285 PHL

Lab # 2
Plasma Proteins
Proteins: Classification
1- Simple proteins
e.g. albumin and globulin
2- Conjugated proteins
Protein + prosthetic gp.
e.g. lipoprotein
3- Derived proteins
Derived from simple proteins (denaturation)
e.g. peptones


Plasma Proteins
Plasma contains both simple and conjugated proteins
Examples
Albumin, globulin, lipoproteins e.t.c.



Albumin Globulin
- Low mol. wt.
- Soluble in sat. soln. of
ammonium sulfate

- High mol. wt.
- Insoluble in sat. soln. of
ammonium sulfate


Determination of plasma proteins

Steps:
1- Estimate total protein
2- Fractionation (e.g. salt fractionation) to ppt. globulin and measure
albumin
3- Globulin = total protein albumin

Determination of plasma proteins
BIURET METHOD
Principle
Cupric ions complex with the groups involved in the peptide
bond. In an alkaline medium and in the presence of at
least 2 peptide bonds, a violet- colored chelate is formed

Methods:
1- Manual
2- Kit

Determination of plasma proteins
Procedure (manual)
1- Total protein stand.
2- Albumin stand.
Pipette 6 ml of
Sulfate sulfite
Soln.
Add 0.4 ml stand.
serum
Invert to mix.
Remove at once
2 ml into t.t
labeled total
Protein stand.
Add 3 ml ether to the rest
of the mix.
Put a stopper
Invert 20 times to mix
Dont shake vigorously
Cap the tube
Centrifuge for 5 mins
The globulin layer will be formed
Tilt the tube & insert Pasteur
pipette into the clear soln.
below globulin layer
(dont disturb globulin layer)
Pipette the clear soln. into clean
t.t.
Take 2 ml of this soln. into anther
t.t.
Label it Albumin stand.
Determination of plasma proteins
Procedure
3- Total protein sample 4- Total albumin sample


5- Blank
In test tube labeled Blank add 2 ml of sulfate-sulfite soln.





Repeat all above steps using serum sample instead of standard sample
Determination of plasma proteins
Procedure




T Ptn St Alb. St Blank Alb. S T Ptn S
-Add 5 ml of Biuret reagent to each tube
-Place in water bath at 37C for 10
- Read the absorbance at 540 nm
Calculations
Serum total protein (gm/dl)=
abs. total protein sample x conc. of std (7 g/dl)
abs. total protein std

Serum albumin conc. (gm/dl)=
abs. albumin sample x conc. of std (3.5 g/dl)
abs. albumin std
Serum globulin conc. (gm/dl)= Total protein albumin



Determination of plasma proteins
Procedure (kit)

Standard Sample
Serum sample
--
0.2ml
Standard 0.2 ml
--
Working reagent 1 ml 1 ml
-Mix and place in water bath at 37C for 15 min.
-Read the absorbance at 540 nm

Determination of plasma proteins
Calculation:
Conc. Of total proteins (g/dl)= A sample x conc. Of st.(7)
A stand.
Normal values


Total protein: 4.2 - 5.6 g/dl
Albumin: 2.3 - 3.5 g/dl
Globulin: 1.9 2.1 g/dl
Clinical significance
Hyperproteinemia
Definition
An increase in total plasma proteins
Causes
Dehydration
Shock




Clinical significance
Hypoproteinemia
Definition
A decrease in total plasma proteins below the reference

causes
Renal diseases
Bleeding
Malabsorption
Malnutrition
Burns
High fever
Liver diseases