EQUILIBRIUM DIALYSIS, MOVING BOUNDARY AND PAPER
ELECTROPHORESIS STUDIES ON THE BINDING OF
HUMAN SERUM PROTEINS HITS GALCIBM,
MAGNESIUM, IRON, AND COPPER IONS

B&r
Henrietta Marie Late

A THESIS

Submitted to the Collage of Advanced Graduate Studies of Michigan
State University of Agriculture and Applied Science
in partial fulfillment of the recrements
for the degree of
DOCTOR OF PHILOSOPHY
Department of Chemistry

1956

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ACOGt&EDGHENT

The author wishes to express appreciation and gratitude
to the late Sr. fi, 1 . M s g laid to ir, ft* A. Billarik efMichigan State University for their guidance and counsel.
The cooperation of Brs. H, D. Anderson and £. B. McCall
of the Michigan Bepartmont of Health in supplying purified
plasma fractions is sincerely appreciated.
The author expresses sincere thanks to Br. P. F. Morse
and the staff ef Harper Hospital for the use ef laboratory
space, apparatus, and materials which made this investi­
gation possible. Financial assistance from a grant given by
the 3. S. Iresge Foundation is gratefully acknowledged.
j
jI
gj
MH
ei
LWMAjIeI
Ma
la'al
'u
Hf
ianmnur

ii

rr$A
Ih@ anther was b o m in Detroit, Michigan on October 26,
1918»

She obtained a MacGregor Scholarship and graduated,

from Ws&m State University
Degree.

3$U7 with a Bachelor of Science

Gradoate studies at Michigan State University com-

timed from 1?£0 to the present.
Professional employment inclwdesj

Besearch chemist.

Harper Hospital, Detroit, Michigan, l9kl~$Qs 19$k~$6$ Besearch
fellow in the Bepiorimant of Chemistry, Michigan State Univer­
sity, 19$0~$hl Chemist, Ethyl Corporation, Feradale, Michigan,
present position.
the anther is a member ef the American Chemical Society!
the American Association for the Advancement of Science! and
an associate masher of Sigma Xi.

ill

. 2WBBI®!, 3 M # M

&C&VM XO&S

Henrietta M b ! LeDoe

AS AS®BNl@l

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and t o alpha* to n , wad p m globulins.

to o h

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to e contains w to a

to a l ion e e a r& to to , toag to e * imm mm e to im , m &um im , im n ,
mem* '^Tprjr 0 m»*#* ■
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followed %

jt

^^lyfrfft^. g*0(pay

selective nefefi^-y^i^

yy < f «x

to dtoraiae t o t o e s sera

(oy ftototf) associated t o k each of the t o r metal

lens*three asnrs&eties to

{1}

m»

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WPwW
ISwkwSBBHf 3*lo IWIRfcMJBmt#

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etedledj

©ooorred*

^gj B»^gwwaartwga
(I4) ©©pper with gwBMt^ giobaHn.#

w s^nyeMoa® ef "the sesrtus proteins m r ®

ssleetive staining proeedarm M m protein and t o reioit
C t o t o m s tort to be pr®»«ni $m all protein eos^oaaaite,

Strip ©lution technics U s t o t o i that albuadn (about 2/3 of* th® total
protein)end p m

glotoalln

2/6 of fee total protein)

eontrtoted equally in binding 2/3 of t o total t o a d ototo,

m & t m m m e ala© fea«K In all searaa proteins.

r

Iron appeared to be

i*r«s«xfc m Igy to

g M & £ % iMIa
nftffitm

«od sine am m i to oocar
fjfty potAAfl^byta^ jpbOAP^a^®'*

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ff Agflljfta 4j0 *j/y»flftu» ffiyygay .tSUS,
S ^blW lil^blW

f f KfeqCtA I NfflflgWi « *3 ifK tly fe a ff g^ y ty ^ |gpgr|($£i|

MAfw&tAMyt jyg th® *MBaM* Ijl^SfflBF -ywy^M-tjiMf' gg
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yi

©OCtET*

TABLE OF CONTENTS
Page
I. INTRODUCTION.
n.

......

HISTORICAL

...............

A. Binding of Metal Ions t© Serum Proteins..............
B. Electrophoresis. ......... .......... ..........*
nj.

m w m m u , . . , , ....... ...........
A. Apparatus........ .....
B. Materials andReagents...............................
C. Experimental P r o c e d u r e s .

IF. DISCUSSION.
.A*
1.
C.
D.
I,

3
3
7
13
13
lb
22

%

....

Moving Boundary Experiments
Paper Electrophoresis Experiments;
....
Exhaustive Dialysis Studies.
.......
Comparison of Findings with Other Work.
The Significance of Protein-Metal Ion Complexes....

F. SUMMARY

1

.............

%
58
6l
63
66
6?

BIBLIOGRAPHY. .......

68

APPENDIX...............

77

vii

ust

of

tm m

TABLE

Page

X Effect of Beep-Freeae Storage on Total Protein and Metal
Ion Content of Human Ssnta.,,.*............

35

XXa Planiaatrie Analysis of Serum Electrophoretic Patterns of
Mormal Human Subjects..**.................................

36

U b Total Protein and S«nut Protein Distribution by Moving
Boundary Electrophoretic Analysis.........

37

XXX Results ©f Metal Ion Analyses..

........

X? ?alues for Normal Human Serum.............................
V

38
39

Coaparison of Moving Scnmdaryand Paper Electrophoretic
Analysis on the Sane Serum........

Uo

VI

Analyses ©f protein Solutions Before and After Dialysis...

itl

VU

Total and Bound Metal Ions................................

kZ

vlii

L »

OF FIGURES

FI«E

Page

X, Location of Component Systems in the Arainco-Stern
Electrophoresis Apparatus........

h3

2, Optical Diagram of the Aminco-Stern Electrophoresis
Apparatus,,...............................................

UU

3. LKB Horisontal Strip Paper Eieetropiioresia Apparatus......

k$

U. I&arajplsa of I-toving Boundary patterns of Human Serum.......

U6

5. Enlarged Ascending Moving Boundary Pattern with Ordinates
Braun.
....

k7

6 . Standard Curves for total Protein and Baptesiti® in Human
Serum. ........
............

k8

7. Standard Curves for Iron sod Copper in Human Serum........

k9

3. Location of Line of Sample Application to Paper...........

$0

9. Cutting Paper for Selective Staining and Elution

51

10. Qualitative Results of Selective Staining

...,.
.......

. 11. Example of Serum Protein Separation by Paper
ELectrophoresis
....

$2

53

12. Distribution of Calcium and Serum Proteins after Paper
Electrophoresis
....

5U

13* Removal of Calcium from Serum by Exhaustive Dialysis...,.,

55

ix

1

I. INTRODUCTION
The chemical literature contains vast evidence that a protein may
form*

(a) a reversible metal complex, (b) an irreversible metal complex,

©r (c) both a reversible and an irreversible metal cosplax.

The

occurrence and extent of such binding have been demonstrated by various
experimental methods such as migration in an electric field, changes in
spectra, etc.
Human blood serum contains various proteins among which are albumin
soul the alpha, beta, and gamma globulins.

Some of the metal ion con­

stituents of this serum have been found to possess biochemical function.
Among these are calcium, magnesium, iron, and copper.

The four metal

ions are all regarded to be protein-bound to some extent in human blood
serum.
The object of this study was to determine and demonstrate the human
serum protein fraction (or fractions) associated with calcium, magnesium,
iron, and/or copper ions.

The investigative approaches involved two

•types of electrophoretic analysis, chemical analysis, and equilibrium
dialysis.

On® method of electrophoretic analysis utilised the moving

boundary technique for serum protein composition together with chemical
analysis for serum metal ion content.

It was assumed that the corre­

lation of results from these analyses would be indicative of specific
protein-ion combinations.

2

The other method of electrophoretic analysis utilized serum
protein separation on filter paper with subsequent selective staining
to locate protein and metal.

If protein and metal were found to migrate

together, it was presumed that they were associated.

Exhaustive di­

alysis ef protein solutions together with chemical analysis were per­
formed to substantiate information on proteln-metal ion association.

3

XI. HISTORICAL
A. Binding of Metal Ions to Serum Proteins
This review is limited chiefly to studies concerning the binding
of calcium, magnesium, iron, and copper with human serum or plasma and
isolated blood proteins.
1.

Qalciam. — ly the method of compensation dialysis (69), it was

reported in 1911 that about i+G# of the total aerum calcium was non-*
diffusible,

the observation was published by Iona and Takahashi (83),

and they suggested that this amount of calcium was in soma way irre­
versibly protein-bound.

In 1931, Green® and Power (35) accomplished

compensation dialysis in vivo by connecting the dialysis tubes to the
femoral veins and arteries of dogs.
to be

bound.

They estimated the blood calcium

A year later, Nicholas (71) reported 36% of the

total serum calcium to be nondiffusible as determined by ultrafiltration,
while Watehorn and MeCance (101) found h3%*
it to be $3%»

Todd (99) in 19kl, stated

These variations in result might be attributed to dif­

ferent experimental conditions involving pressure and time.
Equilibrium studies of isolated human serum albumin and globulins
with calcium were made by m e a n and Hastings (68) in 1935 and by Weir
and Hastings (103) in 1936, while Drinker, ®t al. (21) in 1939, used
©quin® globulins.

All suggested that the complex between calcium and

the respective proteins can, as a first approximation, be described by

k

applying the mass notion law.

The expression as given by Alberty (3 )

to represent such a series of ion ©©raple&ee PAg (x varies from 1 to a)
in equilibrium with free protein £ and interacting ion A is*

In general, the calcium studies indicate that one-third to one-half
of the total serum calcium is irreversibly protein-bound.

Both albumin

and globulin are capable of binding calcium reversibly.
2. Magnesium, — Of the four metal ions harem considered, magnesium
has been the least investigated.

The reason for this may be due to the

assumption that its properties are like those of calcium since the two
have similar electronic configurations. Also the lack of interest in
magnesium with respect to human pathologies and lack of sensitive ana­
lytical methods for its determination may be additional factors,
§y in vivo compensation dialysis, Greene and Power (35) in 1931
ascertained the binding of magnesium to be 35~b5$ of the total, the same
as that obtained for calcium.

However, the next year by ultrafiltration

techniques Watehorn and MeGance (101) determined that only t$% of the
total was bound, in contrast to k3% for calcium.
3 . iron. —

Barkan (?) observed in 192? that less than one-fourth

of the total iron in human plasma was non-dialyaable.

Ten years later,

the same investigator with Shales (8) demonstrated that when serum was
one-half saturated with ammonium sulfate, the iron coprecipitated
quantitatively with tie globulins.

In 193b, Fowweather (28) obtained

5

1.5-2.5 times more iron from ashed serum than from the protein-free
filtrate,

Kitaes, ®t al. (U7)^ noticed in 19kl the equal distribution

of iron between filtrate and precipitate upon trichloracetic acid
treatment of serum.

In the same year, Holmberg and Laurell (U3) (in

contrast to Barkan) reported that the iron-bonding serum component was
not precipitated with ammonium sulfate.

They concluded that iron m s

bound to albumin as did Bisler and co-workers (2U). The latter, in
1936, found iron migrated with albumin daring moving boundary electro­
phoresis of serum proteins.
Cohn (16) showed in 191*8 that serum beta globulin combined with
iron to form a pink complex.

Four years later, Koeehlin (53) demon­

strated that the reaction involved one molecule of the globulin with
one or two atoms of iron.
Many reports in the literature on this subject show contradiction
or confusion.

Although they agree that Iron is partially protein-bound,

it has not bean established whether the protein involved is albumin or
globulin.

Serum beta globulin has been shown to combine with iron.

k. Copper. — Marburg and Krebs (100) discovered in 1927 that
copper is loosely bound in serum and that binding is altered by lowering
pH,

A year later, Abderhalden and Holler (1) stated that copper was

not dialyzable.

In 1937, Boyden and Potter (9), who concluded all

plasm copper was bound, investigated the effect of changing pH with
hydrochloric and sulfuric acids.

They suggested that bound copper

occurred in more than on© form.
Eisler, at al. (2i0, Investigated the migration of copper during
electrophoresis of serum in 1936 and cam® to regard it as bound to

6

albumin.. However, according to the 191*7 report of Holmberg and
laurell (i*3 ), serum copper was coprecipitated with th® globulins by
one-half saturation of serum with ammonium sulfate*
fanford (93) in 1952 analysed bovine serum albumin polarographieally
in the presence of divalent copper and determined complex-formation.
Klotz and co-workers (52) confirmed this finding in 1955 with bovine
albumin by absorption spectra analysis, but could not detect a Ilk©
reaction when human albumin was employed*
By radioactive isotope tracer techniques, Ifollf, #t al. (105)* in
the same year, also found species differences.

They decided that copper

transport in serum involved nonspecific loosely-bound complexes.

An

©xaHdnatiom of human serum protein precipitated by heavy metal ions led
Astrup and associates (5) to the discovery in 19$h that gamma globulin
bound the cupric ion firmly and irreversibly.
lii 1936, Maim and Keilin (66) isolated a metal protein complex
from horse serum (haemoouprein) which had a Q,3k% copper content.

Ten

years later, Holwfoerg and laurell (kh) isolated a similar protein
(ceruloplasmin) from human serum and characterized it as a metalloglobulln.
Cohn (16) demonstrated in 191*8 that a human serum beta globulin
combined with copper to form a blue-green couples.

In 1952, Koechlin

(53) observed that the r©action seemed to involve one molecule of protein
with on© or two atoms of copper.
These studies show that all serum copper is bound is the species
investigated. It may or may not b® associated with albumin.

7

In contrast to bovine, human serum albumin does not react Kith copper
sfeereas beta globulin does.

A eopper-globulin compound has been iso­

lated from human and equine sources.

Gama globulin binds divalent

copper irreversibly.

B. Electrophoresis
1.

Moving boundary. -- Th® need for affective separation and

analysis of serum proteins has been ufilqasly met in electrophoresis.
A chemical method is inadequate to deal with such mixtures whose com­
ponents are similar and whose distinctive properties may be destroyed
by their chemical separation,

fh® physical method, electrophoresis,

uses the characteristic charge on each protein and the electrical forces
applied are assumed to be harmless.
In 1937 j Uselius (95) described an apparatus which made possible
the study of protein mixture®.

Optical systems, utilising the

Schlieren leas, were soon adapted by bongsworth (62), Philpot (7^)> and
Svensson (91) to simplify quantitative analysis.

The extensive use of

electrophoresis in biology and medicine is shown by a bibliography
published late in 1955 which lists 3200 papers (42).
In moving boundary electrophoresis, a sharp initial boundary between
protein and solvent is formed and observed as an electric field is
applied.

If the protein is homogeneous in mobility, th® boundary

remains single.

If a mixture of proteins is present, each may move at

a different speed depending on its charge.

Th® initial boundary may

separate into several boundaries, each representing a protein of

8

different mobility.

The number sad position of the boundaries are

observed by change in refractive index produced, by change in protein
concentration.
In the quantitative Tiseliua electrophoresis procedure, the pro­
teins are separated and the pattern they form is photographed and
enlarged.

Ordinates are drawn from the lowest points between, two peaks

to th® baseline (98) and the areas under each of the peaks are measured.
If th© specific refractive increment of each protein is known, the
concentration of the components in the mixture can be determined.
Application to aeram proteins. In 1937, Tiselius (96), using &
basic phosphate buffer, found purified hors® serum globulin contained
three fractions which h© named alpha, beta, and gamma in the order of
their mobilities.

The next year, Stehhagen (89) showed human serum

contained albumin and three globulins similar to th® ©quin®,
iongsworth {6k), in 19U2, employed a more basic barbiturate buffer to
resolve alpha globulin into two fractions which he designated alpha*
and alpha3,
Cohn and associates (12,13,1U,155 have used electrophoretic analysis
as a control method in fractionation.

They obtained purified fractions

and information correlating chemical and biological properties with
electrophoretic behavior.

Tiselius (9?) found antibody activity in

gamma globulin and noted immune serum had more of this component than
normal.

By correlating chemical analysis with electrophoresis,

Longaworth and Maclnnes (63) showed that serum lipid was associated with

beta globulin,

Seibert, et jl. (86), reported concomitant increases in

polysaccharides and alpha* globulin,
.Seram protein-anion interaction.studies. The mobilities of serum
protein* and too number of fraction* resolved electrophoretically
depend upon ion specie*, pH, and ionic *ireagto of too buffer*

Thusly,

Tiselius (S>6) fractionated alpha, beta, and gamma globulin* in phosphate
buffer of pi 8 .0, toil* longsworth (6h) resolved alpha globulin into
alpha* and alpha* in barbital buffer of pH 8.6. In 1951, Fischer, et al.
(27), shewed beta globulin consisted of two protein entities in a
barbital-eaprylate medium,

these differences in mobility and resolution

have boon attributed to reactions of buffer anions with proteins (3).
Electrophoresis has been used to investigate reactions of proteins
with other substances.

Patterns and mobilities are obtained before and

after equilibration with too reactant and changes indicate reaction.
By these techniques, lessen (78) in 19i*3 reported albumin m s the only
human plasma protein capable ef reaction with diaeo dyes,

Two years

later, Putnam and Heurath (76) showed th® eotoinatioa of isolated eqaine
serum albumin with detergent anions.

Also in 191*5, Ballou and associates

(6) concluded anion-human albumin association from studies with salts of
the lower fatty acids,

liberty and Marvin (2) in 1951 found bovine

albumin combined with ©hlorlde ion.
Their findings confirmed the equilibrium dialysis results of
Seatohard, et a|. (85), to© studied bovine albumin combination with
chloride ion in 191*6, Klots and co-workers investigated the combination
of the same protein with aaosulfonat® dyes (1*9), sulfonate dyes (50),

10

and methyl orange (51).
each ease*

Spectral analysis demonstrated reaction in

In 1950, Karush (1*5), using dialysis, proposed that 22

binding sites per bovine albumin molecule were involved in reaction
with an anionic as© dye.
2.

Paper electrophoresis. Th® high cost of moving boundary

Tiselius electrophoresis apparatus has precluded its use by many in*
vestigators.

However, the development of simple and comparatively

inexpensive equipment using paper saturated with buffer as a separation
medium has made this technique mere widely available.
In 1939, von Klobusitsfey and K#nig (I48) observed the migration of
a yellow- chromoprotein from a snake venom on paper in a low-voltag®
direct, current apparatus.

Several years later, independent and almost

simultaneous studies led to the development of several types of apparatus.
Requisite to toy type arei

two vessels containing buffer and electrodes,

paper which bridges the vessels and dips into the buffer, a source of
potential, and an enclosure to decrease evaporation.

To demonstrate

the practieability of the procedure, in 191*8 bieland and Fischer (IGu)
separated amino acid Mactures with a simple catenary paper suspension,
Burrum (22) extended th® technique two years later to peptides and serum
proteins using a ridge-pole suspension.

Also in 1950, Gremer and

Tiselius (18) described a sandwich-type apparatus in which th® paper
was held between glass plates and immersed in chlorobanaene*

This was

soon modified to eliminate the ehlorobensen® by clamping the paper
between plates and sealing the edges (58).

McDonald, et al. (6?),

designed an apparatus which held the paper taut and horizontal on a
plastic frame.

11

Besides isolating the paper from the atmosphere to decrease chromato­
graphic effects, the sample m s applied to paper pre-veited with buffer.
Liquid levels in the buffer vessels were maintained equal by means of a
connecting tube.
(18,56,57,67).

Some investigators have advocated temperature control

In the electrophoresis procedure, a drop or streak of

serum was placed on the edge of the paper, direct current was applied for
a period of time after which the paper was removed from th© apparatus
and dried*

Separated components were located by a specific protein dye

and estimated by dye elution or direct densitometry of the paper.
Parallel studies of the paper and moving boundary electrophoresis
methods have met with various degrees ©f success.

Some investigators

found it necessary to us® derived factors to achieve comparability
(18,33,56,58,88).

Others reported that rigidly standardised techniques

of saa^le application, drying, and dyeing gave comparable results
directly (30,36,b©,5b,61,65),
Selective staining of parallel strips for protein and for lipid or
carbohydrate content substantiated earlier correlation studies using
moving boundary apparatus and chemical analysis,

the concept ef serum

lipid transport by beta globulin (63) has been confirmed (23,25,55,72,
8b,52),

Similarly, the carbohydratc-alpha globulin association (86)

has also bean demonstrated (55).
Protein ion interaction studies. Isotope tracer techniques combined
with paper electrophoresis have led to some interesting results.
in 1552, Koiw and ObranwaU (55) corroborated the formation of the

Thus

12

iron-beta globulin complex as reported by Cohn (16) in 191*8.

Iodine

transport by albumin and alpha globulin was reported (60,82), while
gold was found to be associated with the alpha and beta globulins (87).

13

HI. EXPERIMENTAL
A description of the apparatus, materials and reagents, procedures,
•Tv

and results will b® presented in this section.

All solutions were pre­

pared with reagent grade or C, P. chemicals, unless otherwise specified.
Sadi solid m s weighed on an Ainsworth chain'weightbalane® and dissolved
in water distilled from ill*glass apparatus.

A H photometric measure*

ments were made with a Oeaco-Sheard-Sanford Photelemeter.

Computations

were mad® with th® aid of a Fridln calculating machine.

A.

tegatus

1. Moving Boundary Electrophoresis
The Tiselius apparatus and accessories were purchased from th®
American Ifostrmaent Company, Inc., Silver Spring, Maryland.

Included

were the Amineo-Stern electrophoresis instrument (Figures 1 and 2),
dialysis units, motor driven compensator with syringe, three-way stopcock
provided with rubber tubing, standard clinical cell with holder, silversilver chloride electrodes, film holder, enlarger-darkroom unit, and the
leuffel and Baser conpoasating polar planimeier.
2. Paper Electrophoresis
The M B apparatus consisting of a power unit and horizontal strip
equipment (Figure 3) was purchased from Ivan Sorvall, Inc., Norwalk,
Connecticut.

A wooden rack held the paper horizontal while it was dried

Ik

with the aid of an infrared lamp.

A 10 x 16 inch Pyrex cake pan was

used to contain the various staining solutions.
3. Exhaustive Dialysis
Dialysis units were the same as those used for the moving boundary
procedure previously mentioned.

Sach dialyzer consisted of a Pyrex

cylinder (k inch diameter x 6 inch height) with a removable stainless
steel cover.

Details of design vers given by Heiner and Fenichel (79).
B.

Materials and Reagents

1* For Moving Boundary Experiments
a, Serum. — Participating subjects sere 21 normal adult humans*
15 female and 6 male, aged 20-5U years.

Seventy-five ml. of blood mas

drawn from each fasting subject by venepuncture into three sterile
syringes.

The blood was transferred immediately to test tube* allowed

to clot two to three hours, and centrifuged 15 minutes at 2,000 r.p.m.
The supernatant serum was siphoned off, centrifuged, and decanted into
sterile test tabes.

If not to b® used immediately, serum was deep-

freese stored at -5°.

According to the data in Table I no significant

change in protein or metal ion content occurred during such storage.
b. Materials for eleotrophcreslB.
Dialysis membrane. — Flaking cellulose tubing 1 V ® inch flat
diameter was used for serum dialysis.
Buffer, — Veronal buffer of pH 8,6 and p . 0.1 described by
Longsworth (6U) was prepared by dissolving 5.526 g, dietbylbarbituric

15

acid (U.S.P.) and 61.85 g. sodium diethyibarbiturate (U.S.P.) in three
liter* of water,
Saturated potassium chloride, — This solution of electrolyte,
introduced into th© electrode vessels, served as a salt bridge.
Film. — Schlieren cylindrical lens patterns were recorded on

k x 5 inch Eastman Contrast Process Panchromatic film.
Photographic solutions. — Kodak 0-8, SB-1, and F-5 were used in
film processing,
c. Protein analysis, «*■* Reagents for the biuret method were prepared
as directed by Kingsley (46) and Wetehselfeau® (102),
Standard protein solution containing 6,61 g, protein per 100 ml.
was prepared ty diluting two ml, Armour’s Protein Standard Solution

(9*91 g. protein per 100 ml. determined by micro-Kjeldahl analysis)
with one ml. water.
Sodium sulfat®, 23% ( s r /v ) , was prepared by dissolving 230 g. of
anhydrous solid is water and diluting to on© liter.

This solution was

stored at 37°*
Stock biuret reagent was made by dissolving U5 g. sodium potassium
tartrate, 15 g. cupric sulfate pentahydrate, and 5 g. potassium iodide
in 0 ,2 R sodium hydroxide made to on® liter.
Working biuret reagent one fifth the concentration of the stock
solution was prepared from 200 ml. by dilution to one liter with 0 .2 N
sodium hydroxide containing 5 %* potassium iodide per liter.
d. Metal jon analytical reagents.
Calcium. — The reagents given in the Clsrk-Gollip modification (11)
of the Kramer-TisdaH method (56) were utied. They are as follows;

16

Ana&enium oxalate, 1$ (w/v), was prepared by dissolving i* g, in
water and (HM ^ g to 100 iid.
Ammonium hydroxide, 1.2$ (v/v) m s

obtained by diluting 2 rol.

concentrated reagent to 100 ml*
Sulfuric acid, 2 I* was prepared by diluting 56.6 ml. concentrated
reagent to one liter.
Potassium permanganate, 0.01 N, mas prepared and standardised
against a weighed amount of sodium oxalato.
Magnesium. ** Solutions described and used by Garner (31) sere
employed in this analysis.
A stock standard (1 mg. magnesium per ml.) m s prepared by dissolv­
ing 10,13 g. magnesium sulfate hepiahydrate in water, adding 0.5 ml.
chloroform, and diluting to one liter*
The w&rkisg standard (0.02 mg, ma@aeslum per ml.) consisted of
2 ml. of steok diluted to 100 ml.
Trichloracetic aeid, 105 (w/v), m s obtained by dissolving 100 g,
in water and diluting to one liter ,
Sum ghatM, ea 0.15 (w/v), was prepared by suspending overnight
1 g. of solid tied in muslin in ©me liter of water.

The residue was

discarded and 2 ml. chloroform added.
Titan yellow (Allied Chemical and Bye Corporation), 0.055 (w/v)>
was made by dissolving 50 mg, in water, diluting to 100 ml., and filter­
ing.

This solution was freshly prepared for each determination.
Sodium hydroxide, & ft, was furnished by dissolving 160 g. in water

and diluting to one liter.

17

|gwa* «u» All reagent solutions except the standard were prepared
according to directions given by Kitsea, ®t al. (1*7)* The standard
Solution was Bade as described by ¥ong (106),
Iron stock standard (0*1 mg. iron per ml.) was prepared by dis­
solving 0,7020 g. crystalline ferrous ammonium sulfate in £0 ml. water,
adding 20 sal. 10# (v/v) sulfuric acid and enough 0 ,1 1 potassium
permanganate t© oxidize the ferrous ion*

Dilution to one liter followed.

Working iron standard (5 pg, iron per ml.) was obtained by diluting
5 ml. of above stock solution to 100 ml,
trichloracetic acid,

t%

(w/v), was mad® by dissolving 50 g. in

water and diluting to 200 ml.
Para-mtrophenol (Potman Kodak Company), 0,1# (w/v), was prepared
by dissolving 100 mg. in 100 ml. water.
Ammonium hydroxide, 6 I, was had by diluting 80 b£L, ©f concentrated
reagent to 200 ml*
Acetate buff®?, 1,6 M, pH h*58 (checked by pH meter), was prepared
by dissolving 33.U g. anhydrous sodium acetate in 27.2 ml. glacial
acetic acid and water and diluting to 250 ml.
Thioglycolic acid (Eastman Practical Grade, “95 ♦

) was used

without dilution.
Alpha, alpha*^bipyridine (Eastman Kodak Company), 0.2# (w/v), was
produced by dissolving 0 .2 g. in 5 ml. glacial acetic acid and diluting
to 100 ml, with water.
Copper. — The reagent solutions described by ©abler and associates
(37) were used for this analysis.

18

Copper stock solution (100 pg. copper per nil,) m s prepared by
dissolving 0.3928 g, copper sulfate pentahydrate in water and dilating
to one liter.
Two standard copper solutions were prepared by diluting G.h ml.
stock to 50 ml* (0*8 pg. par ail,) and 1.0 ml. stock to 50 ml*
(2.0 jag. per ml.).
Eydrochlortc acid, 2 M, m s obtained by dilution of 85.5 ml. concen­
trated reagent to 500 ml.
trichloracetic acid, 20$ (w/v), was prepared by dissolving 100 g.
in water and dilating to 500 ml.
Sodium pyrophosphate, saturated, m s obtained, with 100 g, of solid
and 200 ml. water.
Sodium citrate, saturated, wae prepared by adding 350 g, to 200 ml.
water*
Ammonium hydroxide, 39$ (v/v), was obtained by diluting 200 ml.
concentrated reagent with 100 ml. water.
Sodium diethydithiocsn*bamat8 (Fisher Scientific Company), 0.1$,
was prepared by dissolving 100 mg, in water and diluting to 100 ml.
2, Materials and Reagents for Paper Electrophoresis
a. gerom. — Portions of the same human blood serum as described
for the preceding experiments were used,
b. Buffer. — The Longsworth (6h) pH 8.6, p. 0.1, veronal buffer
was employed.

It was used with Mhatman No. 1 paper cut to 18 x Uh cm.

rectangular sheets as the supporting medium,

19

e, Protein stain. —

The bromphenol blue staining bath and rinse

solutions as described by Durrum and co-workers (23) were used.
The bath was prepared by dissolving 100 mg. bromphenol blue
(Butrltioaal Biochemical Corporation) In $Q ml. glacial acetic acid,
adding 50 g. mercuric chloride, and diluting to one liter kith mater.
Acetic acid, 2% (v/v), rinse solution, mas obtained by diluting
20 ml. glacial acetic acid to one liter.
Sodium acetate, 0.5$ {w/v) in acetic acid, prepared by dissolving
5 g. in one liter of above 2$ acetic acid, mas the final rinse*
Ethanol, 50$ (v/v), was used to elute the fixed stain,
d. Metal ion staining reagents.'
Calcium. — The alisarin red S histoohemical stain developed in
1952 by Dahl (19) was employed to locate Calcium upon the filter paper.
In 1955, latelsom and Pennial! (70) demonstrated that the calciumalizarin cclor in solution obeyed Beer’s law.
Alizarin red S (Sberbaoh f Con Company), 1$ (w/v) was prepared bydissolving 10 g, in a liter of water containing 1 ml, concentrated
ammonium hydroxide.
Ammonium hydroxide, 29% (v/v), obtained by diluting $00 ml. con­
centrated reagent with 500 ml, water, was used for elution.
Magnesium, — The staining solution for the identification of this
metal ion upon filter paper was ©f the following composition*
56 ml, 0,1$ (w/v) gam $iatti

Bk ml* 0.05$ (w/v) Titan yellow
112 ml. h H sodium hydroxide.

20

The#® three reagents wore among those used in the Garner method (31)
for the eolorimetrio determination of magnesium in serum.

The volume

proportions 1 t 1 ,5 * 2 in the above order were the same as those given
In Gamer’s analytical procedure*
Iron, «— A 20% (m/v) , potassium thiocyanaie reagent used as a stain
for iron mas prepared by dissolving $0 g. in 21+0 ml. water and adding
10 ml, concentrated hydrochloric acid.
Copper. -«*• The stain for detecting this ion m s obtained by mixing
the following solutions!
50 ml, saturated sodium pyrophosphate
50 ml. saturated sodium citrate
100 ml, 39% (v/v) ammonium hydroxide
50 ml, 0 ,1$ (w/v) Sodium diethyldithiocarbamate
The above four reagents and the volume proportions of 1 * 1 1 2 * 1
were as used in the Gubler method (37) for serum copper determination.
3. Materials end Eeagents for Dialysis
Dialysis membrane. — Viaking cellulose tubing 11/8 inch H a t
diameter Was used in all experiments.
Buffer. — The Longaworth (61+) pH 8,6, f. 0,1, veronal buffer served
as solvent for the solid protein fractions and as external liquid in
dialysis.
Serum. — Portions of the human sera as described for moving
boundary experiments were pooled both for dialysis and for the prepara­
tion of protein solutions Albumin II, Albumin HI, and Mixed Globulins
which are described subsequently.

21

Altounin I. — A lyophilliaed sample of 99% human albumin made from
the Cohn (15) Fraction V and labeled as rework #127 was obtained from
the Biological Products Section of the Division of Laboratories of the
Michigan department of Health.

A 50 ml, solution, designated Albumin I,

was prepared by dissolving 6,52 g. of the material in veronal buffer.
Albumin XI. — Forty-eight g, ammonium sulfate m s slowly added
with stirring to 96 ml, pooled hums sera.

After 30-minute centri­

fugation, the clear supernatant albumin solution m s siphoned off and
placed in a Tisking dialysis sac,

Parvaporation (9k) for four days

resulted in deposition of salt on the outside of the membrane which m s
removed by washing with water every 12 hours,
remained 52 ml. clear protein solution within,

Ihen finished there
this m s labeled as

Albumin XI solution and found to consist of 83$ albumin by electro­
phoretic analysis,
Albumin III. —

the protein solution referred to as Albumin H I m s

obtained from 100 ml, of human serum, by th® low temperature methanol
precipitation procedure of Pillemsr and Hutchinson (75) to remove
globulins.

Following concentration by pervaporarbion of th® supemate

to u3 ml., electrophoretic analysis showed th® preparation to contain
93# albumin.
Mixed Globulins. — This preparation was mad® by dissolving the
Pillemer-Hutchinson globulin precipitate (above) in a minimum amount of
saline solution (0.9# sodium chloride) and diluting to 50 ml, with
veronal buffer.

22

Gamma Globulin. — A rework sample marked as #10li mads from the
Cohn (15) Fractions II and H I was also generously provided by the
Biological Products Section of the Division of laboratories of the
Michigan Department of Health,
gamma globulin.

Electrophoretic analysis showed 97%

A $0 ml, solution was prepared by dissolving 1.96 g.

of the lyophilliaed material in 0.1 H veronal buffer of pH 8,6,
Paper strip analytical reagents, -*■ The same reagents as previously
described for total protein and metal ion determinations in moving
boundary experiments were used.
€* Experimental Procedures

Results of moving boundary electrophoretic analysis of proteins
have been correlated with certain other serum constituents.

Thus, in

1937 Tiselius (97) observed that serum high in gasman globulin also gave
high values for isamme bodies,

This serum protein fraction has since

been used for protection against poliomyelitis* measles, and hepatitis
and from it several types of antibodies have been isolated (3U)»
Longsworth and Madnncs (63) noted that serum with high beta globulin
content possessed a large amount of lipid and proposed that a lipo­
protein couples: existed.

Seibert and co-workers (86) observed that

serum high in &lphas globulin also had a high polysaccharide content
and regarded the two to be associated.

The existence of these globulin-

lipid and globulin-carbohydrate combinations has been demonstrated
directly by paper electrophoresis (23,25,55*59*72,8^,92),

23

1* Moving Boundary Methods
The experimental approach adopted for indication of protein-motal
ion association involved correlations between amounts of protein
components obtained by moving boundary electrophoresis and metal ions
present in serum,

In this connection, serum of 21 normal human subjects

was used for investigation,
a, Electrophoresis techniques
Dialysis, — * Ten ml, serum was diluted with ten ml, of veronal
buffer to produce an approximate protein concentration of 3.2-3.8 g. per
1C® ail. The diluted serum was osmotically equilibrated by dialysis
against 800 ml. of the same buffer solution for 2k hours at 2°.
Electrophoresis ♦ «*- Buffer, saturated potassium chloride, and
dialyzed serum were introduced into the cell in the prescribed manner.
By visual observation, initial boundaries were set two cm. from the
lateral edges of the screen.

Electrophoresis at 1$ milliamperes and

360 volts was continued until maximum separation of protein components
was achieved,

Th® time varied from 90 to 120 minutes (5U00 to 7200

seconds),
Photography. — Negatives of ascending and descending patterns were
obtained by four to five second exposures of the panchromatic film and
proper processing.

Examples of resulting patterns are given in Figure h.

Enlarged images, 3 I linear magnification, were traced on paper,
(See Figur® £),
Pattern area measurement. — Ordinates were drawn on the enlarge­
ment from the minima between adjacent peaks to the baseline as suggested

by Tiselius «®d Rabat ($3).

Each of these separated areas was measured

planimetrieally.

figure 5 shows an enlarged ascending pattern with

erdinates dram,

The equation for calculation Is given in Appendix I.

Beth ascending and descending patterns were measured.

The values

obtained from the descending were discarded because some separations
ware net distinct.

(See Figure 1*) Ideally, ascending and descending

patterns would be mirror images.

In practice, however, the peaks in the

descending patterns are net as steep.

This may result in iaceiqplete

resolution of albumin (the largest component) and alphax (the smallest)
and of gamma globulin and the epsilon anomaly, a nonprotein boundary
which represents a buffer gradient that remains near the initial
descending boundary.
The average ©f three planlmetri© determinations on the ascending
pattern of each subject is reported in Table 11a.
b, Total protein determination, —

The Kingsley method (U6) with

WQichselbaam’e (102) biuret reagent was used for total serum protein
determinations.

The procedure is based on th© eolor reaction between

peptide linkages and basic copper sulfate.

The determination involves

photometric comparison of the protein content of unknown human serum
with known bovine serum and assumes identical species response.
Suspensions of human and bovine serum were prepared for total
protein analysis by mixing 0 .5 ml* of each serum with 9.5 ml. 23# sodium
sulfate.
following*

Four ml. of working biuret reagent was added to each of the
first, 2 ml. water (blank)j second, a 2 ml. portion of human

serum suspension (unknown)! and third, a 2 ml. portion of bovine serum

25

suspension (known). After 15 minutes, the photometer with g ?eea filter
(cant,ral maximum 525 aft) was set to 100 with the bleak (1^) and values
were obtained for human (%) ami standard bovine (1^) sera,
ware eoasrertsd to sbsorbaacy units by the relationships

The values

A* m log10

The equation for the calculation of total serum protein is given in
Appendix I. . .
Figure 6a shows that the ooncentration-abeorbancy relationship
follows Beer’s law,

The reaulbsef serum protein analyses are reported

in Table p b .
■

Procedures for m r m metal ion determinatioae
Calolum, — The Clark-Gollip modification (11) of th® Kramer-Tisdall

(56) method was uoed.

In this analysis, calcium oxalate was precipitated

directly from dilute serum, converted to oxalic acid, and titrated with
0 ,0 1 N potassium per®!aagaaat® solution (hi) , Besulbs are reported in
Table III.
Magaeaium, ** The method developed by Qaraar (31) for serum or plasma
was employed*

Basically, th® procedar® involves the release of magnesium

associated with serum proteins by acid dan&turation and precipitation.
The formation of a colored complex between magnesium and Titan yellow
in the filtrate allows the photometric comparison with standards,
Four mil, serum was mixed with 8 ml, water and i* ml. 105 trichlor­
acetic acid, allowed to stand five minutes; then the filtrate through
Whatman Ms.* 1*U paper was collected.

Two magnesium standards (corres­

ponding to 1 and 2 mg, per 100 ml. serum) were also prepared! one
contained 1 ml. working standard and 5 ml* water, the other 2 ml. working

26

standard and k ral. water*

A blank consisted of 6 »L. water.

Two ml.

trichloracetic acid mss added to each standard and the blank.

One ml.

G.l# gum $mbti, 1.5 «&, of G.G5'£ Titan /allow, and 2 ml. U S sodium
hydroxide vara added to an S ral. portion ©f collected serum filtrate,
standards, and blank.. After- setting the photometer (green filter) to
IS© with the blank, readings were obtained for serum and standards.
The*# results wereeonverfcsd to absorbanc/ units as previously described.
A linear oust© from th© two standard eolation values was constructed
shewing magnesium ion concentration in mg. per 100 ml. versus eorresponding abscrbaacies,

Since the standards were prepared as above, serum

concentrations within this range wer© read directly from the curve,
figure 6b is a typical standard absorbaucy curve for magnesium.

The

analytical results are given is Table III.
■ , Iron. *- Th® colorimetric method for serum ferric ion analysis
described by Kits®# and associates (1*7) was utilised.

In principle, the

method involves the heat release of iron from protein, pH adjustment of
the protein**free supernata, reduction to th© ferrous state by thioglycclio acid, ami- development of a color by this form with alpha,
alpha*-bipyridin®.
Three ml. water was mixed with irral. human serum in a centrifuge
tube and heated to opacity in a boiling water bath.

After cooling in

a 5-1©° water bath, 2 ml. t % trichloracetic acid was added and contents
mixed.

The tube was next heated in a 90-95° water bath for 3 minutes and

cooled again.

After 5 minute* of centrifugation at 2,500 r.p.m«, the

sruperaate was decanted into a 75 ml. Messier tube pro-calibrated to a

27

15 ml. volume.

The protein residue in the centrifuge tub® was washed

with k ml* water and 1 ml. trichloracetic acid*
described procedure was repeated,
original supernote.

Then the afore-

the washing was combined with the

it this point, -standard ferric ion solutions were

prepared in similar Kessler tubes by introducing 1, 2, and 4 ml. working
standard (containing 5 fig. per ml.) end 3 ml. trichloracetic add.
A blank consisted of 3 d . of the add.
A drop ef 0.1# para*id.ir©phen©l indicator was added to th© serum
filtrate, the 3 standards, and the blank*

ArasnoniUE hydroxide (6 M) was

added dropwia© t© each solution until yellow.

After adding 1 ml. of

pi U.5® acetate buffer, water was supplied to make a thoroughly mined
total volume of 15 ml.

Finally, two drops of thioglycolic acid and 1

ml* 0 ,2$ alpha, alpha*-bipyridin© reagents were added to each tube.
The photometer with green filter was adjusted to 100 for the blank,
and readings for serum and the 3 standards were recorded.

These were

converted to absorhancy units and a standard curve was drawn as is
presented in Figure 7a.

2a order to express serum iron in fig. per 100

ml. serum, the interpolated concentrations were multiplied by 25.
Analytical findings for serum iron content are presented in Table XXI.
Copper. «*, The procedure described by Qubler, et al. (37), m s
adapted for the determination of serum copper.

Essentially, it in­

volves the release of copper from protein by acid and subsequent protein
precipitation.

Pyrophosphate and citrate remove interfering iron and

a colored copper^carbamata complex is produced.

28

Two ni. M m w w i sriLaced with 2 ml. 2 N hydrochloric acid and
allowed to stand 10 minutes.

Upon the addition of 2 ral, 20$ tri­

chloracetic acid, mixing and standing time were repeated.

Following

45 ndimtes of centrifugation at 3,000 r»p.m», the aupemat© was decanted
into a l x ? cm, test tube.

Two ml, of 2 I hydrochloric acid and 2 ml,

20$ trichloracetic acid were also added to 2 mi* of each of two standard
copper solutions (0,8 and 2.0 jag, per ml.) and to a 2 ml. water blank.
All tubes contained a volume of 6 ml,

Four ml, portions of above serum

sapernate, standards, and blank were transferred to photometer cuvettes.
Four-tenths ml. saturated sodium pyrophosphate, 0.4 ml. saturated sodium
citrate, and 0.8 ml, 39% ammonium hydroxide war© added to each cuvette.
Following mixing and setting th® photometer with blue filter (central
maximum 410 mp) to 100 with th® blank, readings in the absence of color
reagent were noted for serum and the standards (1^5 to correct for the
innate color of the serum filtrate.
Four-tenths ml. 0,1$ sodium diethyidithiocarbaraate was added to
all cuvettes and the second set of photometer readings was obtained (I8).
Results were converted to absorbancy units and tabulated as Agl and Aea
values for before and after color development of serum and standards.
The calculation of the quantity (4®jrAg;jf), the color absorbamcy of the
copper-carbamat® complex corrected for volume change, is shown in
Appendix I.

From a plot of (Ag2-ASlf) versus jug. copper/100 ml. which

results in the standard curve, th© serum copper content was read
directly.

Figure ?b illustrates such a standard copper curve.

Results

of determinations of serum copper values in jag./lOO ml. are given in
Table III.

29

8, Procedures for Paper Electrophoresis and Staining
Sines dirset demonstration by isolation of human serum llpo- or
gLyco-protein fractions upon paper had been accomplished, it was deemed
feasible to extend this technique to metal lon-protains. Filter paper
electrophoresis was used to separate the serum proteins,

Seram samples

from the aforementioned 21 normal subjects were used in 38 paper runs.
From each sheet a strip was stained to locate proteins and other strips
were stained to locate metal ions,
a.

Electrophoresis, — Six hundred ml, of pH 8.6, u 0,1 veronal

buffer was placed in eaoh electrode vessel of the paper electrophoresis
apparatus.

A liquid junction, established between vessels by siphon,

maintained liquid levels.

Paper was inserted and wetted by the buffer

Solution due to capillarity.

A 0.2 ml. sample of undiluted serum was

streaked across the end of the paper 8 cm. from the horizontal edge
adjacent to the cathode.

(See Figure 8),

The variable resistance was

adjusted to apply 200 volts and 2.U milllamperes.

In all cases,

electrophoresis was contiimed for lb hours at 5°,

The paper, removed

from the apparatus, was dried in air with the aid of an infra-red lamp.
Of th® original 18 x hi* cm, rectangular sheet, the 10 x 30 cm, mid­
section was retained and cut longitudinally into five strips, each 2 cm.
wide and 30 om, long.

(See Figure 9a),

b * Protein staining, —

One 2 x 30 cm. strip from a run on human

serum was placed in 250 ml. of bromphenol blue staining bath for six
hours with occasional agitation.

Rinsing as described by Durrum, et al.

(23) was accomplished by three 5-minute immersions in 250 ml. 2% acetic

30

acid and A final 10-ralmte immersion in 250 Ml, 0,5# sodium acetate in

2% acetic acid,

The paper m e again dried.

areas on a whit® background,

The strip showed five blue

Figure 10 indicates th® locations of these

colored areas.
On the same strip, as shorn in Figure 5b, th® line of sample appli­
cation and 0.25 ©a, either way wad designated as aero migration distance.
From this 0.5 x 2 cm, area, transverse segments (cross-pieces) 0,5 cm.
wide were marked, assigned migration distances, and cut off for elution.
Th# stain from each segment was eluted by a 30-isiirate immersion in two
ml, of 50# (v/v) ethanol.

After photometry (green filter) of the

segment eluatss, a plot of migration distance versus eluate absorbaney
was mads.

A resulting representative plot frcm the paper electrophoresed

human serum protein® is shown in Figure 11,

The curve is similar to a

moving beuMsry pattern.
Quantitative analysis of the paper electrophoresis pattern for
protein distribution followed th® same procedure as that used in moving
boundary electrophoresis. Ordinates wore drawn from the minima between
peaks to the baseline (zero afesorbauQy), separated area® were measured
with th# planimeier, and th© relative amounts of the serum proteins were
calculated,
c. Betection of metal ions ea paper. -** Staining techniques upon
paper were attempted for numerous of the serum ions.

In addition to

the four metal ions initially reported in this investigation, sine was
also successfully located.

No colors were obtained when stains for

potassium, phosphate, carbonate, chloride, and sulfate were tried.

31

Calcium. — For qualitative work, & strip 2 cm. wide was immersed
two minutes in the 1# alizarin red 3 staining bath, rinsed briefly

OS seconds) six times with water, and dried. Five ©range-pink areas
on a pale pink background resulted,

Figaro 10 shows the positions of

these ealoium-aliaartn areas.
For the quantitative distribution of calcium among the serum pro­
teins, a 6 x SO cm. strip mas stained for calcium as just described.
Migration distances (in 0.5 cm. malts) were marked as in the protein
procedure,

The stain from each 0,5 x 6 cm, segment was eluted by 30-min­

ute immersion in three ml. of 29% ammonium hydroxide,

Photometry (green

filter) of elnates and graphing of results with migration distance were
done as previously described for serum proteins,

The findings are

represented in Figure 12,
Magnesium. ** Several 2 x 30 cm. strips were immersed in the Titan
yellow staining bath for periods of time ranging from two minutes to
four hours.

Mo matter how long the immersion, red spots on a yellow

background were visible only when the paper was wet with staining solu­
tion but disappeared during drying.

Figure 10 shows the locations of

the cclored areas on the wet paper strips.
Iron.

Another strip from a serum paper electrophoresis run was

stained in 2Q% thlocyanat® solution for 15 minutes, rinsed with five
changes of water, and dried.

The position of th© single pink area of

iron-thiocyanate complex that resulted is shown in Figure 10.
Copper. — A 2 x 30 cm. strip was immersed for 15 minutes in the
diethyldithiocarbamate copper staining bath and dried.

The position of

th® one resulting pale yellow area is represented in Figure 10,

32

mm
Historical. la 198b#fault and Sehon (73) demonstrated an associ­
ation of ©qulae albumin with sina ion by conduetdvlty measurements,
la 195©* Gohn and ee-workera (17} reported that the addition of sine
ioa to plasma effected protein precipitation with lass alcohol and that
tha sslne could b© removed ©emplotely from the proteins, Qurd and
Goodman (39) in 1952 showed a totally reversible reaction when human
aapoy aifeiMBin wa« equilibrated with ySnre chloride solutions*

fhe first

precipitate formed in the reaction of bivalent sine with human serum ^
was. found, to contain albumin and gamma globulin by Resalar, et Q , (81),
in 1954* Thus, it has been diwannstrated that albumin and globulins
react revoraibly with sine ions.
the solutions to locate this ion on paper were
prepared from directions given by ©lick (32).
practical Grade),
10$ (w/v), was famished by dissolving 25 g. in water and diluting to
25© »A*
potassium sulfide «M.F.% 2*5$ (w/v), was prepared by dissolving
6.25 g. in- 250 A * water.
Procedure. A paper strip was stoned 15 minutes with 10$ sodium
niiropruaside solution at 5©°.

Ebccess reagent m s removal by washing

15 minutes with naming distilled water.

A one-minute immersion in

2,5% potassium sulfide yielded a single lavender area.
indicated in Figure 10.

Its location is

33

Attearptad spmtro graphic analysis for metal ions on paper.
Attempts wears made to l&entif^r spectrographically the metal ion residues
ia serum jaretslas separated by paper electrophoresis. A 0.2 ml. serum
Bas$l® m s separated on Hh&tmsa So* 1 filter paper, and a 2 cm* strip
m s stained for protein*

Th# remainder of the paper m s cut into fire

transverse segments each known (from the protein stain) to contain a
single protein component.

A blank run (with the same conditions of

buffer, voltage, time, etc*) using no serum m s made and segments were
out.

All papers were dry-ashed and residues transferred to cupped carbon

electrodes*

Emission are spectra of the carbon electrodes (to correct

for electrode impurities), paper blanks (to correct for paper impurities),
and proteins sere obtained with a Bauseh and Lemb medium quartz spectro­
graph.

ixaminatlon of the speetr©graphic plate showed no significant

differences betweenth® protein, paper, and electrodes,

the metal ions

of interest m r e present as impurities in paper and electrodes.
Presuming that failure m s due to th® minute quantities of metals
present in 0.2 lal. of serum, one ml. of serum m s separated on heavy M
Whatauatt paper.

Protein isolation, paper segmentation, and spectrographic

analysis were repeated,

line intensities obtained from blank paper and

proteins were again of the setae magnitude.

Hence this type of analysis

was abandoned,
3, Procedure for Exhaustive Dialysis
To determine the time necessary for exhaustive dialysis, the follow­
ing procedure was used.

Two identical 30 ml. pools of undiluted serum

(labeled 1 and 2) ware equilibrated for seven days at 2° against 650 ml*
of veronal buffer.

Every 2U hours, the volume of pool 1 m s measured

and a two ml. portion removed for analysis and replaced by two ml. from
pool 2.

The outside buffer liquid of each m s changed dally.

Analyses

for residual calcium, corrected for volume changes, compared with dialy­
sis time showed by plot that after five days no farther removal of
calcium occurred;

(Figure 13).

The course of exhaustive removal of the other ions, magnesium, iron,
and copper, was presumed to be the same.

Other studies showed that they,

too, were removed as coaqpletely as possible by five day dialysis.
After these preliminary conditions were established, five-day
exhaustive dialysis was performed upon the following six labeled protein
solutions;

Albumin 1 (99%$ Cohn Fraction ?), Albumin XI (83%, from

ammonium sulfate precipitation of globulins), Albumin III (93%, from
methanol precipitation of globulins), Mixed Globulins (methanol pre­
cipitate), Gaaam Globulin (91%, Cohn Fractious IX and HI), and whole
serum,

A 30 ml. portion of each of th© above listed solutions contained

within a cellophane membrane m s equilibrated at 2° against 650 ml. of
veronal buffer for fiv® days by changing th©

outside buffer daily,

determinations of total protein and magnesium, calcium, copper, and iron
metal ion concentrations were made on dialysed and undialyzed portions
of each protein solution,
Table ?I.

Th© results of these analyses are given in

35

ciaus i
EFFECT OF DEEP-FHEE2E STORAGE ON TOTAL PROTEIN
AND ifflTAL ION CONTMT OF HOMAN SSRUH

Days of
Storage
0

Total
Protein1

Caloiura2

Magnesium2

Iron3

OOJyfT*

¥.01

10.7

1 .U

65?

DiO

69

lijO

69

HiU

12

dmm

35

mm

■mm

60

7.01

mm

80

mm

10.5

.

ig.AOO ral.
Smg./lGO mi.
^ag./lGO ral.

"**

■mm

.M»OW

l.U

•MO

mm

mm

36

TABLE Ila

PLAKIMETRIC AHALX3I3 OF SERUM ELECTROPHORETIC
PATTSRHS POK EACH 0? 21 iH&H SUBJECTS
lelative fractional Composition of Serum froieln Components
dLobulina
Albumin
Alpha3
Beta
Oamra®
ll$hik4''

M.A J>— .^JLBtafcer

I

0.553
0.6O3
©.578
0.466*
0.655##
0.548
0.565

0.039*
0.053
0 .087*#
0.053
0*0514
0.073**
0.042
0.050
o.©51
0 .038*
0.065
0.063
0,©58
0.060
0.043
0.048
0.047
0.064
0.032*
O.O76*#
0.033#

0.085
0.109
0.117
0.105
0.124**
0.096
0.084
0.111
O.O83
0.067*
o.H9*»
0.113
0.107
0.113
0.092
O.09O
0.094
0.114
0.053*
0.095
0.098

0.173#*
0.116
0.123
0,313
0.128
0,116
o.m*
0 .093*
0,134
0 .107*
0,159
0 .175**
0.125
0,149
0.148
0.151
0.170**
0.167**
0.137
0.155
0.122

0.148
0.147
0.145
0.119
0.103*
0 .238**
0.155
0,124
0.141
0,193**
0,152
0.175
0,170
0.152
0.164
0,308*
0,111*
0.188*#
0.125
0.127
0.183

0.561
0.052
0.5090.613

0.054
0.014
0 .0400.068

0.099
0.018
0.0610.117

0.136
0.025
o.m0.161

0.351
0.033

0.555
0.575
0.527
0*609
0.591
04 77*
0 *618««
0*622#*
0*601
0.595
0*506#
0.473*
0,540

a

3
4
5
6
7
8
9

10
u

12
13
14

0.528

15
16
17
18
19

m

21

t
Its

■"

*Valne® lea® than X - a
Values more than 1 + s

0.1180.184

37

TABLE U b
TOTAL HtGTETH G Q N T W AND SERUM PROTEIN DISTRIBUTION
IT MOVING BOUNDARY ELECTROPHORETIC ANALYSIS
lerum GofflDonenta in g./lOO ol.
Subject
RlBdMB?

Total
Proiain
g./lOO ml.

(Eobalins
ATfoipiTai

ITT—

*” Gararaa

i
2
3
k
5
6
7
0
9
10
21
12
13
Ik
1%
16
17
18
19
20
21

6.61
6.70
6*70
6.80
6.97
6,88
6.k3»
6.97
7.17
7.56**
7.01
6*61
6.61
6.70
7*37#*
6,98
6.70
6.61
7.07
7,26m
l.htm

3.67
3.85
3.53
k.lk
k.12
3.28
3.97
k,3k«*
k.31
k.5o»*
3.55
3.13*
3.57
3.5k
k.08
li.21
3.8?
3.08*
k.63#*
3.98
U.21

0.26*
0.36
0.58**
0.36
0.38
0,50
0.27*
0.35
0.37
0.29
©,k6
0.U2
0.38
o.ko
0,32
0.3k
0,31
0,k2
0.23*
o.55»*
0.25*

0.56
0,73
0.78
0,71
0,36**
0,66
0.5k*
0,77
0.60
0.51*
0,83**
0.75
0.71
0,76
0.68
0.63
0.63
0.75
0.37*
0.69
0.73

l.lk#*
0.78
0.82
0.77
0.89
0.80
0.65*
0.65*
0,88
0,81
1.11
1.16**
0.83
1.00
1.09
1.05
l.lk**
1.10
0.97
1.13**
0.91

0.98
0.98
0.97
0,81
0,72
1.6k**
1.00
0.86
1,01
l.k6**
1.07
1.16
1.12
1.02
1.21
0.75*
0,7k*
1.2k
0.88
0.92
1.37**

r

6,91
0.31
6*607.22

3.88
0,k3
3,k5~
k.31

0.37
0.09
0,280.k6

0.68
0.12
0.560.8 0

0.9k
0.1?
0.7k1.11

1,0k
0,2k
0 ,801.28

_ •
I * a

*?alues lass than f * e
values m m than I ♦ •

38

t m m ixi

mmn
Subject
Number

Oalcium ' mg./lOO ml .

1
2
3
U
5
6
7
8
1©
11
12
13
U*
15
16
17
18
19
2©
21

1
a
lie

am kisss

Magnesium
mg,/lGG ml.

Iron
/(g./lOO ml.

Copper
/fg./lOO ml.

18*7
10.2
U .l
1Q.U
11,8
10.8
10,5
1 2 .1 **
10.9
11*9
10.3
9*2*
9.7
10.3
1 2 ,2 **
10*8
10.9
9 ,7 *
11,2
11.7
11.6

1.3
1.3
1.U
1 .5
1,3
1.2
1.3
1 .6 **
1.1*
1*5
1 .6 **
1 ,2 *
l.U '
l.U
1.3
l.U
1.5
1 .1 *
l.U
1.U
l.U

1UU**
138
9h
9U
125
128
U7*
56*
138
69
9U
i )|J|
106
69
119
62
188**
72
125
156**
56*

9k
lUe
128
70
80
18U**
70
lUo
132
20U**
88
76
76
76
76
UU*
106
132
68
76
220**

10,9
0.8
3 0 .1 31.7

l.U
0,1
1 .3 1.5

101
39
621U0

109
U9
60158

*?alu®® 1®»8 than X - ■
?«lue« more than I * s

/

or k m ,

39

TABLE IV
VALUES FOR NORMAL HOMAN SERUM
Electrophoresis
Relative Fractional Composition of Serum Protein Components
Globulins
Albumin

Alpha1

41ph«j

0.533
0.603
0.568
0.560
0.560
0.601
0.561

0.080
0 . 01(0
0.072
0.072
o.ol*5
0,050
Q.Q51*

0 , 101*
0.097
0.087
0.088
0.115
0.093
0.099

Beta

Gemma

0,138
0.128
0.128
0.131
0.160
0.115
0.136

0 . 11*2
0.132
0 . 110*
0.11*7
0.120
0.11*1
0.151

Reference
86
90
77
80
10
29
This work

Total Protein and Metal Ions
Total
Protein
g.1

Calcium
rag.®

Magnesium
mg.®

5,5-8 ,i*

9-11

1-3

Iron
/'g,3

110 f.4
129 m.5
60-200

6,91

10.9

xg. par 100 ml. serum
®£g. per 100 ml. serum
^ag, per 100 ml. serum
4for adult females
®for adult males

1.1*

101

Oopper
/*g«3

Reference

Itl
38
38
26
81*-H*3 f,4 37
68-131* m.® 37
109
This work

ko

TABLE 7
COMPARISON OF MOVING BOUNDARY AMD PAPER ELECTROPHORETIC
AMALISIS ON THE S A M SERUM

Protein

Moving Boundary

Paper

Albumin

0.601

0.602

Alpha^ Globulin

Q.O£L

0.037

Alphas Globulin

0.083

0,083

Bata Globulin

0.15U

0.128

Gamma Globulin

O.U4I

0,150

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Figure

5. Enlarged

Ascending

Moving

Boundary

Pattern

with

Ordinates

Drawn

47

48
v & T)x..ip./'.-L

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49

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50

Figure

Paper

in cm

Selective

•H
•P

pH

•H

CO

•H

Discard

to
distance

for

Staining

and

Elution

10 cmr

Migration

9. Gutting

51

18 cm

Discard

o
iH

iH

52
Figure 10, Qualitative Results of Selective Staining
Line of Sample Application
Protein

Magnesium

Iron
X

Copper

X

Zinc

X

Gamma

Beta

Alp lia^ Unbaj

X indicates presence of dye.

Albumin

53

Figure

12.

Comparative

Calcium

< o

•H

o o

CO PQ

O

O

O

O

O

Xou^qjosqv

O

CV

O

in cm

r— <

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PQ O

Migration

of

distance

Distribution

and

Serum

Proteins

54

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cv

53
P.O
•

to

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CV

55

Figure 13.

3.5

Exhaustive Dialysis
Days of
Dialysis

Volume

Total
Mg. Calcium

0
1

30
32

3.10
0.91

2
3

33.5
34

0.66
0.50

4
5
6
7

35
35
35
35

0.34
0.34
0.34
0.34

3.0

Total Calcium in mg

•

2.5

2.0

1.5

1,0

0,5

4-

-

Q

~------------------------

0

1

2

3

4

6

7

56

IV. DISCUSSION
This section will attempt to evaluate and. interpret experimental
results on metal ion binding by human serum proteins.

A. Moving Boundary Experiments
1.

Statistical considerations. — Electrophoretic analysis yielded

the relative fractional amounts of each serum protein (e.g.. Table Ila,
for subject number 1, showed albumin was 0.555 ©I the total serum
protein).

The total protein in serum was determined to enable the calcu­

lation of absolute amounts (in g. per 100 ml.) of protein components.
A sample calculation is given in Appendix I.
The relative amounts of serum proteins are given in Table II&,

The

results of total protein determinations together with the absolute
amounts of the serum protein components obtained by calculation are pre­
sented in Table lib.
The arithmetic mean (f) and standard deviation (s) for total protein
and for the relative and absolute amounts of serum proteins were calcu­
lated by the statistical equations (20) t

in which X * a single observation and N » number of observations.

The

means, standard deviations, and ranges (f ± s) for each value are in­
cluded in Table II,

Values less than I-s are designated *, while values

57

more than X+s ar® designated. *».

This was den© t© facilitate corre­

lation observation©.
Table H I presents the results of serum determinations of calcium,
magnesium, iron, and copper for the seme subjects as Tables Ila and lib.
Here again, means and standard deviations were calculated and values
exceeding the Its rang© are indicated * and *#,
To demonstrate the validity of results of serum protein distribution
obtained by electrophoresis and the determinations- of total protein and
metal ions in human bleed serum, Table 17 gives the mean values resulting
from this experimental work together with values from other literature
sources (10,26,29,38,1*1,77,80,86,9®),

Examination of the table shows

acceptable agreement of results.
To obtain the data presented in Table I, determinations of total
protein and metal ions ware made on human serum before freesing and
checked after 33-80 days of deep-freeze storage.

The table shows that

no significant changes occurred with cold-storage treatment of serum.
2* Serum metal ion contents and correlation with serum protein
fractions. —

Bata of Tables II and H I indicate the following*

Calcium. — The serum concentration of this metal ion exceeded the
range 10.1-11.7 mg. per 100 ml. for subjects 8, 10, 12, 15, and 18*
In all cases, total protein exceeded the range 6,60-7.22 and/or albumin
exceeded the rang© 3.1*5-U.31 g. per 100 ml.

The coincident increases

(or decreases) in calcium and total protein and/or albumin suggest that
a relatively large amount of bound calcium may be associated with albumin
and that the globulins may also participate in binding.

58

Magnesium. — Seram magnesium contents for subject 6, 8, 11, 12,
and 18 ware outside th® Its rang© of l.l**!^

per 100 blL* Th® serum

albumin was low for the three subjects with low magnesium values
(6, 12, and 18) and high for on© Subject with a high magnesium value (8).
these findings suggest that albumin would be the serum protein primarily
involved in magnesium association.
Iron. — Values less than 62 or more than 11*0 jug. per 100 ml. serum
were obtained for subjects 1, 7, 8, 17, 20, and 21,

For all except th®

last subject, a concomitant change in the same direction occurred in the
beta globulin fraction.

These observations tend to indicate an iron-

beta globulin association.
Popper. —

Popper values

greater than 158 jug. per 100 ml. serum

were obtained for subjects 6, 10, and 21,

Of th© serum proteins, gamma

globulin exceeded 1.28 g. per 100 ml, in each case.

A copper value

below 60 and a gamma globulin content below 0,80 occurred in Subject 16,
These parallel changes suggest the possibility of copper-gamraa globulin
binding.
B, Paper Electrophoresis Experiments
1.

general considerations. ** Figure 11 is a sample electrophoretic

pattern obtained by the horizontal strip procedures for human serum
protein separation, staining, and strip elution.

Alpha* globulin m s

found visible on paper as a separate and distinct blue colored area.
However, when elution data (migration distance versus absorbancy) were
plotted, no separate peak attributable to this fraction appeared.

59

Distortion of th® albumin peak suggested that alpha* globulin •was
included in the albumin.

A calculation of mobility relative to that of

albumin indicated this was true.
Alpha* globulin has bean, shorn to migrate at Q.82 the rate of
albumin under the same experimental conditions.

This value may be

calculated from th® absolute mobilities of the serum proteins (at pH
8.6, p 0.1 veronal buffer) given by Armstrong, et al., (h), in the
following manner!
k.85 ± 0.23 (mobility of alpha* globulin)/5.92 ± 0.21 (mobility
of albumin} » 0.82 i 0.0? (relative mobility of alpha* globulin).
Th® literate® reveals that some investigators (30*36,10,51*,61,65)
have achieved similar results from both moving boundary and paper
electrophoretic analysis.

Other workers (18,33,56,58,88), failing to

gat agreement, attribute this to albumin tailing and th© unetjual stain­
ing of alhsffflfl and globulin©.

On® object in the development of th©

paper electrophoresis procedures in this investigation was to obtain
comparable results with th© two methods.

To compare paper with moving

boundary results, portions of the same sera were examined by both
electrophoretic methods.

A saxq?l® result is given in Table 7.

The

agreement obtained between the two methods tends to sdniraize albumin
(albumin low, globulins high) and unequal dye affinities
(albumin high, globulin® low) ♦ Apparently, con^arability of results
can be achieved with th® proper paper electrophoretic techniques.
2.

Metals found in proteins separated on filter paper. — The

qualitative results of paper electrophoretic separation of human

60

serum proteins end, subsequent selective staining are presented in
Figure IQ.
GaO-giTO. — m e orange-pink areas of the calcium-alizarin complex

on a pal® pink background corresponded to th© five positions of the
serum proteins as shown in Figure 10.

Thus, calcium appeared to be

present in albumin and a n the globulins.
Yisual comparison of color intensity on parallel strips stained for
protein and calcium suggested an unequal distribution of metal ion on
th® proteins.

Figure 12 presents elution data (migration distance

versus absorbancy) for calcium and protein plotted on the same coordinate
system.

Measurement of areas of the two curves showed that*

(a) albumin

(almost two-thirds of the total serum protein) was associated with about
one-third of the total bound calcium and (b) gamma globulin (approxi­
mately one-sixth of the total serum protein) was associated with almost
one-third of the total bound calcium.
Magnesium. — * Th© five red areas given by the magnesium-Titan yellow
complex were in the same positions as the areas stained for all of the
separated serum proteins.
of the serum proteins.

Magnesium, therefore, seemed to occur in
Due to color instability of the complex

and the stron&ty basic medium required for its formation, attests to
obtain magnesium distribution among the five protein coaponente were
unsuccessful.

Iron, — The single pink iroii-ihiocyanat© area corresponded with
th© beta globulin son® on paper strips.

This observation suggested an

association of iron with only this on© serum protein,

61

Copper.

yellow area of th® eopper-earbaraate complex was in

th® gamma globulin position, indicating bound serum copper occurred
principally in that particular globulin.
21330. ** Th© lavender spot attributable to sine mas also discovered
in th® gamma globulin region.

Consequently, sine also seemed to be

present only In this on® globulin fraction.
0, Exhaustive Dialysis Studies

' '**• 3hs change in protein and metal ion contents. — Th® results of
total protein and metal ion analysis before and after exhaustive dialysis
are given in fable 71.

fh© decrease in metal ion content per 100 ml. as

Sheen uas the result of both dilution of the protein solutions and
actual metal ion transfer to the outside liquid.

To eliminate dilution

effects the weight of metal ion per g. of protein mas determined for
calcium, magnesium, iron, and copper.

These results permit the calcu­

lation of percent retention (bound ion) as given in Appendix I.

The

binding of metal ion per g. protein and percent retention values are
reported in Table HI.
As presented in the historical section, it is apparent that dialysis
has been a widely used investigative method for th® study of metal ionprotein binding.

In this set of experiments, the dialysis conditions

were limited to pH 8.6 in 0.1 M veronal buffer.

The purpose of these

exhaustive dialyses mas to demonstrate that protein-metal ion binding
m s actually present under th® conditions of the preceding electrophoresis
experiments carried out in th© earns medium.

62

2»

M m s t a l ions by ssrun proteins.

£ » M 2 S * *“* Albumin I (99%, Cohn Fraction V), Albumin XI (83$,
ammonium aulfat® removal of globulins), and Albumin i n

(93%, methanol

precipitation of globulins) all shewad 10-20$ retentions of calcium.
M&esd globulin* (methanol precipitate) and Gamma globulin (91%, Cohn
Fraction IX and H2)showed 71 and 300+$ retentions, reepeoiively.
these data ware consistent with the findings of paper electrophoresis
in that albumin was associated with less calcium then expected from an
equal distribution of bound calcium on the serum proteins, while gamma
globulin was associated with more.
Magnesium. — Results regarding th® amount of this metal ion
associated with the serum proteins war© inconclusive.
ation of serum

In the determin­

the bound metal was released merely by the

acid precipitation ©f protein.

Because of their mod© ©f preparation,

this loose binding behavior could account for the initial absence of
magnesium in Albumin I, the Mixed Globulins, or Gamma Globulin.

Mien

Albumin XI and Albumin III were exhaustively dislysed, they showed %$%
retentions as could be anticipated since they had no acid treatment
and were th© supernatants from serum,

fhe lack of any retention of

tqpgn^-Mn by whole serum is a discrepancy that may be attributed to an
f$»ien concentration effect (3).
Iron. — After exhaustive dialysis, Albumin II (83$) and JObumin
H I (93%) both demonstrated th® presence of bound iron,

Sine® Cohn (12)

reported th® general order of precipitation of th© serum proteins to b®
gamma, alpha plus beta, and albumin, th© residual iron in these albumin

63

preparations might be associated with beta globulin.

The h3% retention

shown for both the Mixed Globulins and serum suggests that all bound
serum iron was affiliated with th© globulins.
Copper. — Substantiation of earlier findings that copper was
associated with globulin was obtained from th® results of exhaustive
dialysis, It was determined that sero par cent copper retentions were
had by Albumin JJ and Albumin H X whereas >5# retention m s given by th®
Mixed Globulin®, Bis complete retention of the metal ion by both Mixed
Globulins and whole serum pointed out that practically all serum copper
was protein bound,
D* Comparison of findings with Other Work
Calcium, •** Correlation studies Involving moving boundary sad chemi­
cal analyses suggested calcium association with both albumin and globulins.
Investigations of serum proteins separated electrophoretlcally on paper
and selective staining demonstrated that*

(a) calcium was bound to

»Th«ntftt and globulin®, and (b) albumin and gamma globulin contributed
egually in binding two-thirds of the total bound ion.

Dialysis studies

showed that albumin solutions retained calcium to the extent of 10-20$,
Mixed Globulins 71$, Gamma Globulin 100+$, and serum h$% as protein
bound.
irreversibly protein bound calcium in serum has been reported to
vary from 30-50$ of the total (35,71*83,99,101).

Ho studies were in­

cluded to determine if albumin and/or globulin® were specifically involved.
But this investigation suggests th© following with regard to serum
protein-calcium interaction*

6k

1, All five serum proteins examined contain bound calcium,
2* Albumin can form both a reversible complex with calcium as
others indicate (21,68,103) and an irreversible conplex with
calcium, and
3# Gamma globulin tends to bind almost all of its calcium
irreversibly, under the conditions studied.
Magnesium. — Magnesium m s found to b© associated primarily with
albumin from the moving boundary correlation studies.

Paper electro­

phoresis indicated this ion to be present in all five serum proteins.
After dialysis, albumin solutions showed 2$$ of their total magnesium
content to be retained,
leports in th® literature state that 2£-50$ of th® total serum
magnesium say be bound (35,101),

These findings contained herein with

regard to serum p rotsin-magaesium association are that*
1, All observed serum proteins contribute to the irreversible
binding of th© ion, and
2, Gospared to calcium, magnesium binding at pH 8.6 is much less.
Consequently, conclusions regarding calcium-protein inter­
action are not necessarily valid for magnesium.
Iron. —

The results of all experiments indicated iron was bound

only to the beta globulin fraction of human serum.

In correlation

studies, high and low serum iron content was found whenever high and low
beta globulin was observed elactrophoretic&lly• Selective staining for
iron after paper electrophoresis indicated its presence apparently all
in beta globulin.

Dialysis showed that albumin solutions not completely

65

globulin~£ree, mixed globulins, and whole serum had retentions of
the total serum iron.
3n the literature ? there is agreement that iron in serum is partially
protein bound,

Two report® stated that albumin was responsible for the

iron ion binding (25,1(3), whereas another reported that th© globulins
were involved in this association (8). Cohn (16) and Koechlin (53)
demonstrated ©©asplest formation between preparations of beta globulin and
Iren,

fhe results of the present electrophoretic and dialysis investi­

gations show an irreversible binding of iron and beta globulin.
Copper, —* The binding of serum copper principally by gamma globulin,
as suggested by moving boundary studies, teas substantiated by the paper
electrophoresis technique.

Exhaustive dialysis demonstrated no copper

retention by albumin, but collet® retention by th® mixed globulins and
whole serum, which lead to the conclusion that a globulin was th® copperbinding protein,
All serum copper has been known to b@ protein bound (1,9,10°)
without clearly indicating the specific serum protein involved.

One

study indicated albumin binding (25), while another reported globulin
of senna copper (53).

Her® definitely, by using Isolated protein

preparations and metal ion addition, beta globulin was shown to complex
copper (16,53) and gamma globulin to bind the cuprie ion irreversibly (5).
the results of the present investigation demonstrate that a single
serum protein fraction, gamma globulin, is mainly involved in binding
copper.

66

J&ttc* —

Zinc, located by histochemical staining after paper

electrophoretic separation of serum proteins, appeared to be present
only in gamma globulin*

Previous workers reported that albumin and

globulins of human serum reacted with sine ion to produce reversible
complexes (17 >39 *81). Prior to this study it m s not known if sine in
human serum was entirely ionic or partially or totally protein bound*
the occurrence of a colored spot attributable to this metal ion in the
gamma globulin zone suggests that sine is bound to some extent to this
particular globulin,
E, the Significance of Protein-Metal Ion Complexes
Hie metal ions which occur in serum activate certain enzyme re*
actions.

An explanation of this phenomenon is that the associated metal

complex represents th© active form of th® enzyme with the metal serving
as an activating proshtetic group,

Ha© metal proteinates also take

part, to some extent, in the regulation of th® metal ion concentration
of the body fluids.
Specifically, protein bound calcium is believed to function in the
mechanism of blood coagulation, the iron-binding globulin is thought to
be responsible for iron transport, and th© copper protein searns to
function in the formation of hemoglobin.

Th® high concentration of zinc

normally found in leucocytes together with the observation of zinc in
gamma globulin suggest a relationship between these two blood con­
stituents that is concerned with infection.

67

v. m m m
Moving boundary electrophoresis together with chemical determin­
ations of total protein, calcium, magnesium, iron, and copper on normal
human serum were undertaken to obtain information about the specific
protein components concerned with metal ion binding.

The results indi­

cated the following*
1. Albumin and globulins were associated with calcium,
2. Albumin was primarily associated with magnesium,
3. Beta globulin was involved in binding iron, and

k* Gamma globulin appeared to bind copper.
Paper electrophoretic separation of serum proteins and selective
metal ion staining demonstrated*
1. Albumin and th® four globulins all contained calcium.

Although

albumin represented two-thirds of the total protein, this fraction had
only one-third of the total bound calcium*

Gamma globulin, about one-

sixth of the total protein, also had one-third of th® bound metal ion,
2. Magnesium appeared to be present in the five serum proteins.
3. Beta globulin was the only serum protein which contained iron.
iu G a m globulin showed th® presence of both copper and sine,
ibshaustive dialysis studies of various protein solutions in the
same buffer medium as that used for electrophoresis showed proteinmetal ion binding was present.

68

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APPENDIX
Calculations
Relative fraction of total serum protein attributable to any single
protein component ■ Area of pattern attributed to that component/Total
area of pattern (exclusive of boundary anomaly).
g, total protein per 100 ml. serum * Abaorbancy of unknown serum/
Absorbancy of known serum x 6.61 g. per 100 ml. (concentration of
known serum).
(AS2-A31f ) is the absorbancy due to the formation of the colored copper
carbamate complex, corrected for the volume change on th® addition of
the color reagent, ju. and A88 are, respectively, the absorbancies of
the solution before and aftef'uh® addition of O.h ml, of sodium diethyldithiocarbamate, the color reagent. The factor for volume correction f
Is 5.6/6.0 since the volume of solution is 5,6 ml. before the addition”
of the reagent and 6.0 ml. after.
Absolute amount of a single jrotein fraction (g. per 100 ml. serum) *
relative fractional amount of that fraction x g. total protein per 100
ml. serum.
Percent retention of any metal ion « rag. (or /ig.) of that metal ion per
g. protein after dialysis/rag. (or jig») of that metal ion per g. protein
before dialysis x 100.