TEE JOURNAL cm BIOLOWCAL CHEMIBT~Y Vol. 234, No. 12, December 1959

Printed in U.S.A.

Electrophoresis of Human Serum Albumin at pH 4.O*

I. A SYSTEMATIC STUDY ON THE EFFECT OF ORGANIC ACIDS AXD ALCOHOLS UPOK THE ELECTROPHORETIC BEHAVIOR OF ALBUMIN

K. SCHMIDT

From the Department of itledicine, Harvard IKedical School, and the Nedi& &‘ewices of the Massachusetts General Hospital, Boston, .JIassmhusetts

(Received for publicat,ion, June 25, 1959)

Recently we have reported on a hitherto unknown modifica- tion of human serum albumin of which the outstanding proper- ties are electrophoretic homogeneity at pH 4.0 and its transfor- mation to an electrophoretically heterogeneous form (2). In continuation of this study, the question was asked whether the ordinary human serum albumin (Fraction V), known to bc electrophoretically heterogeneous at pH 4.0 (3), could be t.rans- formed to an electrophoretically homogeneous form.

Serum albumin, when studied at pH values acid to its isoelec- txic point, eshibit.s several physicochemical abnormalities, such as the appearance of a multicomponent system on electrophoresis (3-8) ; changes of sedimentation and diffusion const.ants (9-12) and of viscosity and optical rotation (9, 12, 13); and the expan- sion of the molecule (11, 14). In the present study a particular aspect of one of these physicochemical changes was investigated estensively, namely the behavior of this prot.ein in pH 4.0, r/2 0.1 buffers as observed on free moving boundary clectrophoresis. The reasons for selecting these conditions are the pronounced heterogeneity at pH 4.0 and the easier interpretation of the elec- trophoretic analyses performed at ionic strength of 0.1.

The electrophoretic behavior of serum albumin at pH 4.0 has been investigated at low ionic strength and at low protein con- centration by Aoki and Foster (7, 8). The obtained data were interpreted as a configurational isomerism, e.g. the “normal” or “native” form binds H-ions that results in a faster moving, pre- sumably higher charged form of albumin. These two modifica- tions arc in equilibrium with each other within a pH range from 4.5 to 3.5. Phclps and Cann (4-6) studied albumin solutions at higher ionic strengths and protein concentrations and considered the different electrophoresis patterns as a result. of a specific in- teraction between albumin and the undissociated organic acids (5). The apparent paradox that a two-component, equilibrium system can manifest itself in two moving boundaries on free elec- trophoresis, provided t.hat the time of electrophoresis is less than the half-time of the reactions, has recently been resolved (16, 17).

* This is publication No. 261 of the Robert W. Lovett Memorial Unit for the Study of Crippling Disease, Harvard Medical School at the Marsachuset,ts General Hospital, Boston. This study was

aided by grants from the Lillv Research Laborat.ories, Eli Lilly and Company and from the %ational Inst.itute of Art,hrit,is and Metabolic Diseases (Grant no. A-2288) of the IJnit,ed Stat,es Public Healt,h Service. A nreliminarv note of this work has been nub- lished (1).

t This work was done during the tenure of an Established In- vestigatorship of The Helen Hay Whitney Foundation.

The aim of this paper is to present the results of a systematic study on the effect of organic acids and alcohols upon the electro- phoretic behavior of serum albumin at pH 4.0.

EXPERIMENTAL

Materials and Jethods

Human serum albumin was prepared according to Method 6, purified furt,her by Method 9 (18) to remove traces of globulins, and stabilized with sodium ucacetyl-tryptophan and sodium caprylate (0.02 molar with respect t,o each stabilizer).1 All ex- periments were carried out on one preparation referred to as al- bumin in this paper. On electrophoresis at pH 8.6, I’/2 0.1 in cit.rate-diethylbnrbiturate buffer, this albumin appeared homoge- neous.

Each buffer solution used during this investigation was made up of sodium hydroxide and one organic acid with a pK value near 4. The amount of acid was calculated so that, after dis- solving it in a known volume of distilled water and adjusting the pH to 4.0 f 0.02 (Beckman pH meter, model G) with 1 N so- dium hydroxide, a total ionic strength of 0.1 was obtained. The specific resistance of the buffers was measured by the bridge method at 0” and at a frequency of 1100 c.p.s. with the use of a conductivity cell (No. 4917) made by Leeds and Northrup and Company, Philadelphia. Human serum albumin, 40 mg, was dissolved in these buffers, 3.0 ml and dialyzed at 2” against 1 liter of the same solution for 17 hours. Before electrophoresis, the protein solution was adjusted to a concentration of 1.1 to 1.2%.

A P&in-Elmer electrophoresis apparatus, model 38, was used throughout this study. The so-called closed system was pre- ferred over t.he open one because under these conditions the flow of liquid in the electrophoresis cell is minimized. During the electrophoret,ic runs which were carried out at a constant current of 9.6 ma the cell was cooled with ice water. Because of the dif- ferent apparent electrophoret,ic mobilities (19, 20), the time of each clectrophoretic run was varied so that the protein moved in each case approximately the same distance. The apparent elec- trophoretic mobilities were calculated according t,o the conven- tional equation. For the calculat.ion of the relat,ire percentage of the different components, enlarged tracings of the electrophoretic

1 This material was prepared in a 25y0 solut.ion by the Biologic Laboratories, Massachuset.ts Department of Health, Boston, for the American National Red Cross and donat,ed t,o t,he blood bank of the Massachusetts General Hospital, Bost,on, Massachusett,s.

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8164 Blectrophoretic Behavior oj Serum Albumin at pH 4.0. I Vol. 234, n-0. 12

FIG. 1. Electrophoretic patterns of human serum albumin in formate (la, 60 minutes) and itaconate (lb, 100 minutes) buffer of pH 4.0 and p/2 0.1, respectively. Both a and b are ascending patterns; arrow indicates direction of movement of the protein; salt boundaries are designated with 6.

TABLE I

Effect of monobasic, aliphatic acids on electrophoretic behavior of human serum albumin at

Anion

Formate

Acetate

Propionate

Butyrate

Phosphate

NaCl-HCle

-

:ompo”e” No.

1 2

1 2 1

2 1 2

1 2 1

2

-

t (

1 4.0 and F/Z 0.1 (Na+)

Apparent electrophoretic

mobility X 105 Crn.~ sec.-l VOW)

i

wcend- ing

4.7

2.8 3.9 2.5

2.8 2.1 2.2

1.6 4.5 3.4

5.5 4.5

Ascend- ing

5.6

4.0 4.6 3.8

2.7

2.2

5.6 3.2

6.0 4.1

.- c kscend- Ascend-

ing ing

54a

46 38 62

64&

36 2gb 72

8” 92c

70

93c 48” 52

61* 39

Irea of components in relative percentage

1OOd

1OOd 41a

59 46 56

n Well separated.

* Peaks not well separated. c Asymmetric peak, resolved in number of peaks as marked. d Very narrow, slight asymmetry on bottom.

e pH 3.85.

patterns were made and resolved in Gaussian curves. The rela- tive areas were measured planimetrically. If the peaks were

separated, they were usually not marked as such in the tables except in certain special cases where it seemed important. How-

ever, they are marked if the components were not well separated or appeared as asymmetric peaks.

RESULTS

A. Electrophoretic Experiments

1. Monobasic, aliphatic acids-The monobasic, aliphatic acids (Table I) affect the elcctrophoretic behavior of human serum al-

TABLE II

Effect of dibasic, aliphatic acids on electrophoretic behavior of human serum albumin at pH 4.0 and p/2 0.1 (Na+)

Anion ,

Oxalate

Malonate Succinate

Glutarate

Adipated

~om~~~‘“’ T

1

2 1 1

2 1

2 1 2

3

t I

I

Apparent Electrophoretic mobility X 105

:cm.? sec.-’ volt-‘)

kscend- ing

2.7 1.5 2.2

2.1 1.4

2.5 1.6

4scend- wxend- Ascend- ing ing ing

3.2 2.4

3.0 2.0

46 54

100

100

3.3 2.2

424

58 100”

23c 776

17c 830

9 13c 78~

5”

95c 5

95 -

ha of compo”e”ts in relative

percentage

a Peaks not well separated. * Slightly asymmetric.

c Asymmetric peak, resolved in number of peak,; as marked. d Run at 23”, electrophoretic mobilities not indicated.

bumin in an apparent regular mode. The lowest member of this series, formic acid, caused the appearance of two well separated peaks2 (Fig. la). The apparent electrophoretic mobilitirs were relatively high (Fig. 2). In acetate again two peaks were notrd. However, the relative percentage of the faster one was decreased. On the descending limb the two components formed an asym- metric boundary.2 The apparent electrophoretic mobilitirs were smaller than those observed in formate. In propionate thr two components had merged completely on the ascending limb, but a slight asymmetry was noted on the descending side. The appar- ent electrophoretic mobilities were further decreased. Essen- tially the same observations were made by analyzing the butyr- ate patterns. The albumin after incubation in the latter buffer exhibited on ultracentrifugal analysis 91 o/a monomer (s~~,~ = 3.8 at l”/$ protein concentration) and 9% dimer. Valcrate and caproafe were not sufficiently soluble to permit the preparation of the corresponding buffers. For comparison, albumin was also analyzed in phosphate buffer and in a NaCl-HCl solution where this protein seems to be affected in a mode somewhat intrrmedi- ate between formate and acetate. However, the influence of the specific interactions with these ions must be taken into considera- tion in explaining the latter patterns.

2. Dibusic, uliphatic acids-Osalate (Table II) caused albumin to appear heterogeneous. The apparent clectrophoretic mobili- ties of the two components were lower than those observed in acetate. In malonate albumin showed essentially homogeneity. However, the electrophoretic mobility scrmed to coincide with that of the faster component observed in the other buffers of this series (Fig. 2). In succinate apparent homogeneity was noted in the ascending limb only. No change in the apparent tlcctro- phoretic mobilities was observed. In glutarate and adipate al- bumin appeared again essentially homogeneous. The relative area of the main peak of the ascending side was much higher than

2 The area ratio of the two components might decrease in differ- ent experiments to 1: 1. In acetate buffer, however, this ratio was found to be constant, if this albumin preparation was reinvesti- gated, and the obtained patterns were identical with that reported by Lutscher in 1939 (3).

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December 1959 K. Schmid 31G5

that of t,he corresponding peak of the dcsccnding limb, a phe- nomenon generally noted in this series of experiments. Again, the higher members of the dibasic organic acids were not soluble enough to prepare pH 4.0 buffers of I?/2 0.1. Wit.h respect t,o the apparent clectrophoretic mobilities, no simple relationship was observed as a function of the number of the carbon atoms of these acids as had been noted with the fatty acids (Fig. 2).

S. Sz&tituted organic a&Es--The substituted organic acids listed in Table III included in t,he first group hydrosylated, in the second group aminated, and in the third group dehydrogmatcd acids. Hydroxylation in the cr- (e.g. lactic acid obtained from propionic acid), p- (nn-P-hydroxybutyric obtained from n-bu- tyric acid), or in (Y- and /?-position (tartaric obtained from suc- cinic acid) caused increased heterogeneity. Amination of t,he @- (B-alanine obtained from propionic acid), y- (y-aminobutyric obtained from n-butyric acid), or e-position (e-aminocaproic ob- tained from n-caproic acid) eshibited a similar rffcct. Rspartic and glutamic acid were not soluble enough to permit preparation of pH 4.0, I’/2 0.1 buffers. &hydrogenation (fumaric obtained from succinic acid) resulted also in a loss of electrophoretic homogeneity of albumin. The increase in homogeneity was con- siderable, if fumaratc was replaced by maleate. In citrate and, particularly, in itaconate (ethylene-malonate) elrctrophoretic ho- mogeneity was observed (Fig. lb). It should be emphasized that in itaconate, albumin seemed still absolutely homogeneous on both the ascending and descending patterns, even after a pro- longed clectrophoretic run of 4 hours made by continuous com- pensation. On ultracentrifugal analysis this preparation con- sisted of 94% monomer and 6% dimer. Furthermore, itaconatc is the only buffer, as far as investigated, which effects absolute homogeneity of albumin judging from the symmetrical, electro- phoretical patterns. With respect to the apparent electropho- retie mobilities, it was again noted that, if the relative arca of the main peak was high, the values of these mobilities were relatively low.

Further experiments were performed to demonstrate that the chosen conditions (pH 4.0 and I’/2 0.1) offer certain advantages for the interpretation of the electrophoretic patterns. At pH 3.5, where the so-called transition from the native to the fast

t ux105

I

- 01 I I I I I I

I 2 3 4 5 6

NUMBER OF CARBON ATOMS OF ACIDS

FIG. 2. Apparent electrophoretic mobilities of human serum albumin in pH 4.0, I?/2 0.1 buffers prepared from fatty acids, 0, and dibasic organic acids, 0. The apparent electrophoretic mo- bilities of this prot,ein in pH 4.0, I’/2 0.1 acetate in presence of different aliphatic alcohols, n-butanol to n-octanol, remained un- changed.

TABLE III Effect of substituted and modijed organic acids on behavior of

human Serum albumin at pH 4.0 and r/2? 0.1

Anion

Apparent : electrophoretic

mobility X 10’ Comyyit (cm.* sec.-l volt?)

, I

oL-@Hydroxybutyr- ate

Tartarat,e

&Alanine

r-Aminobut,yrate I

c-Aminocaproate

Fumarate

Maleate

Citrate

It,aconat,e ---.

1 2

3 1 2

3 1 2

3 1 2 1

2

3 4 1 2

3 1 2

3 1

2 1 2

3 1

- Descend-

ing

4.2

2.8 3.4 2.7

I 2.1 : 1.2 ! 5.6

4.1

4.3 3.5

2.8

4.5

3.1

2.9 2.4

i 1.7

2.0

1.5 ! 1.5

wend- ing

-__

4.8 3.8

2.9 3.3 2.7

1.8 2.9 2.2

1.3 5.6 4.4

4.6 3.9 3.3 2.4 4.9

3.6 2.8 3.9

3.1 2.4 4.0

1.9 2.5

1.9 1.2 1.8

bra of components in reiatwe percentage

-.~ --

kscend- Ascend- ing ing

- ~_

6 12a / 31b 88a 63

9a 66 91~ 29

6

3 170 11 83a! 86 24~ 1 36”

76a

74 10

16

64

69 10 18

3 78

20 2 4”

18~ 7ga

57 43

gb

9b 83

100

85

15

3a 22”

75

loo

106 166

a Asymmetric peak, resolved in number of peaks as marked. b Well separated.

moving form of albumin is completed (7) and homogeneity is again restored, albumin appears homogeneous in acetate or itaconate at U/2 0.1, but not at U/2 0.05 or below. Further- more, at I’/2 0.1 in pH 4.0, acetate buffer human y-globulin, &metal-combining protein, or-acid glycoprotein, and ovalbumin appear electrophorctically homogeneous,3 whereas at lower r/2 these proteins, as far as investigated, exhibited more than one boundary (21).

/t. Alcohols-Xormal aliphatic alcohols added to pH 4.0, r/2 0.1 acetate buffer to give a concentration of 0.01 M, may effect homogeneity (Table IV). n-lMano1 appears t,o affect only slightly the a.lbumin patterns observed in acetate buffer. n-Oc- tanol of which the solubility corresponds to approximately 0.001 Y, proved to be estremely effective in bringing about electro- phoretic homogeneity. In no experiment was absolute homo- geneity observed. Other alcohols, such as isoamylalcohol and benzylalcohol and amino acids with long hydrocarbon chains, such as norleucine, did not affect the acetate patterns. The apparent electrophoretic mobilities of the albumin components remained unchanged in all experiments.

It should be emphasized that, whereas in the series with fatty acids and certain di- and trivalent organic acids, attainment of

3 Unpublished experiments.

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3166 Electrophoretic Behavior of Serum Albumin at pH 4.0. I Vol. 234, No. 12

TABLE IV the apparent electrophoretic mobilities. This finding suggests Electrophoretic behavior of normal human serum albumin in pH that two types of interactions might take place: one responsible

4.0, I’/2 0.1 acetate buffer in presence of 0.01 M alcohols for the homogeneous behavior and the other responsible for the

Alcohol

n-Butanol

n-Pentanol

n-Hexanol

n-Heptanol

n-Octanolb

set(3)- Pentanol (1)

Benzylalcohol

nL-Norleucine

(

.-

-

hnponent No.

-

(

1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 3 1 2 3

Apparent Electrophoretic mobility X 10b

:cm.2 sec.-’ volt-‘:

kscend ing

3.7 2.3 3.7 2.5 4.1 2.8 4.1 2.8 3.8 2.8 3.7 2.7 3.3 2.3 4.7 3.7 2.5 5.4 4.0 2.8

-

- 1

-

&end- ing

I

apparent electrophoretic mobilities of albumin (for more details Area of components

in relative see subsequent paper). percentage

4.3 3.6 4.0 3.2 4.2 3.4 4.4 3.3 3.8 3.2 4.1 3.4 3.9 3.1 4.8 3.9 3.2 4.9 3.9 3.0

__

I

B. Investigation on the “Electrophoretically Homogeneous” l- Ascend.

ing

2ga 38 72a 62 35 44 65 56 13a 6” 87” 94a 9, 6

91 94 11 11 89 89 17 1Oa 83 9oa 35 43 65 57 3” 4

28” 36 69” 60 2 5

26 32 72 63

_-

0 Asymmetric peak, resolved in number of component.s as indi- cated.

b Acetate buffer saturated with octanol, estimated solubility 0.001 M.

Use of the aliphatic alcohols was suggested by Dr. W. H. Batchelor.

electrophoretic homogeneity of albumin seemed to be paralleled by decrease of the apparent electrophoretic mobility, in this series of experiments with sliphatic alcohols the attainment of electrophoretic homogeneity was not paralleled by a decrease in

Human Serum Albumin

For comparison, additional experiments were carried out with the “electrophoretically homogeneous” albumin.

1. Crystallization-Since the electrophoretically homogeneous human serum albumin has recently been obtained in large, well defined crystals of extraordinary appearance, pictures of these crystals are reproduced here. The procedure for the crystalliza- tion has been described earlier (2). The crystals shown in Fig. 3a had been grown at -2” in a solution originally containing 7 per cent protein, 0.07 M lead acetate, 10 per cent acetone, and 10 per cent methanol. The lead complex of this form of al- bumin proved to be essentially insoluble in water; subsequently the protein was recrystallized from water at +2” again as a lead complex (Fig. 3b).

2. Electrophoresis-The crystallized electrophoretic homoge- neous albumin, after treatment with cysteine at pH 5 and de- ionization which brought about electrophoretic heterogeneity (2), was analyzed in pH 4.0 itaconate buffer r/2 0.1. Under these conditions it proved to be almost homogeneous.

DISCUSSION

The principal finding of the present systematic study on the electrophoresis of human serum albumin at pH 4.0 and r/2 0.1 is the demonstration of the homogeneity of this protein under hitherto unknown conditions. The area ratio of the two ob- served moving boundaries, called degree of homogeneity, could be correlated with the number of carbon atoms of the organic acids and alcohols used.

The most striking result obtained by investigating the effect of the univalent aliphatic acids on albumin, is the gradual trans- formation from the electrophoretically heterogeneous (two com-

a b

FIG. 3. Crystals of the “electrophoretically homogeneous” human serum albumin. Fig. 3a, lead complex from methanol-acetone- water at -2’; X 40; Fig. 36 lead complex from aqueous solution at +a”; X 40.

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December 1959 K. Schmid 3167

ponents) to the homogeneous state as a function of the molecular weight of the acids. The attainment of the homogeneity was paralleled by a decrease of the relative area of the faster moving component and by a decrease of the apparent electrophoretic mobilities (Fig. 2). The latt,er phenomenon is due to a decreas- ing, positive net charge and, this in turn, suggests a capacity of albumin to bind the higher fatty acids or their anions or both with increasing force. Moreover, it follows that the nonpolar portion of the acids is responsible for the binding, because the changes in electrophoretic mobilities are greater proportionately as the carbohydrogen chain of the acids is longer.

At neutral pH value the high molecular weight fatty acids are known to be very tightly bound to albumin (22-24), whereas formic and acetic acid seem to interact very weakly (25).

The dibasic aliphatic acids, as reported earlier (2), revealed a regularity with respect to effecting electrophoretic homogeneity of albumin, similar to that noted in the series of the lower fatty acids. With increasing molecular weight of the compounds, ex- cepting perhaps glutaric acid, homogeneity was observed. The apparent electrophoretic mobilities of the components plotted as a function of the number of the carbon atoms of the dibasic acids differed greatly from that of the fatty acids and might be suggestive of different modes of binding of the two types of acids to albumin (Fig. 2) particularly malonate. Again the impression was gained that the more tight the binding, the higher the degree of homogeneity and that the complex of albumin with the disso- ciated or undissociated acid or both seems homogeneous. (For more detailed discussion see the subsequent paper of this series.) In both series of aliphatic acids described above, the lower mem- bers with one or two carbon atoms effected albumin to appear as a two-component system, whereas members with 3 or more car- bon atoms effected homogeneity. With resped to the type of binding, malonate, citrate, and maleate are probably exceptions, inasmuch as the homogeneity-effect may not be explained by in- teraction due to an increased nonpolar portion of the molecule. The importance of steric factors involved in the interaction be- tween albumin and small molecules has been pointed out by others (5, 26).

The modified organic acids obtained by introduction of hy- droxyl or amino groups or by dehydrogenation resulted in loss of electrophoretic homogeneity and an increase in the apparent electrophoretic mobilities of albumin. This observation could be explained on the basis that the hydroxyl and amino groups decrease the binding between the nonpolar portion of the acid and that of albumin. These findings bear evidence that the binding of the dissociated, or undissociated, or both forms of the investigated acids to albumin are probably due to van der Waal’s forces, rather than electrostatic interaction. The opposite ef- fect, namely the increase in the degree of electrophoretic homo- geneity of albumin at pH 4.0 was observed, if the nonpolar por- tion was increased (acetic to propionic acid; malonic to itaconic acid). Citrate, &hydroxyl+carboxyl-glutaric acid, is an ex- ception to this rule. It causes homogeneity of albumin probably due to a different type of interaction, perhaps similar to that of malonate, based essentially on electrostatic interaction.

Aliphatic alcohols, noncharged molecules, also effect homoge- neous behavior of albumin. n-Butanol in contrast to butyric acid did not exert such an effect because it appears that at least 5 carbon atoms are required to bring about electrophoretic homo- geneity of this protein. Further, it was noted that the longer the hydrocarbon chain the higher this effect. The binding of

these alcohols is probably due to van der Waal’s forces, perhaps similar to those responsible for the interaction of fatty acids. For comparison, it is of interest to note that higher aliphatic alcohols, like ndecanol, stabilizes albumin against heat denatura- tion at neutrality (27).

The prerequisite for the appearance of two moving electro- phoresis boundaries of albumin of almost equal area, in absence of hydrogen-bonding breaking agents, seems to be the pH value of 4 (7, 8). However, the H-ion concentration is not the single factor which determines the electrophoretic behavior of thii protein, since in the presence of substituted organic acids similar effects can be invoked. Therefore, the assumption (28,29) that albumin appears as a multicomponent system in presence of small ions or molecules, like those of formic and acetic acid, be- cause they penetrate easily the albumin molecule, seems un- likely. Indeed high concentrations of acetate (I’/2 0.2, pH 4) effect homogeneity (4). Certain higher molecular weight com- pounds, such as nr@-hydrosybutyric acid and DGUOrhciUe also cause albumin to appear as a two-component system. Thus, interaction between albumin and the undissociated or dissoci- ated form of organic acids and alcohols seems to be of primary importance in modifying the electrophoretic behavior of this protein at pH 4.0. Albumin, commonly thought of as being electrophoretically heterogeneous at pH 4.0, may appear homo- geneous at the same pH in the presence of buffers of certain or- ganic acids.

In this paper the electrophoretic behavior of only one albumin preparation is reported. In a further publication the results ob- tained from investigating different albumin preparations will be presented. Further, the reaction responsible for effecting ho- mogeneity of albumin at pH 4.0 will also be discussed.

SUYY.&RY

1. It is demonstrated that normal human serum albumin may appear homogeneous on electrophoresis at pH 4.0 and I’/2 0.1, depending upon the nature of the buffer.

2. Electrophoretic homogeneity is effected by aliphatic, mono-, di-, and tribssic acids with more than 2 carbon atoms and by normal aliphatic alcohols with more than 4 carbon atoms.

3. Homogeneity is due to binding of the mentioned organic acids to albumin. Modification of these acids by introduction of hydroxyl or amino groups or by dehydrogenat.ion decreases greatly the interaction, so that albumin appears as a heteroge- neous, two-component system similar to that observed in for- mate, acetate, or oxalate.

Acknowledgment-The author is very much obliged to Pfizer and Company and E. I. DuPont de Nemours and Company for the generous gift of itaconic and adipic acid, respectively. The technical assistance of Miss A. Polis is gratefully acknowledged.

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3168 Electrophoretic Behavior of Serum Albumin at pH 4.0. I Vol. 234, No. 12

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K. SchmidTHE ELECTROPHORETIC BEHAVIOR OF ALBUMIN

STUDY ON THE EFFECT OF ORGANIC ACIDS AND ALCOHOLS UPON Electrophoresis of Human Serum Albumin at pH 4.0: I. A SYSTEMATIC

1959, 234:3163-3168.J. Biol. Chem. 

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