PERFLUOROOCTANOIC
HUMAN
BY

(PromGORDON

the Department

I,.

ACID
SERUM
NORDBY*of Chemistry,(Received

INTERACTIONSALBUMINAND

Stanfordfor publication,

J. MURRAY
University,
AugustWITH

LUCK
Stanford,California)

1, 1955)

Materials
A solution of human serum albumin (decanol procedure) was electrodialyzed at 40 volts per cm. against conductivity
water and clarified bypressure filtration through a sterilizing filter pad. The product was lyophilized and stored at 3”; appropriate amounts of the powder were removed
as needed for the preparation of albumin solutions.
Sodium perfluorooctanoate(PF8) solutions were prepared by carefully
neutralizing a 0.5 per cent aqueous solution of the acid with a minimal
volume of sodium hydroxide.
Procedure
Precipitation-A
number of buffered 0.3 per cent solutions of serum albumin in PF8 were prepared.
Each solution was distinctive with respect
either to its PF8 concentrationor to its pH, the latter being determinedby 0.1 M McIlvaine
buffers (3). The pH of each solution was between
4.25 and 5.25. The PF8 concentration of each solution was such that themole ratio (PFS to albumin) ranged between 0 and 200.’ The solutions
were thoroughly mixed, allowed to stand for 15 minutes, and centrifugedin a clinical centrifuge at room temperature for 20 minutes.
For conditions
under which the protein in a given solution was only partially precipitated,opaque supernatant solutions were sometimes formed; in all other cases the* Public
HealthServiceResearch
Fellowof the NationalHeartInstitute.
1 A value of 61,500 was used for the molecularweightof humanserum albumin
399

(4).

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For several years perfluorooctanoic
acid (1) (CFB. (CF&. COOH) has
been used in this laboratory as a protein precipitant (2). Qualitatively
ithas been observed to bind so completely to proteins that little of the precipitant remains in the filtrate; this is an obvious advantage in chromatography and in various analytical procedures.
The purpose of the present study is to define the conditions under whichperfluorooctanoic
acid will effect a precipitationof human serum albumin
and to describe in detail the interaction
between these two reactants.
Human serum albumin was used because of its unusual properties in binding a variety of ions.

400PERFLUOROALBUMIN

PRECIPITANT

supernatant solutions were clear. An aliquot of each supernatant liquid,or solution if no precipitate existed, was then analyzed for protein by theLowry et al. modification of the Folin test (5). PF8 does not interferewith this analysis.
The percentage of albumin that was precipitated fromeach solution under the stated conditions of pH and PF8 concentration
was calculated.
The results appear in Fig. 1.
Anion Binding-A
number of 0.3 per cent albumin solutions were prepared in radioactive PF8. The ratio (PFS to albumin) in each solution
was between 80 and 200. The solutions were then titrated at 25” to about
pH 3 with hydrochloric acid. In the course of the titration between pH

4 and 3, the protein precipitated completely; several 0.005 ml. aliquots
were removed from each supernatant liquid, placed on aluminum disks,
and immediately dried under an infra-red lamp. The disks were thenplaced on an automatic sample changer which operated into a gas flow
counter (6). The time required for an arbitrary number of counts wasrecorded for the sample on each disk. After making suitable corrections
for ethciency and background interference in the counting, the relative activities of the samples were determined and compared with the originalactivities of their respective solutions before the titration was started.
The molar ratio (bound PF8 to albumin) in each solution was then calculated as a function of the molar ratio (PF8 to albumin). The data are
given in Fig. 2.
Hydrogen Ion Binding-A number of 0.3 per cent albumin solutions were

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FIG. 1. The percentage of human serum albumin precipitatedfrom aqueous solution is a function of the pH of the solution and the molar ratio (PFS to albumin) inthe system. For example, at pH 4.75 and a molar ratio (PFS to albumin) of 60, about
48 per cent of the albumin is precipitated.

G.

L.

NORDBY

AND

J.

M.

401

LUCKprepared.
Half of the solutions contained that concentrationof PF8 forwhich the molar ratio (PF8 to albumin) was 196; the remaining solutions
Each solution
contained an equivalent concentration of sodium chloride.
BOUNDPFB
100z

BA, the molarratio(PFS to albumin)in a systemdetermines
theCurve
B, the reciprocal
numberof PF8 anions that bind to each albumin
molecule.
of the numberof PF8 anions boundto each albumin
moleculeis plottedagainstthemolarratio(albuminto free PF8).
FIG.

2. Curve

I
Di

FIG. 3. The numberof hydrogenions bound to an albumin
molecule
is a functionof the pH of the solution.
Curve
A represents
1 mole of albuminin the presence
ofsodium PFS; Curve
B represents
1 mole of albuminin the presenceof sodiumchloride.

was then titrated at 25“ with either hydrochloric acid or sodium hydroxide.
The amount of hydrogen ion bound by the albumin, or dissociated fromit, was calculated quite directly from the total acid or base added to a given
solution and the amount of acid or base actually present in the solution asreflected by its pH. Subsequently, the charge on the albumin molecule
was calculated as a function of pH. The data are presented in Fig. 3.

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A402

PERFLUOROALBUMIN

PRECIPITANTDISCUSSION1-=rY&C;+;

In the above equation, r is the molar ratio (bound PF8 to albumin), n is
the molar ratio (maximal bound PF8 to albumin), c is the molar ratio (freePF8 to albumin) in the solution, and K is a constant proportional
to theequilibrium constant for the PF8 anion-binding
reaction.
The parameters in the above equation are l/r and l/c.
Although there is no a priori
evidence that the equation does apply to PF8 anion binding, it can be as-

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Precipitation-AlthoughFig. 1 indicates a certain critical range of pH
and PF8 concentration which must prevail for the precipitationof humanserum albumin, it also indicates in a qualitative manner the conditions
under which most other proteins would be expected to precipitate.
Threerather striking features are noted for attention.
(1) The conditions ofpH and ionic strength under which complete albumin precipitation occurs
are very mild. (2) The precipitationof albumin is completely reversible
with respect to pH.
An albumin precipitate formed in a PF8 solution canbe completely dissolved by making the solution somewhat alkaline (pH 6to 7) to the pH at which precipitation
occurred.
The minimal pH towhich the solution must be raised will depend, of course, upon the PF8(3) In acidic solutions, the precipitation ofconcentration of the solution.
albumin is irreversible with respect to the PF8 concentration.
Providedthat an albumin precipitate is dialyzed against an appropriately
acidicsolution, it will not dissolve appreciably as the concentrationof PF8 inequilibrium with the precipitate decreases.
However, the PF8 precipitant
can readily be dialyzed from albumin in neutral or slightly alkaline solutions.
Anion Binding-The
scope of Curve A in Fig. 2 is restricted at the upper
limit by the rather low solubility of PF8 in aqueous solution and at thelower limit by the concentrationof PF8 which would adequately precipitate albumin under the conditions of the experiments.
However,
it isclear from the graph that a considerable number of PF8 anions bind to eachalbumin molecule.
The molar ratio (bound PF8 to albumin) increases
quite rapidly as the PF8 concentration is increased.
In contrast, the molarratio (bound PF8 to albumin) decreases very slowly as the molar ratio(PF8 to albumin) is decreased below about 120. In solutions containing
the minimal concentration of PF8 which is effective in completely precipitating a given concentration of albumin, the PF8 is almost completely removed from the solution as part of the precipitatingcomplex.
The data of Curve A are treated by an expression derived by Klotz (7);the results appear as Curve B of Fig. 2.

G.

L.

NORDBYANDJ.

M.

LUCK403

SUMMARY

It has been shown that human serum albumin can be reversibly precipitated from aqueous solution under mildly acid conditions in the presenceof low concentrationsof perfluorooctanoicacid. In the formation of theDownloaded from http://www.jbc.org/ by guest on May 21, 2019

sumed, by virtue of the linearity of a portion of Curve B, that the equation
does apply to the linear portion.
The indicated extrapolation
to infinite
PF8 concentration
reveals that a maximum of 63 PF8 anions bind to analbumin molecule by reactions having nearly the same equilibrium constant.
Additional anions are bound to albumin at high PF8 concentrations
by some less easily described series of reactions.
At very high PF8 concentrations, Curve B of Fig. 2 becomes nearly vertical; therefore the maximal number of PF8 anions that can possibly bind to an albumin molecule
cannot be calculated by the evidence available.
In contrast, a study ofoctanoic acid anion binding by Teresi and Luck (8) reveals that a totalof 36 octanoate ions binds to two types of sites on the albumin molecule.
Preliminary
isotope dilution studies indicate that the PF8 anions are reversibly bound to the albumin molecule.
Hydrogen Ion Binding-Twohydrogen ion binding curves for humanserum albumin are depicted in Fig. 3. These binding curves are completely applicable to reversible titrations
over the pH range illustrated.
It is readily apparent from these curves that PP8 strongly influences thealbumin molecule charge in acid solution.
A similar effect has been observed by Steinhardt (9) for the titration of wool protein in the presence of
2,4,6-trinitroresorcinol,
picric acid, or flavianic acid.
By means of the equation described above, the extrapolationof Curves
A and B to infinite hydrogen ion concentration indicates that the maximal
number of bound hydrogen ions per albumin molecule is 107 in each case.
This number agrees with that found by Tanford (10). Thus, the charge
on the albumin molecule in extremely acid solutions in the presence of thatconcentrationof PF8 represented by a molar ratio (PF8 to albumin) of
196 does not exceed 28; 79 PF8 anions are bound under these conditions.
The dotted line in Fig. 3 represents the difference between Curves A andB as a function of pH.
The maximal difference between the two curves
is near pH 4.25 at which an extra 64 hydrogen ions are bound by the albumin in the presence of PF8.
The significance of the coincidence between
the 64 extra hydrogen ions and the 63 strongly bound PF8 anions which
are bound to the albumin is not known at this time; but the coincidence is
so striking that it is brought to attention.
Finally, it is apparent thatCurves A and B coincide above pH 8. This is additional, although inconclusive, evidence that the PF8 completely dissociates from the albumin
molecule in alkaline solutions.404

PERFLUOROALBUMIN

PRECIPITANT

precipitate, the albumin molecule binds both hydrogen ions and perfluorooctanoate ions to form, under the conditions of the experiments described,
a complex that has a net charge of almost zero.
Acknowledgment
is made to the Cutter Laboratories
for a sample ofFraction V (ll), decanol human serum albumin, and to the Minnesota
Mining and Manufacturing
Company for a sample of perfluorooctanoic
acid. Carboxyl-labeled(C14) perfluorooctanoic
acid was obtained throughthe Atomic Energy Commission from the Minnesota Mining and Manufacturing Company.

(12).
BIBLIOGRAPHY

1. Kauck, E. A., and Diesslin, A. R., Ind. and Eng. Chem., 43,2332 (1951).
2. Luck, J. M., Griffin, A. C., Boer, G., and Wilson, M., J. Biol. Chem., 206, 767(1954).
3. McIlvaine, T. C., J. Biol. &em., 49, 183 (1921).
4. Charlwood, P. A., Biochem. J., 61, 113 (1952).
5. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J., J. Biol. Chem.,
193, 265 (1951).
6. Nye, W. N., and Teresi, J. D., Anal. Chem., 23,643 (1951).
7. Klotz, I. M., Arch. Biochem., 9, 109 (1946).
8. Teresi, J. D., and Luck, J. M., J. Biol. Chem., 194,823 (1952).
9. Steinhardt, J., Ann. New York Acad. SC., 41,287 (1941).
10. Tanford, C., J. Am. Chem. Sot., ‘73, 441 (1950).
11. Cohn, E. J., Gurd, F. R. N., Surgenor, D. M., Barnes, B. A., Brown, R. K.,
Derouaux, G., Gillespie, J. M., Kahnt, F. W., Lever, W. F., Liu, C. H., Mittelman, D., Mouton, R. F., Schmid, K., and Uroma, E., J. Am. Chem. Sot., 72,465 (1950).
12. Klevens, H. B., and Ellenbogen, E., Discussions Faraday Sot., 18, 277 (1954).

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Addendum-Klevens
and Ellenbogen have recently published their research on thevan der Waals association of bovine serum albumin in the presence of perfluoro acids

PERFLUOROOCTANOIC ACID
INTERACTIONS WITH HUMAN SERUM
ALBUMIN
Gordon L. Nordby and J. Murray Luck
J. Biol. Chem. 1956, 219:399-404.

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