Calculate the absorbance of a protein at 280nm from the primary sequence:

The molar extinction coefficient of a particular protein can be calculated quite accurately from the protein sequence and is quite useful to know since it allows you to accurately quantify the amount of protein, assuming you can obtain it in pure form. You can do this by measuring the absorbance of the protein at the ultraviolet wavelength of 280nm, which you can do accurately in a quartz cuvette in a UV spectrophotometer. How much a protein absorbs at 280nm is almost totally a function of the content of the aromatic rings of the amino acids Tyrosine and especially Tryptophan (the aromatic ring of Phenylalanine absorbs well at 260nm, but not 280nm). A solution to Tryptophan will give an absorbance of 5500 M^-1 cm^-1, while a solution of Tyrosine will give an absorbance of 1490 M^-1 cm^-1. The absorbance units are expressed per M per centimeter, so that a 1 M solution of Tryptophan in a typical 1 cm path length cuvette will have an absorbance of 5500. Since the absorbance of Tryptophan is so much higher than Tyrosine, the absorbance of a protein is very heavily influenced by the Tryptophan content. This can be a problem since this is the rarest amino acid, so that the average protein contains only ~1.3% Tryptophan. This means that many proteins contain little or none of this amino acid and so have very low absorbances at 280nm, while proteins which happen to have unususally high amounts of this amino acid will absorb unusually strongly at 280nm. Tyrosine is one of the rarer amino acids, at ~3.25% in the average protein, so the same problem can occur to a lesser degree here also. So proteins of similar molecular weight can have quite different absorbances, since they can have utterly different Tryptophan and Tyrosine content. However if you know the amino acid sequence and can get your protein pure you can correct for this problem and also quickly and conveniently measure the concentration. To calculate the absorbance of a 1M solution of a protein you simply count up the number of Tryptophan residues and multiply that by the absorbance of a one molar solution of Tryptophan. Then add that to the number of Tyrosine residues multiplied by the absorbance of a one molar solution of Tyrosine, and the sum is the absorbance of 1M of the whole molecule. So we use this simple equation;

Molar Extinction Coefficient = (Number of Tryptophan residues X 5500) + (Number of Tyrosine residues X 1490)

The Molar Extinction Coefficient is the absorbance of a 1M solution of the protein, which is not possible to achieve for most normal sized proteins; for example a 1M solution of the 66kDa protein bovine serum albumin would have a density of more than 66kg/L, almost three times that of Uranium, the densest substance on the surface of planet Earth. More useful is the absorbance of a 0.1%, equal to a 1g/L or 1mg/ml solution. This is usually referred to as the absorbance, and can be calculated by simply dividing the Molar Extinction Coefficient by the molecular weight. So this gives you the absorbance that a 1mg/ml solution of your protein would have. So all you have to do is measure the absorbance of your pure protein at 280nm and divide this by the absorbance value which a 1mg/ml solution would have, giving you the mgs/ml concentration of your protein solution.

Three caveats and a caution: The caution is that we used the values for a 1mg/ml protein solution above, which is a 0.1% protein solution. This is used by many authors, textbooks, lab manuals, but some others use a 1% protein solution, or 10mg/ml. You might have to look carefully to see whether a particular, paper, protocol or whatever uses absorbance values for 1% or 0.1% solutions- if the values appear to be about 10X of ours, they are most likely using absorbances at 1%. First caveat, Cystine, in other words two Cysteine residues linked by a disulfide bond, also has an absorbance at 280nm, but the effect is small (110 units for a 1M solution), and to calculate how much this influences the absorbance of your protein you have to know how many of the Cysteine residues are disulfide linked, which you can't determine from the sequence alone. Generally speaking disulfide bonds only occur on extracellular regions of proteins, while most proteins are inside cells. So we ignored the Cystine effect, since Cysteine is also a rare amino acid (~2% in the average protein). Second caveat, contamination by substances with aromatic rings can mess up your calculations; each and every DNA and RNA base contains either one or two aromatic rings which all absorb strongly at 280nm, so contamination by even small amounts of nucleic acids can be a particular problem. Third caveat, the pathlength of your cuvette is assumed to be 1cm, which is standard in most spectrophotometers. If the pathlength is less than this, then the concentration measured should be corrected to take this into account.

Disclaimer: This program was constructed primarily to save time for busy researchers around the world. We hope you find it useful, and we are confident that it is accurate and reliable. However, we cannot be held responsible for any problems which may arise as a result of the use of this program.