Lucie Vyletova

Absorbance of BSA (c=1mg/ml) measured at λ=280nm and λ=260 nm

Absorbance AMEAN SD
λ=280nm 0.592 0.590 0.590 0.592 0.592 0.5912 0.0010
λ=260 nm 0.362 0.362 0.361 0.361 0.360 0.3612 0.0007

Determination of BSA concentration:

ε (BSA) = 0.667 mg-1 cm-1, ε (BSA) = 43,630 M-1cm-1

A
C= = 0.5912 / 0.667 = 0.886 mg/ml
ε ×l

A
C= = 0.5912 /43,630 = 1.355 x 10-5M
ε ×l

Christian-Warburg Method:

CProt ( mg/ml) = 1.550 x A280 – 0.757 x A260

CPROT = 1.550 x 0.5912 – 0.757 x 0.3612 = 0.643 mg/ml

CNA = 62.9 A260 – 36.0 x A280

CNA = 62.9 x 0.3612 – 36.0 x 0.5912 = 1.436 µg/ml

Quantitating of Sulphohydryl Groups Using a Cysteine Standard

Absorbance Absorbance
measurement at λ=412 nm measurement at λ=412 nm
Standard [Standard]/mM (Martin) (Lucie)
A 1.50 1.906 2.025
B 1.25 1.652 1.6
C 1.00 1.204 1.398
D 0.75 0.896 1.038
E 0.50 0.703 0.777
F 0.25 0.305 0.399
G 0.00 0 0
Key: The same samples measured on 2 different spectrophotometers

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Lucie Vyletova

Quantitating of Sulphohydryl Groups Using a Cysteine Standard; A

Graph
2.5 of Cysteine concentration (mM) versus Absorbance Measured
at λ=412 nm

2
Absorbance Measured at λ=412 nm

f(x) = 1.35 x
R²
f(x)==11.27 x
R² = 1

1.5

Lucie's
measurement
0.5

0
0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60
Cysteine concentration (mM)

Quantitating of Sulphohydryl Groups Using a Cysteine Standard by Measurement of Absorbance at λ=412 nm

Absorbance Absorbance (2nd attempt on Martin's spectrophotometer,
(Measured on Lucie’s
Unknown however with reaction buffer from a different group as we run
spectrophotometer, repeated as too out of the stock solution
strange data obtained)
BSA I 0.023 0.034
BSA II 0.029 0.036
BSA III 0.031 0.027
AMEAN 0.0277 0.0323
SD 0.0042 0.0047

Determination of –SH group concentration:

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Lucie Vyletova

ε (TNB2-)= 14,150 M-1cm-1 250µl in 2.8 ml→ DF = 2.8ml/0.25ml = 11.2

A
[-SH] = x DF
ε ×l

[-SH] =0.0323/1x14,150 x 11.2 = 2.283 x 10-6 M x 11.2 = 2.56 x 10-5 M =2.56 x 10-5 mmol/ml

[-SH] = 2.56 x 10-5 M =2.56 x 10-5 mmol/ml = 2.56 x 10-2 µmol/ml

Stoichiometry result

[−SH ] 2.56 x 10−5

= = 1.9 ?????
[P] 1.355 x 10−5

Determination of –SH group concentration from a graph:

A graph equation for a Lucie’s calibration curve: y = 1.3542x & AMEAN=0.0277

[-SH] = 0.0277/1.3542 x DF = 0.020 x 11.2 =0.229 mM = 0.229 mmol/l = 0.229 µmol/ml ????? (a
difference in results in a one decimate place)

A graph equation for a Martin’s calibration curve: y = 1.2708x & AMEAN=0.0323

[-SH] = 0.0323/1.2708 x DF = 0.022 x 11.2 =0.285 mM = 0.285 mmol/l = 0.285 µmol/ml

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Lucie Vyletova