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Measuring Peptide Concentration

Establishing the actual peptide concentration based on the weight of the lyophilized peptide is inaccurate because there may be a significant quantity (10-70%) of bound water, and salts or counter ions. The type of counter ion and the amount is dependent on the peptide sequence and the solvents that have been used in the manufacturing and purification process. The peptide concentration can be estimated based on whether there are Tryptophan or Tyrosine resides present in the sequence.

If your Peptide contains Tryptophan or Tyrosine1, 2

1. Weigh out a known amount of peptide and dissolve in 6M Guanidine hydrochloride, pH 6.5, 0.02 M phosphate buffer.

2. Measure the concentration using UV spectrophotometry and applying the following calculation:

[Peptide concentration] mg/ml = (A280 x DF x MW) / ε

 

A280 is the absorbance of the peptide solution at 280 nm in a 1 cm cell
DF is the dilution factor
MW is the molecular weight of the peptide
ε  is the molar extinction coefficient of Tryptophan or Tyrosine at 280 nm.

Tryptophan ε (Trp) = 5690 M-1cm-1
Tyrosine ε (Tyr) = 1280 M-1cm-1

If the peptide contains more than one Tryptophan or Tyrosine, or a mixture, then the extinction coefficients are added, e.g., if there are two Tryptophan and one Tyrosine, ε= (2 x 5690) + (1 x 1280).

The calculation assumes that the peptide is unfolded in extended conformation. This is achieved by measuring under denaturing conditions.

If your peptide does not contain Tryptophan or Tyrosine3

1. It is possible to determine the concentration of peptides that do not contain aromatic amino acids by measuring absorbance at 205 nm. However this measurement is far more prone to external influence, since many solvents and other chemicals will absorb at this wavelength. A high quality, dual-beam spectrophotometer is required in order to reduce the effects of non-specific absorption and to measure low concentrations. For a high level of reproducibility, a combination of absorption measurements at a range of wavelengths is recommended.

2. If routine analysis is required, then HPLC analysis with measurement at 205 nm would be a far superior option because of its ability to resolve the peptide from the buffer constituents, thus allowing for more accurate concentration determination. Integration of the peak of interest will allow for accurate determination of peptide quantity. Using control peptide solutions to determine the absorbance at 205 nm that corresponds to a fixed concentration of peptide is recommended.

References

1. Edelhoch, H., 1967. Spectroscopic determination of tryptophan and tyrosine in proteins. Biochemistry 6:1948-1954. [PubMedID: 6049437]

2. Gill, S.C., von Hippel, P.H., 1989. Calculation of protein extinction coefficients from amino acid sequence data. Anal. Biochem. 182:319-326. [PubMedID:2610349]

3. Scopes, R.K., 1974. Measurement of protein by spectrophotometry at 205 nm. Anal. Biochem. 59:277-282. [PubMedID:4407487]

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