The Electrophoretic Analysis of Protein Interaction. I. The Interaction of

ELECTROPHORETIC .%SALYSIS O F P R O T E I S REACTIOS. I

33

ELECTROPHORETIC ANALYSIS OF P R O T E I 5 INTERBCTIOK. I THE ISTERACTIOX OF BOVISESERUMALBWMIX AND METHYLORAKGE~ ? RORERT F . SMITH

AXD

D. R . BRIGGS

Dicision of Agricultiiral LZiochemistry, L7niusrsity of Minnesota, St. Paul 1 , Minnesota

Downloaded by GEORGETOWN UNIV on August 23, 2015 | http://pubs.acs.org Publication Date: January 1, 1950 | doi: 10.1021/j150475a003

Received August 22, 1949

During the last few years the investigation of the interaction of proteins with other substances in solution has shown great promise of giving new information on the physical structure of proteins and the mechanism of their action in living systems. Any method which can yield information about the nature of such complexes is therefore worthy of consideration in the light of this fundamental objective. X number of papers have appeared in n-hich it has been demonstrated that for a mixture of components where interaction occurs, the configuration of the Tiselius electrophoresis pattern obtained with the system reflects such interaction when one or more of the interacting components migrates in the electric field. Complex formation of proteins with proteins (8,14),of proteins with nucleic acid ( 5 , 10, 16), of proteins with polysaccharides (4, l j ) ,and of proteins with lower-molecular-xeight ionogenic substances ( 3 , 9, 11, 13) have been indicated from the electrophoretic analyses of such mixtures. In searching for an interpretation of the behavior of such systems in terms of the interaction constants involved it would appear advisable to start with as simple a system as possible. The equilibrium inyolved in the formation of the complex should be one nhich is established rapidly in relation to the rate of separation of the components by electrophoresis, the interaction should be strong enough to be readily detectable at low concentrations of the non-protein interactant and the equilibrium constants should be determinable by independent methods. The reaction of serum albumin with non-aggregating monovalent, dyes seems to fulfill these requirements. In our experiments, therefore, we have used bovine serum albumin lyith methyl orange as the interacting anion-mainly because of the extensive investigations which have already been carried out by Iilotz (7) and coworkers nith this mixture. They have shown that the interaction obeys the law of mass action and can be described by the Langmuir adsorption isotherm in the form:

where r = moles of bound dye anion per mole protein = ([Ao] - [A])/[Po] Presented a t the Twecty-third Sational Colloid Symposium, which was held under the auspices of the Diviaion of Colloid Chemistry of the American Chemical Society a t Minneapolis, Minnesota, June 6-8, 1949. 2 Paper S o . 2499, Scientific Journal Series, Minnesota Agricultural Experiment Station, The contents of this paper constitute a part of the thesis to be submitted by R. F. Smith to the Graduate School of the University of Minnesota in partial fulfillment of the requirements for the degree of Doctor of Philosophy.

34

ROBERT F. SMITH AXD D. R. BRIGGS


n = the limiting value of this ratio as [A] + e ,or the maximum number of binding sites, K = a dissociation constant, [Ao]= the total molar concentration of the dye, [A] = the molar concentration of free dye anions, and [PO]= the molar concentration of the protein. The constants n and K are obtained by plotting [Po]/([Ao]- 1-41)against 1/[A]. Our studies have shown that, when [A]and [PO] are known, the values of [A] in the original solutions may be calculated from the boundary patterns obtained after the protein-dye mixture has been subjected to electrophoresis. A series of experiments made a t varying ratios of [Ao]to [PO]will yield the data required for the plot of l / r oersus 1/[A] and hence the interaction constants, n and K , of the reaction. Two groups of electrophoresis experiments, which are important to our present discussion, were set up as follows: These groups of experiments will be referred to as group 1 and group 2. In both groups the buffer employed was acetate buffer, pH 5.5 (25OC.), ionic strength 0.05. The protein used was crystallized bovine serum albumin (Armour) and the dye employed was twice recrystallized sodium p-dimethylaminoazobenzene-p'mlfonate (the sodium salt of methyl orange). Standard protein, dyestuff, and buffer solutions were made up such that when measured volumes of each were employed and diluted to a proper volume with water the required final solutions would be obtained. In the calculations, the molecular weight of 67,000 was used for this protein. In experiments of group 1 a known amount of protein in a known volume of buffer was placed in a cellophane sac and allowed to dialyze to equilibrium without volume change at 4OC. against a known volume of buffer containing a known amount of dye. In the manner already described by Klotz et al. (7) the equilibrium values of [A], [Ao],and [Po] ivere obtained. From the series of experiments with varying ratios of [PO] to [&I the plot of l / r versus 1/[.4] yields values of n = 22.6 and K = 3.12 X lov4, nhich were in fair agreement with those reported by Klotz et al. for this system (n = 22.4 and K = 4.48 X Figure 1 shows the data from these experiments plotted by the alternate linear form of equation 1, i.e., [A]/r versus [A]. I t is to be noted that for values of [A] below about 2 X lo-' molar the curve is linear and the values of K and n are those given above, but a t higher concentrations of [A] (greater than 6 X lo-' molar) the resultant value of n increases and that of K decreases. This may mean that a second group of sites for interaction becomes important at high equilibrium dye concentrations. This possibility will be alluded to in the latter part of this paper in relation to the characteristics of the Tiselius patterns as obtained at these higher equilibrium concentrations of the dye. For each system, so equilibrated, electrophoresis experiments were carried out a t 4°C. in Xvhich the protein-dye mixture (inside the sac) was used as the lower solution in the U-tube and the equilibrium buffer-dye solution (outside * T h e experiments of Klotz e t al. were done in 0 1 M phosphate buffer, pH 5 67


P?

I-

.

r F I ~U. o n s t i t u e n t mobility of the protein,

tip

(crn.l v

see.-' X loL)versus

T

shows the results obtained where the mobility found for the descending moving boundary in each experiment is plotted against the corresponding value of ([&I - [A])/[Po].I t mill be noted from the linear relationship shown that a unit increase in the moles of bound anion per mole of protein is accompanied by an


36

ROBERT F. SMITH AND D. R. BRIGGS

increase in the constituent mobility of the protein (i.e., the mobility of the boundary in which the protein disappears in presence of the dye (17, 18)) equal to cm.' IT.-' see.-' This is about 0.33 the mobilky increment due to 0.13 X one added electronic charge as indicated from titration data and is in qualitative agreement with the results of Longsworth and Jacobsen (9), mho found that a bound methyl orange anion increased the albumin mobility about 0.4 as much as did one electronic ~ h a r g e . ~ I n the experiments of group 2 the protein-dye mixtures to be used as the underlying solution in the U-tube were prepared by combining measured volumes of the standard protein, standard dye, and. standard acetate buffer solutions with an amount of water needed to yield a final solution of the desired pH, ionic strength, and protein and dye concentrations. -4s the overlying solution, buffer of the same pH and ionic strength as that of the protein-dye solution was used. Under these circumstances no dye was present in the overlying solution and, upon electrophoresis, patterns characterized by asymmetries which have been described by Longsworth and MacInnes (10) were obtained. Figures Sa, 5b, and 5c are tracings of typical scanned patterns obtained lvith such serum albuminmethyl orange mixtures (where the equilibrium value for [A] was less than 2 x IOw4 molar). Patterns are entirely reproducible and the areas and mobilities of the peaks are independent of the time of electrophoresis. The 6- and e-boundaries are not appreciably different in magnitude from those obtained when the protein solution is dialyzed against the overlying buffer. The slow peak on the descending side migrates with the mobility of free protein and the fast peak on the ascending side (when detectable) migrates with the mobility of free dye, but the relative areas under the migrating peaks, the mobility of the leading peak on the descending side, and the mobility of the slower boundary on the rising side all vary with the relative (and absolute) concentrations of the interacting components. The solutions in the Tiselius cell at the beginning of an experiment (group 2) are represented in figure 3a. Z P h i represents a series of complexes from i = 0 to i = n existing in different concentrations in equilibrium with each other. A- represents free dye anion. Protein, dye, and their complexes are all negatively charged a t pH 5.5. I n these experiments u A > 2iA > C p > u p (see belov for meanings of these mobility symbols). Figures 3b and 3c represent the situation in the rising and descending boundaries as developed after electrophoresis. Before attempting to describe the behavior of this system in an electric field we must know something about the influence of the interaction upon the mobilities of each of the components as it migrates in the presence of the other. Tiselius (18) has given an equation for the constituent mobility of a component which exists in a number of forms in equilibrium with one another when the equilibrium is established rapidly as compared to the rate of electrophoretic separation:

ax = 4

alux,

+ azux, +

' '

. + a,ux,

(2)

The result of Longaworth and Jacobsen was obtained in buffers of somewhat higher

ionio strength.

ELECTROPHORETIC ASALTSIS O F PROTEIN REACTIOS. I

3i


where .Gx is the constituent mobilit'y of the component x, or the resultant mobility of any molecule of x taken on a time average, a, is the fraction of total x in the ithform, and uxi is the mobility of the ithform. Let us represent total molar and [&I, respectively, moles per liter concentration of protein and of dye by [PO] of free protein and free dye by [PI and [.I], moles per liter of the ithcomplex by [P.L], moles per liter of bound dye by [Ao]- [A], the constituent mobilities of

I

buffer

+ A- +

ybL.Yi-k f I PAi

-___

ZPAi

1

ljb$;;,i . ..........

FIG.3. Diagranirnatic representation of the Tiselius cell before and after the develoyment of the boundaries, showing the composition of the solutions in different parts of the cell and the designations used for the different regions.

protein and dye by G p and 2iA, and the mobilities of free protein and free dye by up and uA. The constituent mobility of the protein is:

38

ROBERT F. SMITH .4KD D. R. BRIGGS

If we assume that each anion bound increases the mobility of its complex by the same amount (=w), then up,,, = up iw and

+

But


and

so

This means that if the mobility increment per anion bound is constant, the protein in the presence of constant dye concentration should move as though it were all in the form of a single complex of hypothetical composition PA, independent of the nature of the interaction constants describing the formation of the complex so long as equilibrium is established quickly. The results of the experiments of group 1 (figure 2) show agreement with equation 3 up to at least r = 20. The constituent mobility of dye in the presence of protein is:

[

1 u, = - [Alu.,

[A01

+ 2 i[PAil. + iw) (UP

i-Q

1

The second term in brackets can be separated into two series and the constant factors taken out to give

but the series cannot be eliminated completely without the introduction of a further assumption concerning the nature of the binding constants involved. In the present system it may be assumed that the binding obeys the Langmuir adsorption isotherm (figure 1). The derivation of equation 1 depends on the assumptions that all binding sites are equivalent and that there is no interaction between bound anions. It has been shown (6, 12) that when this is the case the binding constant for the ithanion is:

Introduction of this relationship leads to the solution that

ELECTROPHORETIC ANALYSIS O F P R O T E I S REACTIOP;. I

and since from equation 1 [A],I(K

39

+ [A]) = r,/n,


01

Alberty and Marvin (1) derived this equation simultaneously with the authors of this paper. lpparently the dye can be treated as though it moves in two forms only-one with the mobility of free dye and the other with the mobility of a hypothetical complex PAL(l+l-,,n). If only one complex were formed, as would be the case if the free dye concentration ivere infinitely high ( r + n and [PA,] + [PO]), then the bound dye would exhibit a mobility equal to the constituent mobility of the protein. When n > 1 and r < n the expression for mobility of the dye will contain the unknown constant n. And when the binding constants are to be determined from electrophoretic mobilities a series of approximations will have to be carried out to obtain accurate values of n and K . RISING BOUNDARY

I n the group 2 experiments we may picture the process occurring at the rising boundary as follows: When the current starts to flow through the cell the free dye in the ascending leg moves out ahead of the slower moving complexes and travels up the tube with a characteristic mobility. Referring t o figure 3b, a decrease in concentration of all components will occur at the stationary boundary a/3. A small change in pH also occurs across this boundary, which will result in an indeterminate change in up and t&. I n addition, in the case of interacting systems, there n-ill occur a shift in the equilibrium across this boundary resulting from the dilution of the interacting components beyond the boundary. Because of these changes a t the a:@ boundary some error \vi11 be introduced by boundary and the estimation of G: from the observed displacement of the calculated (or measured) value of K’. I n many cases these errors will be negligible. However, from the known values of [PO] and [Ao]in the a: solution and the dilution factor for the cup houndary, the values of [PO]and [Ao] in the /3 solution can be safely calculated. If by any method we could determine [A]’, then the values of rs and [A]’ could be used in the graphical determination of n and K , since the equilibrium in region p is as real as in region a. [A]’ can, theoretically, be derived from [A]’ by use of the moving boundary equation for weak electrolytes given by Svensson (17) and by Alberty and Sichol ( 2 ) :

40

ROBERT F. SMITH . I S D D. R. BRIGGS


where vB is the velocity of the By boundary in ~ m per . ~coulomb and K’ and K’ are the specific conductivities of the solutions in regions p and y. Since the protein disappears in the boundary, the constituent mobility of the protein in p is aj, = UP,@ (6) Prom equations 5 and 6 and the elimination of C i by substitution from equation 4,we can solve for [A]’ to obtain

n n

[A]@=

I’

(7)

where up and uA are obtained directly from the rising boundary pattern as the mobilities of the PY and boundaries, and [A]’ from the concentration change occurring a t the yt boundary. In our experiments it was not possible to measure the [A]’ value because the dye concentrations were usually too low to give a stable boundary a t the -yG boundary. Even when the anion is in higher concentration or possesses a higher equivalent weight, as, for example, nucleic acid (Longsworth and MacInnes (10)) which gives a large stable peak, the calculation of [A]’ by equation 7 cannot yield dependable values since w,n, r , K’, and K’ are unknown. When K’ ?t [ill’. and T n, [A]’ Because of these uncertainties, it appears that the rising boundary pattern is not very useful in any attempt to obtain n and K from electrophoresis data.

=

=

DESCESDINQ BOUNDARY

I n order to devise an equation to describe the relationship between the values of [PO],[.4], and [Ao] - [A] in the original protein-dye mixture (in group 2 experiments) and the pattern of the descending boundary after electrophoresis we have pictured the process at the descending boundary as follobis (see figure 3c). Free anion moves down into the protein solution, the complexes left behind dissociate to restore the equilibrium, and the anions set free by dissociation follow those which were initially free. Eventually all the dye moves out of the upper part of the protein solution (c) and free protein is left migrating with its characteristic mobility. The dye disappears through the boundary bc. There is no change in concentration of any component at ab-the theoretical free dye boundary. If it is pictured that all of the free dye originally present in b , c, and d has passed the point ab, then all the free dye now present in b must originally have been bound, The complexes in b have their original composition, so the free dye in b must have been bound by the protein in c. The amount of dye originally bound by this protein is r[P]”times the volume of the cell occupied by c, [PIcbeing the concentration of protein in c, and r being the original ratio of bound dye to total protein. The amount of free dye in b is [A] times the volume of b. Since the cell is of


ELECTROPHORETIC ANALYSIS O F PROTEIN REACTION. I

41

uniform cross section, these volumes are proportional to the distances moved by the boundaries or, since all boundaries move Trith constant velocities, to the mobilities of the boundaries. The mobility of boundary bc is the constituent mobility of the dye, tiA, and that of boundary cd is the apparent mobility of the free protein up,, which differs from the true mobility of the protein, up, by the factor K ~ / K ' . If K' g K ~ then , the mobility of boundary cd = up. The mobility of the hypothetical boundary ab is that of the free dye u . ~which , may be determined by separate experiment in the same buffer if it is assumed that the presence of protein has negligible effect upon the free dye mobility. Equating the amount of free dye in volume b to the bound dye which has been set free from the protein in T-olume c, we get

anti

n-hcre [Ao] and [PO] are the original concentrations of dye and protein, cAand up, may be determined from measurements on the scanned pattern, and is the known mobility of the anion. The ratio [P]'/[PO] can be approximated closely from areas of the descending pattern by the equation:

n-here I c d Ibc

:ire& of thc cd peak, area of the bc peak, I d r = area of the e-boundary, and Z.40 = area due to total dye obtained from known value of [Ao] and the refractive increment of dye in terms of the units of area measured. = =

Alberty and Marvin (1) have shown that the concentration of protein in c may be calculated from mobilities in the rising and descending legs of the tube without making aiiy area measurements. Their equation is :

being approximated from the mobility of the 0-y boundary and the dilution factor correcting for the dilution which occurs at the ap boundary. For reasons already given under the discussion of the rising boundary, the value of ti" may better be obtained as the mobility of the mol-ing boundary on the descending

42

ROBERT

~. SMITH

AND D. R. BRIGGB

side in a separate experiment of the type described in group 1 above. Solved for this equation is:

a;

-

=

e)

+

6

Combining equations 8 and 9 and assuming that


(sa)

upe K'

E K~ they get:

They have also shown that equation 10 can be derived from the definitions of (equation 2) without any reference to the mechanism of the process a t the descending boundary by assuming ( 1 ) that only one complex is formed, and (2) that all the protein is combined with dye, or (3) that the average mobility of bound anion is the same as the constituent mobility of the protein. Equation 3 above indicates that in calculating the constituent mobility of the protein assumptions (I) and ( 2 ) are allowable. Equation 4 indicates that assumption (3) is not strictly allowable if n > 1 or r < n. Values of [A] and [ A 0 1 - [A] calculated from the data of table 1 by equations 8 and 10 (both of which include assumption ( 3 ) )agree within 2 per cent (on the basis of r ) . Deviations are greatest, as illustrated in figure 4, in those experjments where the values of [A] are loivest. This is the condition (see above) under which the true value of I$ will be expected to deviate most from &, This observation exmphasises the desirability of determining tZp by separate experiment of the group 1 type. If it is not assumed that tZp is the same as the average mobility of bound anionwe obtain from equations 3 , 4 ,and the relationship that r = ([Ao]- [A])/[Po], tZA and G p

[&I

[GA

- UP

[AI = u*

+ nl -

-

(UP

-

2ip

-

+-

1

UP)

(lip

or, eliminating 2ip by equation 9a and assuming [&l(2iA

[AI =

- up)

1

K*

- [pPl(cP - UP) __-

E E',

(Tn - 1 e + '> - [ P , I ( ~- e)(a, - up)

(a, - up) -- e + (,,I

(11)

- up)

(12)

'>

n

+ u A - t i A

In figure 4, the values of l l r versus 1/[A], as obtained from the experimental data presented in table 1 with the aid of equation 8 (or equation 10) on the one hand and with the aid of equation 12 on the other, are plotted for comparison. Values of n obtained from these graphs for the two cases are n = 22.0 and 25.2, respectively. Values of K are found to be nearly the same (2.83 X lo-' and 2.92 x IO-', respectively). These values of n and K as obtained from electrophoresis data are in fair agreement with those obtained from dialysis experiments (vide supra) (n = 22.6 and B = 3.12 X at equilibrium values of [A] less than 2 x IO-' molar.

43

ELECTROPHORETIC ANALYSIS O F PROTEIN REACTION. I

TABLE 1 111

I

E X P E R I M E K T KO.

PROTEIN CONCEYTPATiOK


8 9 2 3 4 5. 6

l

~

VI TIYE OF R U K

moler/lile~x I@

molrr/lilcr X I@

1.14 1.14 1.14 1.14 1.14 1.14 1.14 0.762 1.52

0.765 0.765 0 765 0 765 0 765 0 765 0.765 0 510 1.02

~

V

PROTEIN CONCENIRATIOS

per C""l

1 7

IV

uollrlcm

rccrndr

6.59 6.94 6.88 6.92 6.66 6.70 6.53 6.99 6.63

12,000 14,000 14,000 14,000 12.300 10,000 10,000 7,000 12,oM)

1.5 1.5 2.25 3.0 3.0 5.0 10.0 5.0 10.0

_ ___ _ Mobzlitzes of boundarzes

I

I

__

EXPERIMEYI bo

__

VI1

_ _ _ _ _ __

--

I 1 5" 9

1 7 8 9 2 3 4 5 6

1 I

~___ _ ____ _ _

_

_

~

M

I

-

"

u scc

-1

x 10s.

-1

cm 1 "

I

2 74 2 76 2 76 275 2 64 2 70 2 59 2 75 2 60

1

VI11

_ -1

sec-1

x

3 82 3.72 3 76 3 85 3 72 3 98 4 23 4 29 3 95

I

I

x

cm: v -1 rcc-I

105

lob

2 90 2 85 2 80 2 90 2 85 2 95 3 34 300 3 23

1 I ~

~

__

Areas of peaks zn the Tzselzus patterns (planzmeter unzts) -

I -~ EXPERIMENT h O

1

l

1 1

7 8 9 2 3 4 5 6

_I_-

l

11

~

___

x TOTALRISIhF

1 88 1 84 1 98 206 1 89 196 200 128 2 76

' ~

1

1 1

XI

XII

TOTAL-

dr

DESCEhDiNO

1 86 1 84 1 94 206 1 86 191 2 03 129 2 66

XIII

i

XIV

rg

1

cd

1

1 58 155 1 58 142 1 29 1 03 0 98 1 33

________

___--

'

0 02 0 02 003 006 0 04 0 04 0 03 0 09

~

'1

' I

015 0 12 0 16 0.15 013 0 14s 0 14 0 07 0 30s

1

__

* Calculated from distance moved by the cd boundary and Kb t Calculated from distance moved by the O r boundary and P times the dilution fnctor a t the or8 boundarj cm v -* sec -' Mobility of methyl orange taken as 10 50 X Refractive index increment of methql orange in planimeter units = 164 per mole.


44

ROBERT F. SMITH .4ND D. R. BRIGGS

It is of interest to note that, in the bovine serum albumin-methyl orange systems, when the equilibrium concentration of free dye [A] in the mixture exceeds a value of about 2 X lo-' molar (corresponding in our experiments to a [Ao]of approximately 1.5 X IOv3M ) there appears a double peak in the bc boundary (the boundary in which dye disappears) in the descending pattern. The faster segment of this double peak increases in relative area as the dye concentration is increased (relative to the protein), as is illustrated by the series ofdescending boundary patterns shown in figure 5 . The analysis of the patterns to yield values of n and K through the use of equations 8, 10, 11, and 12 is applicable only to patterns from mixtures containing dyestuff' concentrations so Iov that the second

I

'.Ot

01

'

'

I

'

. .

2

' 3

4

1

5

6

7

8

I

FIG.4 . l / r FersiLs 1/[A] (X10-') from electrophoresis data. Points m:irkcri x ivere calculated b y means of equation 8, points marked 0 by equation 10. and points marked 0 l)y equation 12 (using a value of n = 25 in the calculations involving equHtion 12).

peak does not occur. The occurrence of this second peak, when higher dye concentrations are present, may mean that a second group of sites of interaction is present, and that the K-value involved is such as to indicate a much weaker binding at these sites than is characteristic of those sites upon \\-hich the initial binding of the dye occurs. The n-value for these second sites \vould appear to be greater than that for the initial interaction. This phenomenon is being further studied at the present time. The data contained in the paper by Longsworth and MacInnes (10) on the interaction of ovalbumin and yeast nucleic acid upon treatment according to equation 8 yielded values of [P,]/([S,] - [SI) and 1/[S] which are plotted in figure 6. The points fall close to a linear relationship which indicates an n-value

45

ELECTROPHORETIC ANALYSIS O F PROTEIN RESCTION. I

Protein conc. 0.765 % p H 5.5

Time -

MeOrang

cone.

4


I

A

I

I

I 4

I

I I

I

I 1

I

I. 5 x I 0%

I

I

d -

: '

I

8,000sec.

1. '

I

A I , FIG.5 . Tracings of scanned pictures of Tiselius patterns obtained in experiments of group 2 w i t h 0.765per cent serum albumin and yarying amounts of methyl ornnge.

of 1.1 (grams of nucleic acid per gram of protein) and a K-value of 2.0 (grams per liter).

46

ROBERT F. SMITH A X D D. R. BRIGGS

It seems likely that the study of interaction by electrophoresis techniques offers wide possibilities and, in some cases, distinct advantages over other methods of such investigation.

12. Concentrations


i n grams X

O

'

2

4

6

i

'h,I Concentration of ovalbumin (in grams per cent) _______ i ' e m i s the reciprocal of Concentration of bound nucleic acid (in grams per cent) the concentration of free nucleic acid (in grams per cent). Calculated from the data of Longsworth and hlaclnnes (10) by equation 8.

FIG.6.

SCXMARY

The interaction of bovine serum albumin and methyl orange in solution at pH 5.5 in acetate buffer of 0.05 ionic strength has been studied by the Tiselius electrophoresis technique and methods have been devised by which the constants of the interaction may be calculated from the boundary pattems obtained after electrophoresis of the solution. Constants thus obtained from electrophoresis data are found to agree quite satisfactorily with those obtained from dialysis equilibrium experiments on the same system. The relationships which are demonstrated to apply betbyeen the electrophoresis patterns and the interaction constants may be expected to be utilizable in the study of interaction in any system in which the formation of complexes between components follows the mass action law describable in the form of the Langmuir-type adsorption isotherm, where the rate of attainment of equilibrium is fast compared to the time required for development of the electrophoresis pattern and where a t least one of the interacting components bears an electric charge under the conditions of the experiment.

ELECTROPHORETIC STUDY O F PROTEIX-IOX

INTERACTION

47

The authors wish to thank the Kational Institutes of Health for a Public Health Service Research Grant under which the studies reported in this paper were carried out. They are pleased to acknowledge the very stimulating discussions and interchange of ideas which they have had iyith Dr. R. A. Alberty of the University of Wisconsin during the preparation of this paper.


REFERESCES (1) ALBERTY, R . A., AND MARVIN, H . H.: J. Phys. & Colloid Chem. 64, 47 (1950). (2) ALBERTY, R . A , , ASD SICHOL,J. C.: J . Am. Chem. SOC. 70, 2297 (1948). P. D., BALLOU, G. A . , AXD LUCK,J. M.: J. Biol. Chem. 167, 407 (1947). (3) BOYER, E., ZIFF, bl.,A N D MOORE,D. H . : J. Biol. Chem. 139, 383 (1941). (4) CHARGAFF, E., ASD PUTNAM, F . W.: J. Phys. & Colloid Chem. 64, 79 (1950). (5) GOLDWASSER, (6) KLOTZ,I. M . : Arch. Biochem. 9, 109 (1946). (7) KLOTZ,I. M., WALKER, F. M.,ASD PIYAX,R . B . : J. rlm. Chem. SOC.68, 1486 (1946). 18) L. G.. CANNAX, R . K.. A N D MACINNES. D . A , : J . Am. Chem. SOC. 62, ~, LONGSWORTH. 2580 (1940). (9) LONGSWORTH, L. G . , A N D J.4coBsES, C. F . : J. Phys. & Colloid Chem. 53, 126 (1949). L . G., A N D BIACISNES,D. .4,: J. Gen. Physiol. 26, 507 (1942). (10) LOXGSWORTH, 13. P . , ELAN,D. >I,,A N D O'COSNELL,R. A , : J . Biol. Chem. 149, 183 (1943). (11) LUSDGREN, (12) MURALT, A. L . V O NJ. : Am. Chem. SOC.62, 3518 (1930). F. W . , A N D S E C R A T HH, . : J. Biol. Chem. 169, 195 (1945). (13) PCTNAM, G . w., P r T s A n , F. W . , A N D BRIGGS,D . R . : Arch. Biochem. 4, 371 (1944). (14) SCHWERT, F. E., A N D WATSON, D. W . : J. Biol. Chem. 140, 55 (1941). (15) SEIBERT, E., AND TEORELL, T.: Trans. Faraday SOC.36, 743 (1939). (16) STESFIAGEN, H.: .4cta Chem. Scand. 2, 841 (1948). (17) SVENSSON, (18) TISELIUS.A . : S o v a Acta Regiae SOC.Sci. Upsaliensis 141 7, S o . 4 (1930).

STCDY OF PROTEIS-IOS I S T E R A C T I O S BY T H E MOVING BOUNDARY METHOD

THEORY OF

THE METHOD'

ROBERT A. ALBERTY h N D H E K R Y H. MARVIN, JR.* Departmenl of Chemistry, Cniuersity of Wisconsin, Madison, Wisconsin Received August 88, 19@ ISTRODUCTION

number of investigators have cited electrophoresis experiments as qualitative evidence of protein-ion interactions. In the preceding article Smith and Briggs (11) have shoivn that the same quantitative information concerning the interaction of bovine serum albumin with methyl orange may be obtained by the moving boundary method as by the dialysis method of Klotz ( 5 ) . The moving boundary method for the study of protein-ion interactions is of interest because 1 This paper was not presented a t the Twenty-third Sational Colloid Symposium but was a n outgrowth of discussions and correspondence with R . F. Smith and D. R . Briggs concerning their studies of the binding of methyl orange by bovine serum albumin (11). * United States Rubber Company Fellow, 1949-50.

The Electrophoretic Analysis of Protein Interaction. I. The Interaction of

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