Bioch.

1
Lab 8
Determination Of an Unknown
Protein Solution
 Tests of protein
solution
1. Biuret’s test
2. Heat coagulation test
3. Xanthoproteic test
4. Rosenheim’s test
5. Full saturation test
6. Half saturation
 BIURET’S TEST

Biuret’s test
3 ml of the protein solution + 2 ml of 40% NaOH

+ Max. 3 drops of 1% cuso4 soln. Drop by drop  A rose to violet color

Test Observation Result

Rose to Violet color Solution is Protein

Biuret’s Test Solution is NOT Protein
No Violet color
(Carbohydrate)
 HEAT COAGULATION TEST

Procedure:
Add 10 ml egg white solution heat the upper part of the solution till boiling for few
minutes.
Result:
Notice the formation of the coagulum particularly at the top of the solution compared to
the rest of the solution.

Test Observation Result

Coagulum formation It is egg white (albumin)

Heat Coagulation
Not egg white , may be casien or
Test No coagulum formation
gelatin
 XANTHOPROTEIC TEST

2 ml of protein solution + 1 ml conc. HNO3 (SLOWLY ON THE SIDE WALL)

 yellow color or precipitate  cool  add 5 ml of 40% NAOH
 deep orange color or ppt.

Test Observation Result

Yellow ppt. Protein is egg white, caseinogens

Xanthoproteic Test Yellow Color Protein is Peptone

No Yellow color
Protein is Gelatin
(No color change)
 ROSENHEIM’S TEST
3 ml of the protein solution + 2 drops of ferric chloride formaldehyde reagent +
3 ml of conc. H2SO4 (run slowly on the wall )
 A violet or purple ring at the junction of the two fluid

Test Observation Result

Protein is egg white,
Violet Ring
caseinogens or peptone
Rosenheim’s Test
No Violet Ring Protein is Gelatin
 FULL SATURATION TEST

Principle :
Examine the effect of full saturation of egg white (albumin) .
Procedure :
Add (NH4)2SO4 solid salt directly to 10 ml of the egg white
solution with vigorous shaking until excess of the salt remains
undissolved.
Result :
A white precipitate of albumin is obtained in all the tube .

Test Observation Result

full saturation test white ppt in all the tube It is egg white (albumin)
 HALF SATURATION TEST

•Principle :
Examine the effect of half saturation of egg white (globulin and gelatin).
• Procedure :
To 10 ml of the protein saturation, add an equal volume of saturated
(NH4)2SO4 solution .
• Result :
A white precipitate is obtained in the middle of tube.

Test Observation Result

white ppt in middle the It is egg white (globulin) or
half saturation test tube gelatin
Biuret’s Test Xanthoproteic Test

3 ml of the protein solution 2 mL of protein solution

+ +
2 ml of 40% sodium hydroxide 1 mL conc. HNO3
+ (SLOWLY ON THE SIDE WALL)
Max. 3 drops of 1% CuSO4 soln. drop by drop
Heat
A rose to violet color
A Yellow color or precipitate

Rosenheim’s Test Cool  add 5 mL of 40% NaOH

3 mL of the protein solution

+ Deep Orange color or ppt.
2 drops of ferric chloride formaldehyde reagent
+
3 mL of conc. H2SO4
run slowly on the wall

A violet ring
at the junction of the two fluid
Half-saturation test
Procedure :

5 ml of the protein + equal volume of saturated (NH4)2SO4 solution  white precipitate in the middle
of tube.

Full-saturation test
Procedure :

7 ml of the egg white solution + (NH4)2SO4 solid salt  vigorous shaking until excess of the salt
remains undissolved  white precipitate in all the tube .

Heat Coagulation test

Procedure:

7 ml egg white solution  Heat the upper part of the solution till boiling  formation of
coagulum at the top of the solution
 Biuret’s Test

Blue Pink Violet

Is carbohydrate Is peptone Egg white or casein or gelatin

Confirmatory Heat
1- Xanthoproteic
2-Rosenheim coagulation