8/21/2019 Dterminación Viscosimétrica Del Punto Isoeléctrico de Una Proteína.

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  ohn

E enron

Viscometric

Determination

of the

Gettysburg College

Geftysburg, Pennsylvania Isoeiectric Point of a Protein

This experiment was designed to present

students with an opportunity to do an experiment of a

quantitative nature in macromolecular chemistry, an

area commonly neglected in undergraduate laboratory

work or studied only by means of short qualitative ex-

periments. The experiment can be performed in a.

3-hour laboratory period by students working in pairs

and is suitable for use by chemistry majors or pre-

medical students in courses a t several levels. The use

and operation of viscometers and pH meters are

illustrated, and the experiment is an excellent test of

technique since the results obtained depend very much

on manipulative care and cleanliness.

Mobility, conductivity, solubility, osmotic pressure,

stability toward coagulating agents, and viscosity of

proteins are a minimum a t the isoelectric point. The

minimum in the case of viscosity is quite sharp and

easily detected by preparing protein solutions of

various pH s, determining the viscosities of these solu-

tions, and graphically finding the pH for minimum

viscosity.

The structure of proteins, the meaning of the terms

zwitterion and isoelectric point, the principle and tech-

nique of operation of a

pH

meter, the measurement of

viscosity1 and viscosity units2 are discussed in advance

of the experiment, either in class or in a written intro-

duction to the ex~eriment. The students should be

Presented at the eleventh annual meeting of the Pennsylvania

h c i a t i o n of College Chemistry Teachers, Diokinson College,

Carlisle, Pemylvania , March, 1962.

DANIELS,., ET AL., Experimental Physical Chemistry,

6th ed., McGraw-Hill

Book

Co., Inc., New York

1962, pp.

147-

=

8 .

J m a ~ ~ s o N s ... AND STRILUMANIS.. E..

A

Short Textbook

of Colloid

he-tj,

John Wiley and sons, Inc., New York,

1954,pp.

1434.

warned of the fragility of the pH electrodes, the danger

of the electrodes becoming blocked by dried protein

solutions, the inadvisability of clamping viscometers

across both arms and the absolute necessity for cleanli-

ness. The technique of rinsing and filling viscometers

and of measuring viscosities in minimum time should be

discussed.

viscometer of the Ostwald or Cannon-Fenskef type

with a drain time for water of around 2-3 minis suitable

for this experiment. It is convenient to keep the

viscometer in a detergent-filled graduate from period to

period. The pH meter need not be very sensitive; an

instrument graduated in 0.1 pH unit and capable of a

reproducibility of

*

0.02

pH

unit is adequate. The

use of a constant temperature ba th a t 25°C is ad-

visable, but a stirred large volume of water a t 25'C is

suitable. 1 by weight stock gelatin solution

( Knox unflavored will work very nicely) has been

most cornmouly used by us; it should be prepared the

day before to allow it

to

age to constant viscosity.

The Experiment

Six 0.5% solutions of gelatin of pH values approxi-

mately every 0.5 pH unit from

3.5

through 6.0 are pre-

pared by dilution of an aged 1% gelatin stock solution.

This operation is conveniently done by pipetting 50

rn

portions of gelatin solution into six 100-ml volumetric

flasks, diluting with water to about 20

ml

short of the

marks, adding dropwise some previously prepared

dilute H CI (p H S 1) until each solution has a pH near

the desired value and finally diluting

to

the marks.

Some drift in pH will be observed and sufficient time

CANNON, . R. ,

AND

FENSKE, .

R., Ind

Eng Cham. Anal

Ed.

10,297 (1938).

68 Journal of hemical Education

8/21/2019 Dterminación Viscosimétrica Del Punto Isoeléctrico de Una Proteína.

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must be allowed for the electrodes to come to equilib-

rium with the solutions before reading the pH. Some

difficulty with foaming may cause trouble in diluting to

the mark, but the error is slight.

The times for flow through the capillary of the vis-

eometer at 25°C are measured for water and the six

gelatin solutions. Drying of the viscometer with

acetone, the usual procedure recommended in texts,

is not necessary so long as the viscometer is carefully

and thoroughly rinsed with the solution to be used.

Drawing the solution up through the capillary into the

storage bulb a t the top and allowing it to drain down

once of i ts own accord before final measurement usually

insures that the solution is a t the bath temperature.

Large bubbles in the capillary side of the viscometer

can usually be avoided by carefully forcing the solution

in and out of the capillary.

Results

The relative viscosities of the six gelatin solutions are

calculated and a plot of relative viscosity versus pH

prepared. The isoelectric point is determined from

the point of intersection of the two nearly linear

branches of the plot and is compared with a literature

value.' The plot diagram presents typical results on a

0.5

gelatin solution at 5°C; the experimentally de-

termined isoelectric point was a t

pH

4.8,

the com-

monly given literature value being

4 7 6

Lack of

sharpness in the minimum or a large error usually indi-

cates impurities in the protein, dirty glassware, or the

presence of fairly large amounts of salts.

'Most standard texts on colloid and surface chemistry list

the isoelectric points of the more common proteins with references

to the original literature.

ABRAMSON,.

A,, J . Den. Physiol.

13

69 1930).

O t h e r A p p l i c a t i o n s

With suitable changes in the conditions proteins

such as albumins, fibrins, and hemoglobins may be

used. Casein is not suitable because of its tendency to

precipitate a t its isoelectric point. An interesting ex-

periment is the determination of the changes in viscosity

of a fresh gelatin solution due to aging. Although

viscometry is perhaps the simplest method to use, other

methods such as conductance, mobility, and sensitivity

toward coagulating agents may he used to determine

the isoelectric point. The isoelectric points of purified

amino acidse may be conveniently determined by con-

ductance or electrophoretic mobility.

The author is indebted to several students for work-

ing out details of the experiment and to Professor

Charlotte

I

Damerel of Wilson College for her helpful

suggestions.

HODGMAN,. D. , edit, Handbook of Chemistry and Phys-

ics, 38th ed., Chemiml Rubber Publishing Co. Cleveland, Ohio,

1956,p.

1649.

Volume

40

Number

9

September

963

/

469