© 2003 by The International Union of Biochemistry and Molecular Biology BIOCHEMISTRY AND MOLECULAR BIOLOGY EDUCATION

Printed in U.S.A. Vol. 31, No. 4, pp. 249 –252, 2003

Laboratory Exercises

Salt Fractionation of Plasma Proteins

A PROCEDURE TO TEACH PRINCIPLES OF PROTEIN CHEMISTRY

Received for publication, October 31, 2003, and in revised form, January 27, 2003

A. C. C. Spadaro‡, A. I. Assis-Pandochi, Y. M. Lucisano-Valim, and Z. Rothschild

From the Departamento de Fı́sica e Quı́mica, Faculdade de Ciências Farmacêuticas de Ribeirão Preto,
USP Av do Café s/n, CEP 14040 –903, Brazil

A two-step laboratory exercise in biochemistry is proposed, comprising salt fractionation of plasma

proteins and protein quantification. This exercise targets mainly undergraduate students, namely in the
second year of Pharmacy, who are up to this point more used to deal with physical and chemical properties
of micromolecules than macromolecules. The exercise requires simple equipment usually available in basic
laboratories. Students work in small teams or alone depending on the laboratory conditions. Questions are
proposed to check and reinforce the essential concepts involved as a preparation for detailed compre-
hension of protein chemistry. After salt fractionation, protein samples can be stored before quantification,
allowing a two-period schedule for the laboratory work. Exercises as proposed here are very useful to guide
students to a detailed analysis of fundamental aspects determining structure and physicochemical
properties of proteins.
Keywords: Biochemistry education, undergraduate education, protein properties, salt fractionation.

The current teaching methodology at universities has
proven to be inefficient in the face of advances in science,
technology, and human knowledge [1]. Therefore, in Bio-
chemistry, as in any other area, a learning process built
from research, where students are stimulated to analyze
experimental data that lead to established concepts, is
each time more necessary.
Here we propose a simple laboratory exercise to guide
students to the comprehension of basic aspects in deter-
mining structure and physicochemical properties of pro-
teins. Based on the type and quantity of amino acids of
some specific proteins, the establishment of correlation
between structure and properties constitutes an opportu-
nity for training conceptual operations such as analysis,
comparison, classification, etc. that are fundamental steps
of the learning process.
At the Pharmacy and Dentistry Schools of Ribeirão
Preto, University of São Paulo, this sort of practical exer-
cise has been applied for many years [2, 10] and the
achievement of satisfactory results justifies the present
proposition. This exercise targets undergraduate students,
particularly those attending in the second year class from
the Pharmacy course who are up to this point more used
to dealing with physical and chemical properties of micro-
molecules than macromolecules.
The use of simple equipment, usually available, should
allow the application of this sort of practical exercise in
basic laboratories. In the present case, the laboratory work
include salt fractionation of bovine plasma proteins, pro-
‡ To whom correspondence should be addressed. E-mail:
cespada@usp.br.
This paper is available on line at http://www.bambed.org
tein quantification, and discussion of obtained results as
well as related questions.
EXPERIMENTAL PROCEDURES
STEP 1: Fractionation of Plasma Proteins by Salts
Ten milliliters of bovine plasma are diluted 3-fold in phosphate-
buffered saline and given to each student or team, together with
a flow chart (Fig. 1) for the experimental procedure. A nomogram
(Fig. 2) to calculate the salt weight required in each step and a
table containing information about protein solubility (Table I) are
also furnished. SDS-PAGE analysis can be conducted to confirm
the protein size of each fraction. As it would be expected from this
fractionation process, fractions will contain preferentially the pro-
tein groups as in Table I, despite of the presence of other con-
taminants. In this context, it should be emphasized that washing
each precipitated fraction with ammonium sulfate solution at the
respective salt concentration will improve the results.
The first set of questions may be discussed at this stage with
the help of additional information given in Fig. 3, Table II, and
Fig. 4.
Questions to the Students: Set 1
1. Fig. 3A: Verify what happens to fibrinogen solubility in
solutions of increasing ionic strength (i.e. containing
variable amounts of ammonium sulfate). Explain why it
happens. Compare amino acid composition of fibrino-
gen, myoglobin, and albumin (Table II) and their solu-
bility in salt solutions (Fig. 3B). Does the variation in
type and amounts of amino acids present justify the
difference in solubility of these proteins? Would you
have an alternative suggestion to explain this differ-
ence? And also the difference in the solubility of these
proteins and the totally insoluble silk fibroin (Table II)?
Molecular weight and relative dimensions of some pro-
teins are given in Fig. 4. Compare: a) hemoglobin to
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250 BAMBED, Vol. 31, No. 4, pp. 249 –252, 2003
TABLE I
Solubility of plasma proteins in (NH4)2SO4 solutionsa
Saturation by
Protein fraction Salt required
(NH4)2SO4
precipitated (g/100 mL)b
(%)
0–20 Fibrinogen 11 g
20–50 Globulins 19 g
50–100 Albumins 38 g
a
Adapted from Ref. 11.
b
This column should be completed by students with the help of the
nomogram in Fig. 2. After dialysis against phosphate-buffered saline,
2 ml each the starting material, fractions, and Solution 3 (Fig. 1) are
mixed with 2 ml of 2 N NaOH and 8 –10 drops of 0.5% CuSO4 to
detect the presence of proteins (biuret reaction). If convenient, protein
samples can be stored to be quantified in the second period.

FIG. 1. Flow chart for fractionation of plasma proteins by

precipitation with ammonium sulfate. *, Each fraction should
be washed at least once with salt solution at the same concen-
tration. All fractions (1, 2, 3) and Solution 3 should be dialyzed
against phosphate-buffered saline to eliminate excess of salt. At
least five groups of students perform this same fractionation FIG. 3. Solubility of proteins in ammonium sulfate solutions.
process at the same time, allowing comparison of the results. A, fibrinogen; B, fibrinogen compared with other proteins. (Adapt-
ed from Ref. 3.)

TABLE II
Amino acid composition of some human proteins and silk fibroina
Amino acid content (%)
Amino acid
Fibrinogen Serum albumin Myoglobinb Silk fibroin
Glycine 5.6 1.6 6.08 42.80
Alanine 3.7 – 5.82 33.50
Valine 4.1 7.7 4.64 3.30
Leucine 7.1 11.9 13.67 0.89
Isoleucine 4.8 1.7 5.27 1.11
Proline 5.7 5.1 5.40 0.52
Phenylalanine 4.6 7.8 8.22 1.33
Tyrosine 5.5 4.66 2.19 11.90
Tryptophan 3.3 0.19 3.40 0.88
Serine 7.0 3.7 4.43 16.3
Threonine 6.1 5.0 2.85 1.38
Cysteine 0.4 0.70 0 0
Cysteine/2 2.3 5.58 – –
Methionine 2.6 1.28 2.69 0
Arginine 7.8 6.15 2.47 1.04
Histidine 2.6 3.5 7.79 0.36
Lysine 9.2 12.3 19.09 0.60
Aspartic acid 13.1 10.4 8.27 2.22
Glutamic acid 14.5 17.4 16.17 1.92
a
Adapted from Ref. 7.
b
As an example of globulin.
FIG. 2. Nomogram for determining the amount of ammo-
nium sulfate needed to attain respective percentages of sat-
uration. The initial and final concentration of ammonium sulfate, would you consider to be the main factor determining
expressed as percent saturation, are on the vertical and horizon- the difference between the solubility of these proteins?
tal scales, respectively. The point of intersection of two lines 2. Which features of protein structure act jointly to deter-
drawn from these points indicates the number of grams of salt to mine their solubility properties?
be added to each littler of solution at the initial concentration to 3. Other reagents such as ethyl alcohol and acids can
lead to the final concentration required. (Slightly modified from also be used to precipitate proteins from solutions.
Ref. 4.) How do they work?
4. What should be considered before choosing between
serum albumin; b) serum albumin to fibrinogen; and c) the use of these reagents and salt?
hemoglobin to myoglobin. Return to Fig. 3B to analyze 5. Why can Cu2⫹ in an alkaline medium be used to detect
their solubility in solutions of salt. In each case, what the presence of protein in solution?
 251
TABLE III
Protein quantification by the biuret method: protocol for preparing the
standard curve and samplesa
BSA 10
H2O BSA BSA Biuret solution
Tube mg/ml
(ml) (mg/tube) (mg/ml) (ml)
(ml)
Blank – 2.0 – – 2.0
1 0.1 1.9 2.0 1.0 2.0
2 0.2 1.8 4.0 2.0 2.0
3 0.4 1.6 8.0 4.0 2.0
4 0.6 1.4 12.0 6.0 2.0
5 0.8 1.2 16.0 8.0 2.0
6 1.0 1.0 20.0 10.0 2.0
FIG. 4. Relative dimensions of some human proteins. (Adapt- Samples: 2.0 ml 2.0
ed from Ref. 12.)
a
Standards and samples should be analysed in triplicate and pro-
cessed at the same time, using same bath of reagents and assay
STEP 2: Protein quantification
conditions. Samples may require dilutions to fit in the range of ab-
In this step, students quantify the protein yield in each fraction. sorbance values of the standard curve.
A standard curve is built for this purpose, using solutions of
bovine serum albumin, according to the method of Gornall et al.
[6] (Table III).
Students are instructed to:
• choose one of the tubes of the protocol to scan the visible
range of the absorption spectrum and find a suitable wave-
length (␭) to use (Fig. 5A);
• record the absorbance of each tube at the wavelength
chosen;
• determine linear regression (A ⫽ ␣c ⫹ b, where A is the
absorbance, ␣ is the specific extinction coefficient, c is the
protein concentration, and b is the linear coefficient, whose FIG. 5. A, absorption spectrum of an alkaline bovine serum
value is nearly 0) using the obtained data (protein concen- albumin (BSA) solution in the presence of Cu2⫹. B, a standard
tration and corresponding absorbance) as well as an appro- curve for BSA by plotting absorbance versus protein amounts.
priate computer program (Fig. 5B);
• follow the same protocol to measure protein concentration in
total plasma and in each of the protein fractions (Fig. 1) using tween amino acid composition, the different levels of pro-
the equation obtained from linear regression and calculate tein structure, protein size and shape, and solubility. Stu-
the yield in each step, as a percentage of total protein in dents are reminded to consider the influence of the
plasma. dielectric constant of the solvent in the solubility of pro-
Students should discuss the questions in Set 2 at this point. teins and the effect of salts (and organic reagents such as
ethyl alcohol and acids) on protein solubility and function
Questions to the Students: Set 2 [9]. At this point, there is a chance to discuss denaturation
6. Why can serum albumin be used as a standard in the and different purposes of protein precipitation.
quantification of proteins by the biuret method? Which In discussing properties that can be used to quantify
structure character of protein allows the use of Cu2⫹ in proteins (Question 6), it may come to light the advantage of
an alkaline medium for its quantification? Do you know using the biuret reaction [8] instead of the simple meas-
of any other protein property that can be used for its
urement of absorption at 220 nm (absorption of the pep-
quantification? Explain.
7. Justify your choice of the specific wavelength selected tide bond). The same comments apply to the measure-
for protein quantification in this method. ment of the absorption of aromatic amino acids at 280 nm
8. The range of protein concentration in your standard or at another wavelength when in association with other
curve is 1–5 mg/ml. Is it possible to quantify protein reagents such as Folin-Ciocalteau’s reagent [5]. The pro-
solutions having higher or lower concentrations using tein quantification procedures can be discussed based on
the calculated coefficient? Explain.
9. After calculation of the total plasma protein and protein the students experimental data and on the answering of
recovery (mg/ml) from each step, analyze your data Questions 7–9.
and comment on possible sources of errors. In our experience, laboratory exercises as proposed
here are useful to guide students to a detailed analysis of
COMMENTS fundamental aspects of protein structure and physico-
This project allows for a practical exercise on the phys- chemical properties, instead of a passive memorization of
icochemical properties of proteins that are a reflection of concepts that wouldn’t add to their real learning process.
their structure. The solubility of a protein depends on the
proportions and distribution of polar hydrophilic groups Acknowledgments—We acknowledge the help of Ana Cristina
Morseli Polizello and Maria Regina de P. Raphaloski in the prep-
and nonpolar hydrophobic groups in the molecule and the
aration of the manuscript; the students that lead them to a reval-
resulting protein dipole moment [12]. Therefore, precipita- uation of their role as mediators in the learning process; and to
tion of a particular protein or a group of proteins in a given Professor Zuleika Rothschild for the example as a teacher whose
salt concentration helps to illustrate the correlation be- priority in academe life was always the students.
252
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