Biomedical Engineering Reference
In-Depth Information
1.2.5 Determination of Nucleic Acids by UV Absorption
The UV absorption of nucleic acids depends strongly on their na-
ture and solvent conditions as pH, ionic strength, and temperature.
Approximate absorption coefficients are given in Tables 1.8 and 1.9.
Using these coefficients, yield is calculated during chromatography,
ultracentrifugation, and other preparative processes with sufficient
precision.
According to Wa r b u r g and Christian the total amount of
nucleic acids and protein is calculated using the following equation
(the factors F and T are given in Table 1.10 in conjunction with the
ratio A
280
/A
260
):
P
1−
T
=
T
·
A
280
·
F
·
T
=
N
(1 −
T
)
N: concentration of nucleic acids (in mg
d
·
/
ml); P: concentration of
/
protein (in mg
ml); d: optical path (in cm); T: [nucleic acid ex-
pressed as part of the total (protein + nucleic acid)]; and factor F
from Table 1.10.
References
Warburg O, Christian W (1941) Biochem Z 310:384
Webb JM, Levy HB (1958) In: Glick D (ed.) Methods in biochemical analysis,
vol. 6, pp. 1-30, Wiley, New York
Table 1.8.
Absorption coefficients of nucleotides (nucleic acids)
A
1mg
/
ml
260
ε
1cm
260
10
−3
×
pH 2
pH 7
pH 12
Ribonucleotides
10.55
10.85
10.3
28.5
Deoxyribonucleotides
10.3
10.45
10.1
22.4
Table 1.9.
Conversation factors for nucleic acids
=
=
µ
/
1O.D.
260
double-stranded
50
g
ml
0.15 mM nucleotides
DNA (dsDNA)
1O.D.
260
single-stranded
=
=
µ
/
33
g
ml
0.1 mM nucleotides
DNA (ssDNA)
1O.D.
260
single-stranded
=
=
µ
/
40
g
ml
0.11 mM nucleotides
RNA (ssRNA)
1
=
µ
g DNA (1000 bp)
1.52 pMoles
nucleotides
=
µ
1 pMol DNA (1000 bp)
0.66
g DNA
=
1000 kD dsDNA
1 520 000 bp
=
=
1000 bp DNA
333 amino acids
37 000 D protein
=
50 000 D Protein
1350 bp DNA
