Decrease in exctinction coefficient of Cy-dye after binding to protein?

Joachim Goedhart jgoed at MPC186.MPIBPC.GWDG.DE
Tue Apr 2 02:32:41 EST 1996


As a student I'm involved in research concerning the EGF-receptor.
Recently, we've labeled murine-EGF with Cy5.
Only one Cy5 can be attached to mEGF (at its N-terminus).
The labeled EGF is purified by gel-filtration followed by reversed-phase HPL=
When the dye/protein ratio is determined from the absorption spectrum, a
dye/protein ratio of approx. 0.5 is observed. This is reproducible, and is
also true for labeling with Cy3.
To correct for this, other people in our lab use an absorption coefficient
for "reacted" Cy5 which is lower than the value found in literature, or as
specified by the company (biological detection systems), which is 250,000
M-1 cm-1 in both case.
Another strange thing is that in the data-sheet of the company, they show
an exampe, of how to calculate the dye/protein ratio. In this example they
use 200,000 as an exctinction coeefficient, without explanation!

My question is the following:
Is it possible that the absorption coefficient of a dye, especially
Cy-dyes, decreases (by a factor of approx. 1.5-2) upon binding to EGF or
any other protein?
If not, are there suggestions for factors which might suggest a low
dye/protein ratio?

Another question concerns the following. The absorption spectrum of unbound
Cy5-dye consists normally of a maximum at 650, and a shoulder at 600 nm.
After labeling avidin with an estimated dye-protein ratio of 1 the shoulder
became a small peak. When the avidin was labeled with a dye/protein ratio
of 4, the absorption spectrum showed two peaks with almost equal absorption
values (the peak at 600 nm still somewhat lower than at 650 nm). Thisam
effect, i.e. the shoulder becoming a peak, is also observed when antibodies
are labeled at dye/protein ratios of higher than 3.

Is this effect caused by a change in local environment of the dye, by which
the e.g. the S2-S0 transition becomes more significant (or the S1-S0
transition becomes less significant)?


Joachim Goedhart
MPI f=FCr biophysikalische Chemie
Abteilung Molekulare Biologie
Am Fa=DFberg 11
D-37077 G=F6ttingen

phone: +49-(0)551-201 1388
fax:    +49-(0)551-201 1467

More information about the Cellbiol mailing list