plasma protien

8/11/2019 Plasma Protien

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285 PHL

Lab # 2

Plasma Proteins


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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


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Plasma Proteins

Plasma contains both s imple and con jugated p roteins

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


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Determination of plasma proteins

Steps:1- Estimate total protein

2- Fractionation (e.g. salt fractionation) to ppt. globulin and measurealbumin

3- Globulin = total proteinalbumin


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BIURET METHOD

Principle

Cupric ions complex with the groups involved in the peptide

bond. In an alkaline medium and in the presence of atleast 2 peptide bonds, a violet- colored chelate is formed

Methods:

1- Manual

2- Kit


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Procedure (manual)

1- Total protein stand.

2- Albumin stand.

Pipette 6 ml of

Sulfatesulfite

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.


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Procedure

3- Total protein sample 4- Total albumin sample

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

Repeat all above steps using serum sample instead of standard sample


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Procedure

T Ptn St Alb. St BlankAlb. ST 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


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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 proteinalbumin


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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


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Calculation:Conc. Of total proteins (g/dl)= A sample x conc. Of st.(7)

A stand.


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Normal values

Total protein:4.2 - 5.6 g/dlAlbumin:2.3 - 3.5 g/dl

Globulin:1.92.1 g/dl


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Clinical significance

Hyperproteinemia

Definition

An increase in total plasma proteins

Causes

Dehydration

Shock


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Clinical significance

HypoproteinemiaDefinition

A decrease in total plasma proteins below the reference

causesRenal diseases

Bleeding

Malabsorption

MalnutritionBurns

High fever

Liver diseases

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