CHEM 160.

1
ESTIMATION OF PROTEIN
CONCENTRATION BY
SPECTROPHOTOMETRY
2
nd
semester 2013-2014
DETERMINATION OF PROTEIN
CONCENTRATION
WHY?
1. Report progress of purification by
estimating the amount of protein
recovered in each step
2. Identify significant amount of
contaminating proteins removed in the
sample
3. Estimate the amount of protein to be
loaded in an assay for test of purity,
biological activity or structural stability
CHOOSING THE APPROPRIATE
METHOD
Mass and volume of protein sample
Specificity of the assay to particular
proteins of interest
Chemicals in the sample that
might interfere in the
measurements
Repeatability and reproducibility
of standard data
Time to complete the method
ESTIMATION OF PROTEIN
CONCENTRATION BY
SPECTROPHOTOMETRY
SPECTROPHOTOMETRY
Transmittance (T) = I/I
o

Absorbance = -log T = abc
T, |A, | c or [ ]
SPECTROPHOTOMETRY
VISIBLE RANGE:
400 nm (violet ) 700 nm (deep red)
At 540 nm,
A : green
T : violet/ purple
HOW THE WAVELENGTH OF
LIGHT IS CONTROLLED
Light source monochromator sample holder
detector read-out
BIURET REACTION
1.5 g CuSO
4
5H
2
O
6.0 g NaKC
4
H
4
O
6
H
2
O
Prevents reduction of Cu
2+
Cu
+

500 mL distilled H
2
O
300 mL NaOH
Enhances complexation of Cu
2+

with peptide N
General test for peptide chains of
AT LEAST three (3) amino acids;
i.e. 2 peptide bonds
Cu
2+
R CH
O=C
H-N
H-N
O=C
HC R
C=O
N-H
N-H
C=O
PURPLE COMPLEX
More intense purple color
More # of peptide bonds
More protein in sample
Higher [protein]
LIMITATIONS OF BIURET METHOD
1. low sensitivity and accuracy
2. high presence of interferences
Buffers that are peptide in nature
Reducing sugars
NH
3
forms complex with Cu
2+

(NH
4
)
2
SO
4
precipitated proteins do
not give accurate results
SOLUTIONS TO INTERFERENCES:
Addition of KI
Protein precipitation by TCA
STANDARD CURVE
A
540

Concn (mg/mL)
STANDARD solution of known [ ]
PROTEIN STANDARD FOR ESTIMATING
PROTEIN CONCENTRATION :
BOVINE SERUM ALBUMIN (BSA)
STANDARD SOLUTIONS
mg BSA/mL mL BSA mL NaOH
0 0 1
2 0.1 0.9
4 0.2 0.8
6 0.3 0.7
8 0.4 0.6
10 0.5 0.5
SAMPLE : EGG ALBUMIN ISOLATE
600 mg
120 mg/mL
- dissolve in 5 mL 0.5 M NaOH
DILUTION OF SAMPLES
1 in 2
1 in 5
1 in 7
1 in 10
DILUTION OF SAMPLES
e.g. 1 in 5 dilution or 1: 4 dilution
If total volume is 5 mL :
120 mg
mL
1 mL albumin soln
+ 4 mL NaOH
DF = 5 mL
1 mL
BIURET ASSAY
1 mL of standard or diluted sample
Add 4 mL of Biurets reagent
Read Absorbance at 540 nm
DETERMINATION OF PROTEIN
CONCENTRATION
Prepare standard
BSA solns
Biuret assay
Construct standard curve
(A
540
vs. concn)
Prepare albumin
isolate solns
(stock and diluted)
Biuret assay
Determine concn of
each soln from
standard curve
DETERMINATION OF PROTEIN
CONCENTRATION
A
5
4
0

Concn (mg/mL)
0.2
0.4
0.6
0.8
2 4 6 8 10
Abs = 0.5
1 in 5 dilution
gave an
[ ] = 6 mg/mL
Then, from std curve
DETERMINATION OF PROTEIN
CONCENTRATION
mg protein/mL isolate = [ ] x DF
For a 1 in 5 dilution
= 6 mg 5 mL
mL 1 mL
= 30 mg/mL
g protein = 30 mg/mL x 100
100 g isolate 120 mg/mL
= 25 % protein
Sample Calculations
The protein content of an unknown sample was
determined using BSA as standard. The absorbance
values of the standard solutions and samples are as
follows. Determine the protein concentration of the
sample in g/mL.

BSA
concentration
(g/mL)

Absorbance

Samples

Absorbance
0.00 0 1 in 2
dilution
0.779
0.20 0.057
0.40 0.115
0.60 0.270 1 in 10
dilution
0.315
0.80 0.330
1.0 0.400
1.2 0.443
LIMITATIONS OF BEERS LAW IN
SPECTROPHOTOMETRY
Use of only dilute solns
Monochromatic light
Light that reaches detector should only be
transmitted light
Only one absorbing species should be
present
SOURCES OF ERRORS IN THE EXPERIMENT
Instrumental error
Voltage fluctuations
Operator errors
Lack of experience in experiments
Impurities in the sample
Degradation of stored protein standards
Absorbing species
Interfering substances during measurements
OTHER METHODS
OF DETERMINING
PROTEIN
CONCENTRATION
WARBURG-CHRISTIAN METHOD
Based on the relative absorbance of
proteins at 280 and 260 nm
Presence of UV-absorbing compounds
DISADVANTAGE/ INTERFERENCE
ADVANTAGES
Fast
Non-destructive to sample
LOWRY METHOD
Biuret + Folin-Ciocalteau reagent
Time-consuming since slow color devt
DISADVANTAGE
ADVANTAGE
Sensitive; gives accurate results
1. Formation of Biuret complex (Cu
2+
Cu
+
)
2. Folin rgt: (Cu
+

Cu
2+
)
Reduction of Trp by phosphotungstic and
phosphomolybdic acid
Yellow to blue (detection at 750 nm)
BRADFORD METHOD
Dye-binding method
Coomassie brilliant blue G-250
Color absorbed by glassware
Rapid color development
DISADVANTAGES
INTERFERENCES
Strongly alkaline buffers
Detergents (SDS)
ADVANTAGES
Fast; sensitive and easy
QUESTIONS
What is the effect of the following on the
absorbance of sample?
Presence of precipitate
Turbidity of mixture
Dirty cuvet
Stray radiation Absorbance
| Absorbance
| Absorbance
| Absorbance
A 0.1 M soln of compound Z (MM=160 g/mol)
has an absorbance of 0.460 at 540 nm. What
is the [ ] in % w/v of this compound if it has
an absorbance of 0.623 at 540 nm?
A = abc
A
a =
bc
0.460 0.623
=
0.1 x
x = 0.135 M
| | | |
=
| |
\ . \ .
mol g g
0.135 160 21.6
L mol L
( )
| |
|
\ .
21.6g
100 = 2.16%(w/v)
1000mL