histidine. See our VIP research overview.

Sequences are from the product listings.

What histidine means for peptide analysis

Mass spectrometry

Histidine’s residue mass is 137.0589 Da monoisotopic, and two mass shifts are worth knowing. Oxidation of histidine to 2-oxo-histidine adds one oxygen atom (about +16 Da); Schöneich’s review describes this metal-catalysed reaction in peptides and proteins. A bound metal ion shifts the mass too, so the reported mass of a copper complex depends on the peptide-to-copper ratio and salt form. Our guide to reading a peptide mass spectrum explains how to match observed and expected masses.

Chromatography and buffers

At pH 2, four units below the pKa, histidine’s ring is almost fully protonated; at neutral pH it is mostly neutral. Retention and solubility change with it, so a purity result is tied to its method. Our HPLC testing guide covers how purity is measured and reported.

What’s established and what isn’t

Sourcing histidine-containing peptides in Canada

For a histidine-containing peptide, particularly a copper complex, a few documentation questions are worth asking:

• Does the identity result match the expected mass? For a copper peptide, check whether it is the mass of the free peptide or of the complex. Our COA guide explains what a useful certificate contains.
• Is purity reported with its method? Histidine’s charge depends on pH, so an HPLC purity figure should come with the conditions used.
• Does the batch number on the certificate match your vial? A certificate for a different lot tells you little about the material you hold.
• How is it stored? Keep lyophilized material cold, dry and dark; see the peptide storage guide.

Pure Peptides Canada is a Canadian-owned supplier based in Toronto, established in 2023, that sells research peptides online in CAD and ships tracked from inside Canada. How testing is documented is explained on the Lab Testing page.

GHK-CuGly-His-Lys with copper(II) held by the amine, an amide nitrogen and the histidine ringView product AHK-CuAla-His-Lys copper complex, with histidine in the same position as in GHKView product GHRP-6Hexapeptide that begins with histidine: His-D-Trp-Ala-Trp-D-Phe-Lys-NH2View product VIP28-residue neuropeptide whose sequence starts with histidineView product

Frequently asked questions