calculating extinction coefficients at 214 nm for proteins

[grumbledook]

Although published in a rather odd journal, I foundthis paper
quite useful. When I compare integrated peak areas at 280 nm
with peak areas at 214 nm for different proteins using calculated extinction coefficients
according to this paper or Pace et.al (1995), resp. , I get pretty accurate and similar results, w/o
using a calibration curve. (The equation from Buck et.al 1989 is rather useless.) So if you
are dealing with a lot of different proteins and would like to estimate puritiy and quantity
after purification by HPLC, you may find this paper useful as well. The interesting thing is,
that the extinction coefficients at 214 differ quite remarkably between proteins (>30%!). So
just using BSA for calibration may not be very helpful. And I found that the determination of
the so called instrument response factor is indeed unnecessary if you use a modern
detection system reporting (m)AU instead of (m)V and calculated coefficients, as one
would assume. (You don't calculate response factors for you spectrophotometer, do you?)
But since I did this only with a small set of proteins so far I
would be interested in the experience of other users in this forum.

[bendicks]

hello grumbledook !
Thank you for your post. I have just found it as I was looking for a way to calculate the protein content from the peak area. But sorry to ask this question, but what kind of equation do you use to calculate the protein content
Thanks in advance for your answer.

[DSP007]

yes, it is useful
IMHO

This effect is associated with various concentrations of tryptophan and phenylalanine in different proteins.

[HW Mueller]

grumbledook,
we just discussed the Beer-Lambert law and UV-spectrophotomers/detectors. One can easily make a mistake here if one is not very familiar with the subject.
DSP007,
at 280 nm the aromatic ring amino acids absorb, at 214 nm it is mainly the peptide linkage.

[DSP007]

DSP007,
at 280 nm the aromatic ring amino acids absorb, at 214 nm it is mainly the peptide linkage.

Yes, of course.
At 214 optical dencity of aromatic ring and amides summarized. The ratio of optical densities for 2 or more wavelengths is a good way to confirm the identity .

[HW Mueller]

Why would the absorption of aromatics and peptide be summarized (summed?) at 214 nm?

[DSP007]

Why would the absorption of aromatics and peptide be summarized (summed?) at 214 nm?

Well .
http://www.chem.asu.ru/org/fcmi/fcmi04.pdf
I think the lecture is clear and without translation.
Aromatic has absorption also in the short wavelengths

[HW Mueller]

OK, what I tried to say is that the Beer-Lambert law holds very well only for dilute solutions, a protein is a highly concentrated arrangement of chromophores which are so close together that they can interfere, etc. Now, some people publish on how to use the absorption idosyncracies for determining structue, matrix, chemical and physical conditions, . . . . , while others show how linearily additive the absorbances are. In a scenarium like that you know that one or the other is pushing things a bit.

[grumbledook]

OK, what I tried to say is that the Beer-Lambert law holds very well only for dilute solutions, a protein is a highly concentrated arrangement of chromophores which are so close together that they can interfere, etc. Now, some people publish on how to use the absorption idosyncracies for determining structue, matrix, chemical and physical conditions, . . . . , while others show how linearily additive the absorbances are. In a scenarium like that you know that one or the other is pushing things a bit.

Interesting to see my post floating up again

Of course one needs to know the nature of the beast to some extend.
But to come back to my original message:

The calculation of absorption coefficients for proteins at 280 nm is pretty well accepted in the protein science community
and has been shown to give pretty accurate values as long as the protein contains Trp's and not only Tyr's.
This has been validated by orthogonal methods. And although the error might be high in some cases, it is on
average surprisingly low. And furthermore, for many projects there is NO alternative. Even the orthogonal
methods need extensive validation and require rather high amounts of protein or a standard which is in many
cases not available. So UV absorption is the method if choice for most research projects.

I had the chance to compare the measured and calculated A280 and A214 (according to Kuipers and Gruppen)
for a pretty large set of very different proteins using RP-HPLC and a DAD. Taking IPA at 280 nm as internal reference
I plotted the calculated eps214 vs measured eps214 (ranging from 14 to 24 for 1 mg/ml) and obtained
a correlation coefficient of 0.96. This means that the Kuipers and Gruppen method is as reliable
as the 280 method according to Pace, which I find a pretty striking result.

[HW Mueller]

Well, there are some differences in the working methods of a biochemist and a physical organic chemist.

[grumbledook]

Well, most of the people involved in developing spectroscopic methods for protein quantitation
were/are (physical) organic chemists by training (e.g. Nick Pace).

The problem with proteins is that it is almost impossible to
determine their dry weight because there will be always residual
water bound after lyophilization which is hard to quantify. And if you use
harsh conditions to get rid of the water completely the protein is "dead".
And in many cases you just don't have enough of it. So, although
the A280 method may come with an average error of 10 % or less, it
is still the best method one can use when dealing with a purified(!) protein.
This has nothing to do with "working methods" but with the nature of the
beast. So get down off you high horse.

[HW Mueller]

If you call it guesswok or estimations, ok. But often it would be better to just do other research were you don´t need to guess.
Incidentally, I wonder where you take the right to place me on a high horse.

[grumbledook]

You're still up there, aren't you?

Anyway, everything I wanted to when I started this topic was to make other
protein scientists aware of this new method for "estimating" protein
concentrations using far UV detection.

Note: good discussions of UV spectroscopy of proteins can be found
in Cantor & Schimmel "Biophysical Chemistry" Pt. 2
and in Alexander Demchenko "Ultraviolet Spectroscopy of Proteins"

[tom jupille]

Well, there are some differences in the working methods of a biochemist and a physical organic chemist.

As I recall from my undergraduate days, the definitions ran something like this:
- a physical chemist makes precise measurements on impure compounds.
- a physical organic chemist makes precise measurements on pure compounds
- an organic chemist makes imprecise measurements on pure compounds
- a biochemist makes imprecise measurements on impure compounds.
- a biologist asks "What's a measurement?"

My biologist friends used to rearrange those definitions somewhat !