Table Of ContentThe Pennsylvania State College
The Graduate School
Department of Agricultural and Biological Chemistry
A STUDY OP THE EFFECTS OF TIME, BUFFER COMPOSITION,
SPECIFIC I OHS, AND I01TIC STRENGTH OH THE SURFACE TENSION
OF SOLUTIONS OF @-LACTOGLOBULIN
A Thesis
*y
Gertrude Haspeslagh Spremulli
Submitted in partial fulfillment
of the requirements
for the degree of
Doctor of Philosophy
August, 1942
Approveds
(L^j.
//__»
1942
^
Professor of Biophysical Chemistry
.-<1
L / ? » 1942
Assistant Professor of Agricultural
and Biological Chemistry
2
1942 ,G
Hea.d of Department of Agricultural
and Biological Chemistry
i.
TABLE OF CONTENTS
Page
LIST OF FIGURES - - - - - - - - - - - - - - iii
LIST OF TABLES - - - - - - - - - - - - - - v
ACKNOWLEDGEMENTS ~ ~ - -----
T±
I. INTRODUCTION - - 1
II. REVIEW OF LITERATURE
A. Effect of Proteins on Surface Tension
and. Its Variation with the pH of the
System - - - - - - - - - - - - - - - £
B. Change in Surface Tension of Protein
Solutions with Time - - - - - - - - 8
G„ Effects of Buffer Composition, and
Ionic Strength on the Property
Measured - - - - - - - - - - - - - - l6
D* Resume^ - -- -- -- -- -- -- -- 18
III. THE PROBLEM - -- -- -- - .............- 20
IV. EXPERIMENTAL PROCEDURE
A. Preparation of the f^-Lactoglobulin 21
B. Preparation of Stock Solutions of
Protein - - - - - - - - - - - - - - 26
Co Preparation of the Buffer
Solutions - - - - - - - - - - - - - 27
D0 Surface Tension Meastxrements - - - 28
, V. EXPERIMENTAL RESULTS
A. Change of Surface Tension with
Time - - - - - - - - - - - - - - - 35
ii
TABLE OE CONTENTS (CONTINUED)
Page
B* Effect of pH on Surface Tension. ^3
C0 Effect of Buffer Composition
and of Ionic Strength on Sfurface
Tension - - - - - - - - - - - - - 62
D« Comparison of the Surface Tension
of Solutions of the Two
Crystalline Forms of /^-Lacto-
globulin - - - - - - - - - - - - 68
E* Effect of ^-Lactoglobulin
Concentration on Surface Tension
Lowering - -- -- -- -- -- - 71
VI* DISCUSSION QE RESULTS
A* Introductory - - - - - - - - - - 75
B* Influence of Time on Surface
Tension Values - - - - - - - - - 78
G* Variation of the Surface Tension
with pH - - - - - - - - - - - - 92
D* Effects of Ionic Strength, and
Buffer Composition on Surface
Tension of Solutions of
@ -Lactoglobulin - - - - - - - 102
E« Difference, in Surface Tension of
Solutions of the Two Crystalline
Eorms of ^-Lactoglobulin - - - 112
E, Conclusion - - - - - - - - - - - 113
VII* SUMMARY -- - - - - - - - - 115
VIII* BIBLIOGRAPHY --- 119
iii
LIST OF FIGURES
Figure Page
globulin
a. Tetragonal - - - - - - - - - - 24
b* Tetragonal with Polarized
Light - - - - - - - - - - - - 24
c * Grthorhombic - - - - - - - - - 25
2. Change of Surface Tension with Time 36
3« Variation of Log Surface Tension, with
Log Time - - - - - - - - - - - - - - - 38
4. Change of Surface Tension Lowering
with Time 42
5* Variation of (r* (from equation 1, = <*-
+ P log t) with pH T
a. Sodium Acetate Buffers of
Ionic Strength Equal to 0*02 - - 44
b* Sodium Acetate Buffers of
Ionic Strength Equal to 0.06 - - 45
c. Sodium Acetate Buffers of
Ionic Strength Equal to 0.10 - - 46
d* Magnesium Acetate Buffers of
?
Ionic Strength Equal to 0.02 - - 4
e. Magnesium Acetate Buffers of
Ionic Strength Equal to 0*06 - - 48
f* Magnesium Acetate Buffers of
Ionic Strength Equal to 0.10 - ~ 4-9
60 Relation Between Surface Tension
and l/Time - - - - - - - - - - - - - - 50
?• Effect of Flaslc Aging on Surface
Tension - - - - - - - - - - - - - - - 54
8* Effect of pH on Surface Tension Lowering
a* Sodium Acetate Buffers of
Ionic Strength Equal to 0.02 - - 56
b« Sodium Acetate Buffers of
Ionic Strength Equal to 0„06 - - 57
c* Sodium Acetate Buffers of
Ionic Strength Equal to 0.10 - - 58
iv
LIST OF FIGURES (COHTIHUED)
Figure Page
d* Magnesium Acetate Buffers of
Ionic Strength Equal to 0*02 ——* 59
e* Magnesium Acetate Buffers of
Ionic Strength. Equal to 0*06 - ~ 60
f * Magnesium Acetate Buffers of
Ionic Strength Equal to 0*10 - - 6l
9« Influence of Buffer Composition on Surface
Tension Lowering 64
10. Influence of Ionic Strength on Surface
Tension Lowering - -- -- -- -- -- -- 65"
11* Relation Between Ionic Strength and the
Minimum in the Surface Tension Lowering-
pH Curve - - - - - - - - - - - - - - - - - 66
12* Relation Between Ionic Strength and the
Maximum (Acid) in the Surface Tension
Lowering-pH Curve - - - - - - - - - - - - 67
13* Variation of h (from log = a - h log t)
with. pH - -- -- -- -- -- -- -- -- 70
14« Effect of -Lactoglobulin Concentration
on Surface Tension Lowering - - - - - - - - 72
15* Variation of Surface Tension Lowering
with Log Log Concentration of # -Lacto-
globulin - - - - - - - - - - - - - - - - - 74
LIST OF TABLES
Table
1* Values of the Constants a and h To
Be Used in the Equation V = ^ ,
for 0*1 Per Cent Solutions •
of -Lac to globulin (Ortho rhomb ic
Crystals) -
2* Effect of Flask Aging on the Surface
Tension of 0*1 Per Cent Solutions of
P -Lactoglobulin (Orthorhombic
Crystals) ~ -
3* Values for Surface Tension Lowerings
in 0*1 Per Cent Solutions of
-Lactoglobulin at Low pH Values -
4* Comparison of Results from the Study
ojA Both Crystalline Forms of
r -Lactoglobulin in Sodium
Acetate Buffers of Ionic Strength
Equal to 0.1 » * - - - - - ~ - - - -
AC KNO WLED GEME1TT S
writer gratefully acknowledges her
. , ^ sb to Professor M. W. Lisse for suggesting
indebtedn^
and for his advice and heir throughout
the probl^
_ ^-fTessor A, H, Palmer for his suggestions and
and to Pr<7
, .--ularly in the preparation of the ^-lacto-
aid parti<? J * *
Both gave valuable assistance during the
globulin*
jr the manuscript*
writing o£
.
1
I. I33TRODUCTIOH
The importance of proteins in all phases of
"biological chemistry and particularly their importance
as components of living matter has made the study of
their physical and chemical properties of the greatest
interest. Adequate knowledge of their "behavior is.,
essential to an understanding of the chemistry of life.
Ideally, of course, they should he studied under the same
conditions as those existing in vivo. Since as yet no
entirely satisfactory methods are available for
accomplishing this, the alternative is to study them in
systems which may be completely characterised in terms
of known variables, with the hope that eventually the
number and nature of the variables can be made to approach
very closely those present in the living cell.
That the physical properties of proteins vary with
variation in the hydrogen ion concentration (H^O*) of
their environment was early noted (Ostwald, 190? {^5T) •
This is to be ejected from the fact that proteins are
amphoteric electrolytes, existing in solution as" dipolar
ions with a net charge which is positive, negative, or
zero, depending upon the pH of the medium surrounding
them. This variation of physical properties with pH is
.
2
so marked that for many years the tendency has heen
to explain or interpret the physical behavior of a
protein entirely on the basis of the pH of the system*
In spite of the fact that the specific effects of added
electrolytes have long been recognized, the influence
of these same ions when part of a buffer system used to
obtain a definite pH lias generally been ignored, and the
pH values alone have been used to define the system.
Inevitably there has come, especially in recent
years, a recognition of the important part played by
these ions, other than the hydrogen or hydroxyl, which
have had to be introduced into the system in order to
create the measured pH, As a result of the important
part played by these other ions it is necessary, in
order to completely characterize a protein system, to
know not only the pH but also the nature and concentration
(activity) of the ions introduced to produce that pH*
The work reported here was designed to show 7/hether or
not the value of an e:cperirnentally determined physical
property of a protein is a function only of the pH of
the buffer system or whether the specific components of
the buffer and its ionic strength also play a part in
determining the value of the measured property.
Most of the early studies on the physical
properties of proteins, such as viscosity, swelling,
osmotic pressure, etc*, have shown that they vary in
a well-defined, and fairly consistent manner with
variation in the pH of the system (39) •
generally found that the value of the property was a
minimum at the isoelectric point of the protein* A
review of the literature revealed, however, that there
is a lack of agreement with regard to the variation of
the surface tension of a protein solution with pH and
that no attention has "been paid to the specific effects
of the ions used to obtain the pH. Hence, it was
decided that it would he of value to use surface tension
as the physical property to he studied. Furthermore,
it was felt that knowledge of surface tension values
would he of importance from many theoretical points of
view - adsorption phenomena, orientation and reaction
at interfaces, foaming properties and others.
Since it was believed that much of the work on
the stirface tension of protein solutions was invalidated
by the fact that the proteins used were not pure or
homogeneous substances, a protein which would avoid that
difficulty ms sought* All data at present indicate that
the crystalline lactoglobulin C ^ -lactoglobulin (iiQ.
