Peptide content
Ioannis Papayannopoulos (iap@iname.com)
Thu, 23 Jul 1998 09:47:25 -0400
For proteins UV measurements, typically at 280 nm, are used for
quantitation. This requires, of course, that the extinction coefficient
for the particular protein is known. It can be determined experimentally
by measuring the UV spectrum of a protein solution and then obtaining a
quantitative amino acid analysis of a known amount of the sample. It then
becomes a simple numerical calculation, using Beer's law, to obtain the
extinction coefficient. If the amino acid sequence is known one can also
calculate the extinction coefficient based on the number of groups that
absorb at a particular wavelength, and in my experience the calculated and
measured values are usually quite close (within 5-15%). For most research
applications the calculated extinction coefficient probably is quite
adequate, but one must have an experimentally determined value for
estimating the active pharmaceutical ingredient (protein) concentration in
a final formulation of a phramaceutical (oddly referred to as "strength" in
some certificates of analysis).
There is no reason why one can not do this (AAA of a "standard" or
calculation of an extinction coefficient, e.g. by adding those of all amide
bonds) for peptides as well. However, if there is no absorbance at 280 nm
and one must use 214 (or 220) nm, there is the possibility that
"impurities" (e.g. buffers) may also abosrb at that wavelength (the
presence of salts, incidentally, is also the reason why one can not just
weigh the lyophilized peptide and use the weight and the peptide MW to make
a solution of known concentration). Therefore, measuring the UV spectrum
of a peptide solution may not be adequate due to interefences, and one may
have to use HPLC with UV detection to separate the peptide of interest from
any UV-absorbiong impurities.
Ioannis Papayannopoulos
CytoMed, Inc.
Cambridge, MA