Biomedical Engineering Reference
In-Depth Information
and the disulfide bond absorb at about 280 nm. The peptide bonds
absorb light below 215 nm with much higher absorption coeffi-
cients, but in this sector a lot of substances also absorb. Nucleic
acids show absorption with a broad band with a maximum around
260 nm. Since for most proteins specific absorption coefficients are
unknownandthisdataarewithoutvalueinmixturesofseveralpro-
teins, some equations are developed to overcome this problem. It
should be kept in mind that none of these equations gives a “right”
value if protein mixtures are measured.
The measurement has to be done against the protein-free sol-
vent (buffer). If a single-beam photometer is used, the absorbance
of this blank has to be subtracted from that of the protein solution.
Pay attention to the path of the cuvettes! Equations a) to d) are
made for
10.0 mm
.
a) Wa r b u r g and Christian equation (cf. Protocol 1.2.5):
mg protein
A
1cm
260
This estimation of protein concentration is valid up to 20% (w/v)
nucleic acid or an A
280
/A
260
ratio
<
0.6.
b) Kalckar and Shafran equation:
mg protein
/
=
A
1cm
ml
1.55
·
280
−0.76
·
/
=
A
1cm
280
A
1cm
260
ml
1.45
·
−0.74
·
c) Whitaker and Granum equation:
mg Protein
280
:2.51
d) Concentration of immunoglobulins (IgG; cf. Table 8.11 for di-
vergent absorption coefficients)
A
1cm
235
/
=
−A
1cm
ml
/
=
A
1cm
280
mg IgG
ml
:1.38
e) Beer-Lambert law
log
10
I
0
I
λ
=
=
ε
λ
·
c
·
d
A
λ
A
: absorbance at wavelength
;I
0
: intensity of incident light;
λ
ε
λ
: (molar) absorption coeffi-
I: intensity of transmitted light;
λ
cient at wavelength
; c: concentration; d: length of optical path
within the cuvette.
If solutions of pure proteins with known amino acid sequence
or composition are measured, the concentration c (mol/l) is cal-
culated from the absorbances at 280 nm (A
280
), 320 nm (A
320
),
350 nm (A
350
), and the number of tryptophan (n
Tr p
)andtyro-
sine residues (n
Ty r
) and the number of disulfide bridges (n
S−S
)
according to equation f):
f)
E
280
−10
(2.5
·
lgE
320
−1.5
·
E
350
)
=
c
5540
·
n
Tr p
+ 1480
·
n
Ty r
+ 134
·
n
S−S
