UNIT: Proteins
16tproteins.wpd
Task
Determination of Total Protein, Albumin and Globulins
Objectives
Upon completion of this exercise, the student will be able to:
1.
2.
3.
4.
5.
Explain the ratio of albumin and globulin in total protein.
Determine the total protein by Biuret Method.
Determine albumin by the BCG Method.
Determine globulin content.
Explain the clinical significance of abnormal protein values.
Introduction
Blood is a complex mixture of cells suspended in a fluid medium, plasma. 92-93% of this fluid
medium is water and the remaining 8% is dissolved proteins, minerals, glucose, etc. By far, the
largest amount of the total solutes are the plasma proteins, collectively referred to as TOTAL
PROTEIN. In serum, one of the plasma proteins, FIBRINOGEN, is missing, but it only constitutes 36% of the total protein in plasma. To standardize normal values, measurement of total protein has
been confined primarily to serum samples.
On the basis of their solubility properties, the serum proteins can be divided into two major fractions,
ALBUMIN and GLOBULINS. The globulins can be further subfractionated into four major fractions:
α1 globulin, α2 globulins, β globulins, and δ globulins. Serum proteins serve a number of different
functions. They constitute a portion of the amino acid pool of the body. They can be de-aminated
to give ketoacids which can provide caloric energy, or be transformed into carbohydrates and lipids.
Some serum proteins are also transport agents, carrying many vital metabolites, metal ions,
carbohydrates and lipids. Enzymes, antibodies, and certain hormones are also proteins. Serum
proteins are important in maintaining the osmotic pressure and the pH of the blood. Liver produced
fibrinogen and prothrombin proteins play a major role in coagulation. Fibrinogen constitutes
0.2-0.4 gm/dL of the protein in plasma. There is a small diurnal variation of total plasma protein of
about 0.5 gm/dL.
Increased protein concentration:
dehydration B all fractions affected equally
multiple myeloma (and related diseases) B one fraction greatly increased, usually gamma
Decreased protein concentration:
kidney damage extensive bleeding
severe burns
inadequate intake
deficient absorption
Albumin makes up about 60% of the total plasma proteins. It has a relatively small size (MW =
69,000). It is synthesized exclusively in the liver and acts as a carrier for many ions and watersoluble substances (calcium, bilirubin, fatty acids). It also acts to maintain osmotic pressure.
Albumin is increased in dehydration. It is decreased in renal disease (nephrotic syndrome with
protein lost in urine), liver insufficiency with decreased albumin synthesis, severe malnutrition, acute
and chronic inflammation, malignancy, pregnancy, and burns.
MLAB 2401 - Clinical Chemistry Lab Manual  H 137
UNIT: Proteins (continued)
The globulin fraction of protein can be obtained from electrophoresis or calculated by subtracting the
albumin from the total protein. The quantity of the globulin fraction is required to calculate the A/G
ratio.
Methods of Determination
1.
2.
TOTAL PROTEIN
a.
Kjeldahl Method C In this determination, the total nitrogen (protein nitrogen plus nonprotein nitrogen) is determined in the serum by Nessler's reaction after the proteins are
converted to ammonium sulfate. Calculations are needed to compensate for non-protein
nitrogen measured and to convert nitrogen measurement to protein concentration. Each
gm of nitrogen represents 6.25 grams of protein.)
b.
Folin-Ciocalteu C In this reaction, a deep blue colored compound is formed when a
phenol reagent is added to alkalinized protein solution.
c.
Refractometer C The refractive index of an aqueous solution increases directly with an
increase in solute concentration. Even though changes in concentration of different
substances may affect the refractive index unequally, the concentrations of electrolytes
and small organic molecules in serum usually do not vary greatly; hence, a change in
refractive index is indicative of a change in protein concentration. A rapid and direct way
of measuring the serum total proteins requiring only one drop of serum. Values obtained
by refractive index correlate well with those found by the Biuret method, but discrepancies
should be expected when abnormal serum proteins (paraproteins) are present or in
specimens that are lipemic, icteric, or hemolyzed.
d.
Biuret Method C All soluble proteins, on addition of a copper salt in alkaline solution
(NaOH), give purple colored complex which is generally known as Abiuret.@ This method
uses a standardized alkaline copper tartrate solution. The reaction occurs in the peptide
bonds of tripeptides or larger.
ALBUMIN
a.
Electrophoresis
b.
Biuret Method C The globulin in serum is precipitated by the addition of 23% sodium
sulfate (salting out). Ethyl-ether is added to help separate the precipitated globulin by
centrifugation. The chemical determination of albumin is based on the formation of a
purple colored complex (biuret) between alkaline copper sulfate and the albumin.
c.
Direct Dye-Binding C In recent years, the ability of albumin to combine with dyes has
been utilized. Methyl orange, bromcresol green (BCG) and 2,4-hydroxyazobenzene
benzoic acid (HABA) are the colored dyes most commonly used. Under ideal conditions,
the dye binds to serum albumin, but not to other proteins in serum.
H 138  MLAB - Clinical Chemistry 2401 Lab Manual
UNIT: Proteins (continued)
2.
GLOBULIN - Globulin is calculated by subtracting the measured albumin from the measured
total protein.
Globulin = Total Protein - Albumin
Equipment
1.
2.
3.
Spectrophotometer
Cuvettes & Pipets
Biuret Reagent
4.
5.
Albumin Color Reagent
NaCl solution (0.85%)
References
Kaplan, Alex. Clinical Chemistry.
Sigma Technical Bulletins, #540, #630
Bishop. Clinical Chemistry, pages 180-184.
Procedure
Total Protein - Sigma 541-2
Reagents
Biuret Reagent - A standardized alkaline copper tartrate solution (copper sulfate and sodium
hydroxide)
Specimen
Fresh serum
Steps
1.
Label test tubes for blank, standard(s), controll(s) and patients.
2.
Add 0.05 ml sample to appropriate tubes.
3.
Add 2.5 ml Biuret reagent to each tube.
4.
Allow to stand 10 - 15 minutes at room temperature.
5.
Read absorbance at 540 nm using blank to zero the spectrophotometer.
Results
Serum total  Atest x Concentration
protein (gm/dl)
of Standard
Astandard
1.
If a series of standards are analyzed, a standard curve can be used to determine the
concentration of the unknowns.
2.
But if only one standard is analyzed in the test run, the Total Protein is calculated as follows:
MLAB 2401 - Clinical Chemistry Lab Manual  H 139
UNIT: Proteins (continued)
Expected Values
Adult normal = 6.0 - 8.2 g/dL
Albumin
Reagents
Albumin reagent B bromcresol green
Specimen
Fresh serum
Steps
1.
Make 1:5 dilution of each standard, control, and sample using 0.85% NaCl (normal saline)
solution.
2.
Label test tubes for blank standard, control and tests.
3.
To each tube, add 3.0 ml albumin color reagent.
4.
Add 0.1 ml of diluted sample to appropriate tubes. To blank tube, add 0.1 ml NaCl.
5.
Mix well and allow to stand at room temperature for 10 minutes.
6.
Read absorbance of each test at 630 nm using blank to zero the spectrophotometer.
Results
Albumin (gm/dl) 
Atest
Astandard
x Concentration
of Standard
Expected Values
Normal Range = 3.8 - 5.1 g/dL
H 140  MLAB - Clinical Chemistry 2401 Lab Manual
UNIT: Proteins (continued)
Name
Date
Protein Worksheet
I.
Total Protein
Wavelength
_____________
Linearity
_____________
Spectrophotometer Used ______________________
Absorbance
Identification
Concentration
Refractometer
(units)
Reading
Standard _____________________
Standard _____________________
Standard _____________________
Control 1 _____________________
Control 2 _____________________
Calculation formula(s) and examples
Total Protein Quality Control
Your Results
Controls= range of expected results.
In control?
Yes / No
MLAB 2401 - Clinical Chemistry Lab Manual  H 141
UNIT: Proteins (continued)
Total Protein Quality Control
Level 1 ID______________
Level 2ID_______________
Accepting Patient Results?
Reason
H 142  MLAB - Clinical Chemistry 2401 Lab Manual
UNIT: Proteins (continued)
II.
Albumin
Wavelength
_____________
Linearity
_____________
Spectrophotometer Used ______________________
Identification
Absorbance
Concentration (units)
Standard _____________________
Standard _____________________
Standard _____________________
Control 1 _____________________
Control 2 _____________________
Calculation formula(s) and examples
Albumin Quality Control
Your Results
Controls= range of expected results.
In control?
Yes / No
Level 1 ID______________
Level 2ID_______________
Accepting Patient Results?
III.
Reason
Globulin
MLAB 2401 - Clinical Chemistry Lab Manual  H 143
UNIT: Proteins (continued)
Identification
Concentration (units)
Control 1 ____________
Control 2 ____________
Calculation formula(s) and examples
H 144  MLAB - Clinical Chemistry 2401 Lab Manual
UNIT: Proteins (continued)
Name
Date
Study Questions
Instructions: Based on information presented in lecture and lab notes, answer the following
questions. Legibly write your answers in the space provided. Unless otherwise indicated, each
question is worth one point.
1.
Plasma proteins are synthesized in the ___________________________.
2.
What are the two major fractions of serum proteins?
3.
Explain why serum rather than plasma is the preferred specimen for total protein
determinations.
4.
Which blood protein group is the smallest in molecular weight?
5.
The biuret method measures the presence in the protein molecule of
A. nitrogen
B. peptide bonds
C. tyrosine
D. tryptophane
6.
Common ingredients of all biuret reagents are
A. sodium potassium tartrate and NaOH
B. sodium tungstate and sulfuric acid
C. copper sulfate and sulfuric acid
D. copper sulfate and sodium hydroxide
E. cuprous oxide and sodium hydroxide
7.
What is the physiological role of albumin?
8.
The globulin concentration of a patient with a total protein of 6.9 gm/dL and an albumin of 3.4
gm/dL is ___________________________.
9.
In the Kjeldahl Method, what is actually being measured?
10.
List three (3) dyes that have been commonly used in the quantitation of albumin.
MLAB 2401 - Clinical Chemistry Lab Manual  H 145
UNIT: Proteins (continued)
11.
The absorbance of the patient's sample in an albumin procedure is 0.376. The 5.0 gm/dL
standard gave an absorbance of 0.355. What should be reported to the doctor?
12.
Briefly explain why normal saline rather than distilled water is used for protein or albumin
dilutions.
H 146  MLAB - Clinical Chemistry 2401 Lab Manual