Determination of serum proteins concentration

THEORETICAL BASIS

Proteins in serum are found in high concentration. They have different functions. Proteins don't have unique molecular weights because they are a mixture of different albumins and globulins. That's why their concentration unit is g/L instead of mol/L or mmol/L.

PROCEDURE

Serum proteins are determined using the Biuret method. The measured reaction product has a violet colour. The name Biuret arises from the word biurea, which is a compound produced when urea is warmed at 180 °C.

urea and biurea

Cu²⁺ ions in the Biuret reagent react with biurea at alkaline pH and form a violet complex. The same colour appears when proteins and peptides are present in the sample because they have two peptide (-CONH) bonds.

The intensity of colour and the serum proteins concentration are directly proportional.

Reference interval serum proteins: 65 - 80 g/L.

EQUIPMENT AND REAGENTS

pipettes,
beakers,
test tubes,
alcohol marker,
serum sample,
control bovine serum for medical biochemistry, ordered by Sanolabor, manufactured by Randox,
standard protein solution,
Biuret reagent (undiluted)

PREPARATION:
4.5 g KNa tartrate
40 mL 0.2 M NaOH
1.5 g CuSO₄ · 5 H₂O
0.5 g KJ
0.2 M NaOH do 100 mL,

Diluting solution

PREPARATION:
5 g KJ
0.2 M NaOH do 1000 mL,
spectrometer SPEKTRA^TM

Working reagent is diluted Biuret reagent. The dilution of Biuret reagent is: 1 part of Biuret reagent and 4 parts of Diluting solution of (1 : 5 or 1 + 4).

HAZARDS

	NaOH: causes severe skin burns and eye damage, may be corrosive to metals R: 35 S: 26, 36/37/39, 45
	CuSO₄ · 5 H₂O: harmful if swallowed, causes serious eye irritation, causes skin irritation, very toxic to aquatic life with long lasting effects R: 22, 36/38, 50/53 S: 22, 60, 61

PROCEDURE

Appropriate volume of working reagent has to be prepared.

In labelled test tubes add:

Reagents	Blank sample (B)	Standard sample (St)	Sample (vz)
working reagent	400 µL	400 µL	400 µL
standard sol. of. prot.	-	10 µL	-
sample (serum)	-	-	10 µL
purified water	10 µL	-	-

Mix contents of test tubes well and let stand at room temperature for 30 min until the chemical reaction has run. Spectrometer is adjusted with sample blank to 100 % transmittance, then the standard and samples are measured. The measuring wavelength is 546 nm (the green light). The absorbance of all samples must be calculated.

Equation for calculation of sample concentration: C_vz = A_vz ·	C_st
	A_st

C_vz - concentration of sample
A_vz - absorbance of sample
C_st - concentration of standard
A_st - absorbance of standard

DATA, MEASURED VALUES AND CALCULUS

C_st = _____________

T_st (measured)= _____________ A_st (calculated)= _____________

T_vz (measured)= _____________ A_vz (calculated)= _____________

Calculation of protein concentration: _____________

RESULT: C_vz = _____________

THE ACCURATE VALUE ( offered by manufacturer of control serum, ordered by Sanolabor, manufacturer Randox): _____________

% of inaccuracy = _____________

Prepared by : Sabina Povhe Lenart, Secondary school for pharmacy, cosmetics and health care, Ljubljana