Again, the method described by Gill & von Hippel is based on statistical averages. Mach et al. (Anal. Biochem. 1992, 200, 74) later revised these values. Pace et al. (Protein Science, 1995, 4, 2411) again re-determined these averages, so if anything, the values from Pace should be used. Pace also refers back to the Edelhoch method as the most reliable method.
Here is the abstract from Pace's paper: The molar absorption coefficient, epsilon, of a protein is usually based on concentrations measured by dry weight, nitrogen, or amino acid analysis. The studies reported here suggest that the Edelhoch method is the best method for measuring epsilon for a protein. (This method is described by Gill and von Hippel [1989, Anal Biochem 182:319- 3261 and is based on data from Edelhoch [1967, Biochemistry 6:1948-19541.) The absorbance of a protein at 280 nm depends on the content of Trp, Tyr, and cystine (disulfide bonds). The average epsilon values for these chromophores in a sample of 18 well-characterized proteins have been estimated, and the epsilon values in water, propanol, 6 M guanidine hydrochloride (GdnHCI), and 8 M urea have been measured. For Trp, the average epsilon values for the proteins are less than the epsilon values measured in any of the solvents. For Tyr, the average epsilon values for the proteins are intermediate between those measured in 6 M GdnHCl and those measured in propanol. Based on a sample of 116 measured epsilon values for 80 proteins, the epsilon at 280 nm of a folded protein in water, epsilon(280), can best be predicted with this equation: epsilon (280) M-1 cm-1 = (#Trp)(5,500) + (#Tyr)(1,490) + (#cystine)(125) These epsilon(280) values are quite reliable for proteins containing Trp residues, and less reliable for proteins that do not. However, the Edelhoch method is convenient and accurate, and the best approach is to measure rather than predict epsilon. Cheers! MM On Jun 16, 2011, at 8:05 PM, Scott Pegan wrote: Here is also a very effective method: 1 Gill, S. & Hippel, P. v. Calculation of protein extinction coefficients from amino acid sequence data. Analytical Biochemistry 182, 319-326, (1989). On Thu, Jun 16, 2011 at 5:56 PM, Filip Van Petegem <filip.vanpete...@gmail.com<mailto:filip.vanpete...@gmail.com>> wrote: A convenient fast way is the earlier mentioned Edelhoch method, as described in this paper which is referenced on the popular Protparam tool: http://onlinelibrary.wiley.com/doi/10.1002/pro.5560041120/pdf Filip On Thu, Jun 16, 2011 at 4:45 PM, aaleshin <aales...@burnham.org<mailto:aales...@burnham.org>> wrote: Mischa, You intrigued me. What is the experimental technique for the Extinction Coefficient measurement (which requires knowledge of protein concentration)? Let me guess, Bradford? Protein evaporation and weighing? Alex On Jun 16, 2011, at 4:22 PM, Machius, Mischa Christian wrote: With respect to the Edelhoch method and the ProtParam server, I would strongly recommend determining extinction coefficients experimentally and not rely on the ProtParam values. The reason is that the underlying extinction coefficients in the formula used by ProtParam and referenced there are statistical averages. They may or may not be valid for a given protein. I have seen differences of more than 20% between the "theoretical" and "experimental" extinction coefficients, particularly for proteins with few Trp and Tyr residues. When relying on relative concentrations, this inaccuracy is not detrimental, but when absolute concentrations are needed (CD, AUC, ITC, any binding experiment, etc.), such a difference would be considered huge. Determining an extinction coefficient experimentally takes but a few minutes. Cheers! MM On Jun 16, 2011, at 6:22 PM, Petr Leiman wrote: Totally support the statements below. We have had several proteins with A280 absorbance of 0.1 or less (at 1 mg/ml). You _have_ to use Bradford in the Nanodrop or whatnot to measure the concentration. Before purchasing the Nanodrop we used a Hellma TrayCell and a "normal" UV/Vis instrument. Similar to the Nanodrop, the sample volume in TrayCell is 2-3 ul. Traycell works well at a fraction of the Nanodrop cost, but Nanodrop is a lot more convenient to use for high concentration quick measurements (especially if you need to measure several things in succession), so you get what you pay for. Petr P.S. Expasy's Protparam tool has been around for ages (10-12+ years?). That plus the Nanodrop are two essential and synergetic tools of a protein chemist/crystallographer. On Jun 16, 2011, at 10:31 PM, Edward A. Berry wrote: Bradford is an assay, Nanodrop is a spectrophotometer. Both the A280 and Bradford methods are strongly dependent on amino acid composition, so unless you correct A280 for that as mentioned by Filip, either one is semiquantitative. Occasionally you come across a protein with no tryptophan which will have a much lower extinction coefficient. Try making a 1 g/l solution of gelatin (collagen?) and see what its A280 is! I noticed recently the "protparam" tool at http://ca.expasy.org/cgi-bin/protparam estimates the extinction coefficient given a sequence. David Briggs wrote: ~~~ I wouldn't touch Bradford with a barge-pole. I've found it to be wildly inaccurate for certain proteins I've handled, where as the OD280 measurements have been fine. One wonders what does "fine" mean, like same as with Biuret or Kjeldahl nitrogen, or solution made up by weight? ----------------------------------------------------------------------- Mischa Machius, PhD Director, Center for Structural Biology Assoc. Professor, Dept. of Pharmacology Member, Lineberger Comprehensive Cancer Center University of North Carolina 4079 Genetic Medicine CB#7365 120 Mason Farm Road Chapel Hill, NC 27599-7365, U.S.A. tel: +1-919-843-4485<tel:%2B1-919-843-4485> fax: +1-919-966-5640<tel:%2B1-919-966-5640> email: mach...@unc.edu<mailto:mach...@med.unc.edu> -- Filip Van Petegem, PhD Assistant Professor The University of British Columbia Dept. of Biochemistry and Molecular Biology 2350 Health Sciences Mall - Rm 2.356 Vancouver, V6T 1Z3 phone: +1 604 827 4267<tel:%2B1%20604%20827%204267> email: filip.vanpete...@gmail.com<mailto:filip.vanpete...@gmail.com> http://crg.ubc.ca/VanPetegem/ -- Scott D. Pegan, Ph.D. Assistant Professor Chemistry & Biochemistry University of Denver Office: 303 871 2533 Fax: 303 871 2254 ----------------------------------------------------------------------- Mischa Machius, PhD Director, Center for Structural Biology Assoc. Professor, Dept. of Pharmacology Member, Lineberger Comprehensive Cancer Center University of North Carolina 4079 Genetic Medicine CB#7365 120 Mason Farm Road Chapel Hill, NC 27599-7365, U.S.A. tel: +1-919-843-4485 fax: +1-919-966-5640 email: mach...@unc.edu<mailto:mach...@med.unc.edu>
