1.

Sample preparation

1 gram sample

1. Crushed
2. Added 10 mL aquades
3. Centrifuged at 3500 rpm for 10 minutes
4. Decantated

Filtrate Residue

2. Making of the standard

1 mL protein standard 1 mg/mL; 2 mg/mL; 3 mg/mL;
4 mg/mL; 5 mg/mL

1. Added 5 mL biuret reagent

2. Incubated at 37⁰C for 10 minuntes
3. Let in room temperature for 10 minutes
4. Measured absorbance at 540 nm
Absorbance

Reaction :
 CuSO4.5H2O (aq) + 2NaOH (aq) → Cu(OH)2 (aq) + Na2SO4 (aq) + 5H2O (l)
 Cu(OH)2 (aq) Cu2+ (aq) + 2OH- (aq)

O OH
O OH
C
C
R CH
R CH
H N
H N
O C
O C
R CH
R CH

H N
H N

O C O C

R CH R CH

NH2 (aq) + Cu2+ (aq)  NH2 (aq) +

3. Determination the absorbance of blanco solution

1 mL aquades

1. Added 5 mL biuret reagent

2. Incubated at 37⁰C for 10 minuntes
3. Let in room temperature for 30 minutes
4. Measured absorbance at 540 nm
Absorbance

4. Determination the absorbance of sample

1 mL protein filtrate

1. Added 5 mL biuret reagent

2. Incubated at 37⁰C for 10 mins
3. Let in room temperature for 30 minutes
4. Measured absorbance at 540 nm
Absorbance

Reaction :
 CuSO4.5H2O (aq) + 2NaOH (aq) → Cu(OH)2 (aq) + Na2SO4 (aq) + 5H2O (l)
 Cu(OH)2 (aq) Cu2+ (aq) + 2OH- (aq)

O OH
O OH
C
C
R CH
R CH
H N
H N
O C
O C
R CH
R CH

H N
H N

O C O C

R CH R CH

NH2 (aq) + Cu2+ (aq)  NH2 (aq) +