Amino acid composition
How the numbers work
A protein is a chain of amino acids joined by peptide bonds, and every peptide bond forms by removing one water. So the mass of the chain is the sum of each amino acid's residue mass (the amino acid minus a water) plus a single water added back for the two free ends. The molecular weight here uses average isotopic masses, so it matches what you would report for a purified protein.
The extinction coefficient at 280 nm comes almost entirely from aromatic side chains. Using the Pace/Gill/Edelhoch method, each tryptophan adds about 5500 M-1 cm-1, each tyrosine about 1490, and each disulfide bond about 125 when cysteines are oxidized. Dividing that by the molecular weight gives the absorbance of a 1 mg/mL solution, so a spectrophotometer reading at 280 nm converts straight into concentration.
The theoretical pI is the pH where the summed charge of the N-terminus, C-terminus, and the ionizable side chains (Asp, Glu, Cys, Tyr, His, Lys, Arg) crosses zero. Different pKa tables shift it by a few tenths, so read it as an estimate.
Related tools: Peptide charge & pI · DNA & RNA concentration (A260/A280) · Beer-Lambert calculator · Amino acid titration curve · Amino acid chart (all 20) · Codon chart (genetic code) · all biochem tools.
Turning an A280 into a concentration
The whole point of the extinction coefficient is quantification. Once you have the A280 of a 1 mg/mL solution (call it the reference), any reading converts in one step:
This is exactly what a NanoDrop does when you enter a protein's extinction coefficient instead of using the generic "1 Abs = 1 mg/mL" setting.
FAQ
How is molecular weight calculated?
Sum every residue mass (amino acid minus a water) and add one water for the free ends. Average isotopic masses give the average MW in daltons.
Where do 5500 and 1490 come from?
They are the per-residue absorptivities at 280 nm (Pace/Gill/Edelhoch): 5500 M-1 cm-1 per Trp, 1490 per Tyr, plus 125 per disulfide bond when oxidized. Proteins with no Trp or Tyr barely absorb at 280 nm.
Reduced vs oxidized extinction coefficient?
Reduced = free cysteine thiols (most intracellular proteins). Oxidized = disulfide-bonded cysteines (many secreted proteins). They differ only by 125 per disulfide, so with few cysteines it hardly matters.
Why does the pI differ from another calculator?
Different pKa tables. The same sequence can shift by a few tenths of a pH unit. It is a theoretical estimate, not a measured value.
Does this handle a FASTA sequence?
Yes, a leading header line starting with ">" is ignored, and so are spaces, line breaks, and residue numbers. Non-standard letters (X, B, Z, U) are skipped and flagged.
Sources and how to cite this page
Molecular weight is the sum of average-isotopic residue masses plus one water molecule for the free termini. The molar extinction coefficient at 280 nm is calculated by the method of Gill and von Hippel (Gill, S.C. & von Hippel, P.H., “Calculation of protein extinction coefficients from amino acid sequence data,” Analytical Biochemistry 182(2):319–326, 1989), which sums per-residue contributions of 5500 M−¹cm−¹ per tryptophan, 1490 per tyrosine, and 125 per cystine (disulfide-bonded cysteine pair).
How accurate it is. The sequence-based estimate is typically within a few percent for proteins containing tryptophan, which is why it is the standard way to convert an A280 reading into concentration. It is less reliable for proteins with no tryptophan, where the tyrosine and cystine terms dominate and the relative error is larger. Note also that the calculation assumes the protein is folded and free of absorbing contaminants; nucleic acid contamination inflates A280 badly.