THEJOURNAL OFBIOLOGICAL CHEMISTRY

Vnl. 254, No. 3. Issue of February IO, pp 1665-1670, 1983

Printed in 11.S.A

Charge Determination of Proteins with Polyelectrolyte

Titration*
(Received for publication, June 16, 1982)

Dieter Horn and Claus-Chr. Heuck

From the Hauptlahoratorium der BASF AG, Ludwigshafen/Rhein,GFR and the Klinisches Znstitut fur
Herzinfarktfo;schung, Heidelberg, GFR

A recently developed photometric version of polye- Interaction of synthetic polyelectrolytes or biological micellar
lectrolyte titration was applied for the determination colloids with polyelectrolyte properties andcells may contrib-
of the number of charged residues on globular proteins. ute toproliferation (13) and/or regulation of metabolic events
Based on the observation that oppositely charged poly- (14, 15). Altered binding of macromolecules after ionic deriv-
electrolytes form, in general, stoichiometric polyelec- atization may cause drastic changes of intracellular metabo-
trolyte complexes, the protein solutions were incubated lism (16, 17).
in excess with an oppositely charged polyelectrolyte, This arbitrary selection of observations demonstrates the
andtheresidualamount was back-titrated using o- fundamental importance of cooperative electrostatic binding
toluidine blue for end point detection. It was found that in biological systems. Therefore,a variety of experiments has
within the range pH 2 to pH 9 the interaction of the been conducted to characterize binding between oppositely
polyelectrolytes, potassium polyvinylsulfate, polydi- charged synthetic and biological polymers (18-21).
allylammonium chloride, and N-methylglycolchitosan As to the importance of the state of charge in itself physi-
iodide, with various proteins of known amino acid com-
cochemical theory postulates that thesurface charge density
position (ribonuclease A, trypsin, chymotrypsinA, pep-
sin, cytochrome c) occurs stoichiometrically through of colloids determines their stability in disperse systems. In
1:1 ion pair interaction, irrespective of thespatial dis- biochemical studies it was observed that an alterationof the
tribution of the interacting ionic sites. The close cor- surface charge density of biopolymers may result in a change
respondence between the experimental data for the net of the turnover of the macromolecules (22). Hydrogen ion
charge and the calculated balance of ionized residues titration and conductometry are well established techniques
fortheproteinsata given pH indicates that in the for the quantitative assessment of the surface charge of bio-
native structure of these proteins oppositely charged polymers (23-25). However, both methods require relatively
ionic functions are largely neutralized by the formation high concentrations of the substratesfor reliable results. Fur-
of intramolecularsalt linkages. thermore, it is necessary to transfer the polymers to their
It is concluded thatpolyelectrolyte titration offers an isoionic point prior to titration in order to evaluate the exper-
easy access to the determination of the surface charge imental data in terms of charge density. Both prerequisites
of proteins and other biopolymers. The data further hamper the application of these techniques in cases where
support the notion of the importance of electrostatic only limited amounts of material are available.
cooperative interactions in biologicalsystems. Alternatively, the numberof charged residues on biological
polyelectrolytes wasrecently measured turbidimetrically with
colloid titration (26). In fact, already 30 years ago Terayama
It has been shown in a variety of cases that cooperative proposed the determination of ionic polymers in solution on
electrostatic interaction may significantly contribute to the the basis of polyelectrolyte interaction (27).Based on this
stability and function of native structures. Biological poly- principle,a photometrictechnique was developedfor the
anionssuchas desoxyribonucleicacids are neutralized by measurement of synthetic polyelectrolytes with high precision
highly basic proteins (1,2). The modulationof this interaction at low concentration levels (28). The following data demon-
is important for reduplicative processes. Complex formation strate that this method offers convenient meansfor the quan-
between glycosaminoglycans and collagen determines thecol- titative determination of the surface charge of globular pro-
loidal properties of connective tissues (3). Modifications inthe teins. By way of example, this is demonstrated with highly
synthesis of acidic mucopolysaccharidescontribute to changes purified proteins with known amino acid composition.
in colloidal stability of the tissue,which is known to be altered
EXPERIMENTALPROCEDURES
in genetic diseases (4) and which may also possibly be the
cause of geriatric diseases (5). Heparin exhibits quasi-catalytic Chemicals and Biochemicals-PDDA’ ( M r= 2 X IO”) was kindly
properties on various enzymatic reactions (6-10). This is in provided from Chemviron (Bruxelles, Belgium). MGC ( M , = 17,000)
part a consequence of its high negative charge density. The and KPVS ( M ,= 320,000) were purchased from ICN Pharmaceuticals.
o-Tb was ordered from Fluka (Neu-Ulm, GFR). The reagents were
mechanism of muscle contraction has been found to result used without further purification.
from changes of cooperative ionic binding of the actomyosin Crystallinebovine pancreatic trypsin (EC 3.4.21.4), bovine pan-
complex (11). Recently, it was postulated that clot formation creaticchymotrypsin A (EC 3.4.21.11, bovineribonuclease A , (EC
results from ionic interaction between fibrin monomers (12). 3.1.27.51,porcine pepsin (EC 3.4.23.1),and equine heart muscle cyto-
chrome c were products from Boehringer (Mannheim, GFR). The
* Thisstudy was supportedinpart by theGermanResearch
Council, Project SFB 90-H9. The costs of publication of this article ’The abbreviations used are: PDDA, polydimethyldiallylammon-
were defrayed in part by the payment of page charges. This article ium chloride; MGC, N-methylglycolchitosan iodide; KPVS, potassium
must therefore be hereby marked “aduertisement” in accordance polyvinylsulfate; o-Tb, ortho-toluidine blue; PI, isoelectric point; eq,
with 18 U.S.C. Section 1734 solely to indicate this fact. charge equivalent.

1665

This is an Open Access article under the CC BY license.

1666 Determination
Charge of Proteins
purity of these proteins of more than 99% was assessed by sodium Chargeequivalency of
dodecyl
sulfate-polyacrylamide gel electrophoresis (29). Pepsin PDDA resp.MGC with KPVS
needed to be purified by gel-permeation chromatography on Bio-Gel eqon,,n/eqc,,io"
P-150.
Polyelectrolyte Titration of Polyelectrolytes-MGC and PDDA
I
were titrated with KPVS for the evaluation of charge equivalence.
Polystyrenecuvettesor polymethyl methacrylatecuvettes witha
volume of 4 ml were used as reaction vials. The measurements were
conducted with an automated photoelectric green-red double beam
titrator system previously described (28). The polycations were dis-
solved in double-distilled water at concentrations between 2 mg/liter
and 20 mg/liter. o-Tb was dissolved a t a concentration of 20 mg/liter
of double-distilled water to serve as indicator solution. One hundred
microliters of the indicator solution were added to 2.7 ml of the oPDDAtitratedwith KPVS
solution of polycation. The pH of themixture was adjusted by x MGCtitratedwithKPVS
addition of 0.01 M hydrochloric acid or potassium hydroxide, respec-
tively. KPVS wasdissolved in double-distilled water (20 mg of KPVS/
liter = 124 X lO-"eq/liter) and added automatically as titrant solution
in aliquots of 1 or 2 pl to the stirredpolycation mixture a t intervals of
5 s. The titration was photometricallyrecordedwhereby the end
point was marked by a pronounced inflection due to the metachro-
matic shift of the indicator absorption spectrum as caused by the FIG.2. Charge equivalence of the complexes PDDA/KPVS
cooperative binding of the dye to the excessive amount of the poly- and MGC/KPVS at different pH. Titrationexperiments were
anion (28). The charge equivalence of the polycation was calculated conducted as described for Fig. 1 under changing conditions of pH.
from the amount of KPVS needed to reach the point of steepest Each point represents theaverage of 4 determinations obtained from
slope, indicating complete 1:l ion pair interaction in the polyelectro- titration of a dilution series of the polycation.
lyte complex formation. The calculations were based on the assump-
tion that KPVS is completely dissociated above pH 2. Consequently
the negative charge of the polymer is 6.17 meq/g of polyanion above Titration of Rlbonuclease A
pH 2.
Polyelectrolyte Titration of Proteins-The charge of purified pro-
teins was measured after preincubating a definite amount of protein
(10-40 pg) in 2.7 ml of polyelectrolyte solution for 5 minat 23 "C. The
pH was adjusted with 0.01 M KOH or HCI to the appropriate pH
between pH 2 and pH9. One hundred microliters of indicator solution
were added. Thereafter, the mixture was titrated a t 23 "Cunder
continuous stirring.
For experiments below the isoelectric point of the protein a KPVS
solution was used containing 6 X lo-" eq of anionic sites. The counter
polyelectrolyte was always in excess of the amount of protein on the
basis of charge equivalence. The free titratable portion of the poly-
Y I

anion was retitrated with a solution containing 160 peq/liter of poly-

cation (MGC or PDDA). For experiments above theof PI the protein

?W
the samples were preincubated with 2.7 ml of a polycation solution
(= 5 X IO ~"eq) for 5 min at 23 "C. Subsequently o-Tb solution (IO0
pl) was added. The pH was adjusted prior to the incubation, and the
solution was retitrated with KPVS as described above.
All experiments were performed with dilution series of protein at
a fixed pH to improve the accuracy of determination. The numberof Opg * Ribonuclease
charge residues per protein molecule was calculated from the differ- I
I I I I I I
ence of the added and retitrated amount of polyelectrolyte preincu- 100 200 300 100 500 600 700 860 pl MGC (O,l5g/O
bated with the protein sample. (=3&3,3peq/l)
Theoretical charge density uersus pH curves were calculated with
FIG. 3. Titration of ribonuclease A at pH 3. Ribonuclease A (4
Tllrotlon o f K P V S w l l h MGC
to 32 pg) was preincubated with KPVS (20 pg) and o-Tb (2 pg) in 2.7
pgMGC at vorkous pH ml of double-distilled water and adjusted to pH 3. Titration was
conducted by automated stepwise addition of 1 pl of MGC (160 X
I eq/liter). Light absorption was recorded with a double beam
200-
photometer at 550 nm and 635 nm.
lheoretlcal expectallon

the assumption that charge neutralization occursbetween ionized

pj
oppositely charged amino acid residues within the protein molecule.
100- Data were obtained by subtracting the numberof oppositely charged
apH 3 residues a t a given pH using the pK values of the corresponding
isolated amino acids.

20-
FIG. 1. Titration ofPDDA and MGC with KFVS in the pres-
ence of o-toluidine blue in aqueous solution at pH 7. PDDA or
MGC was preincubated with o-Tb (2 pg) in 2.7 ml of double-distilled
water. The pH of the solution was adjusted to pH 7 with HCI (0.01
M).Automated titration was performed by stepwise addition of 1pl of
KPVS (124 X IO-' eq/liter) at intervals of 5 s. The relative change of
light absorption was recorded with a double beam photometer at 550
nm and 635 nm.
RESULTS
Measurement of Charge Equivalencebetween Polyelectro-
lytes-The pattern of titration curves obtained from measure-
ments of relative change of absorbance a t 635 nm with respect
to theisosbestic point a t 550 nm issimilar to thatof common
acid-base titrations of strong acids and strong bases (28, 30).
It is almost independent of pH with quarternary ammonium
salt polymers(Fig. 1). However, depending on the type of
polycation used, slight deviations from the theoretical charge
equivalence are obtained (Fig. 2).
 Determination
Charge of Proteins 1667

KPVS KPVS and retitrated with MGC. The amount of retitratable

Crsl negative charges of KPVSdecreases inverselywith the
amount of protein, indicating that the complex between pro-
0- Ribonuclease A tein and KPVS is not disrupted after additionof the polycat-
ion. The binding between KPVS and protein at increasing
concentration and at different pH is shown in Fig. 4. The
6- linear dependence between the amount of titrant and the
amount of preincubated protein indicates astoichiometric
interaction. Obviously, the effect of pH may be explained as
4- due to changes in the number of chargedresidues on the
protein, since the charge density of the polyanion does not
alter significantly between pH 2 and pH 10. The number of
chargedresidues isevaluated by essuminga one by one
2- pairing interaction of oppositely charged ionic sites. The num-
ber of surface charges per protein rnolecule at different pH
values for trypsin, chymotrypsinA, pepsin, cytochrome c, and
ribonuclease A is shown in Figs. 5-6. mhey are comparedwith
theoretical curvescalculated fromthe aminoacid composition
of each individual protein as listed in Table I.
FIG. 4. Titration of a dilution seriesof ribonuclease A at pH At pH 2-3 all carboxylic groups within the proteinmolecule
2 to 7. Measurements were made as described for Fig. 3, except that are expected to be undissociated. Therefore, the number of
the pH was changed. The difference of added and retitrated charge
equivalents of KPVS is marked on the ordinate.
positive charges should correspond to the number of basic
amino acids within the molecule. For cytochromec the excess
C ~ y ~ y . p s A# lnb o v l n e P.ncle.sJ l r y p s ~ n(bovmc p a n c I c . s )
is 24 basic amino acids, for chymotrypsin A it is 19 amino
n,.lmol acids, for trypsin 19 amino acids, and for ribonuclease A it is
",./rnO'
18 amino acids (Table I). Since at pH 2 one positive charge
from the protonated amino group at the NH2-terminal end
must be added (for chymotrypsin A which is formed from 3
\ '1 peptide chains linked together by disulfide bridging there are
3) the number of positive charges increases by one, or 3 for
chymotrypsin A. Dissociation of the COOH-terminalcarbox-
ylate group occurs above pH 2 (31). Therefore, at pH 3 the
number of basic charges is reduced by the number of carbox-
ylate groups forming a COOH-terminal end at the peptide.
Titration at pH 2 revealed protonation of 24 amino acids for
cytochrome c,21 amino acids for chymotrypsin A, and 18
amino acids for ribonuclease A, independent of the cation
(MGC or PDDA) used as titrant. Corresponding data were
n, /mol
2L- ;& determined a t higher pH. Thedifferences of degree of proton-
LO-

35-
22-
20-
18-
) ocolculafed
IKPVSIPDDAI
bovine Ribonuclease A1
nlKPVSlMGCI
30- 16-
c IL-
25- I / ,
"""Imo1
20-
x calculated
18- v t i t r a t e d w i t h MGC
16-
14-
I
n".lmol
12-
FIG. 5. Polyelectrolyte titration of cytochrome c, chymo-
trypsin A, trypsin, and pepsin betweenpH 2 and 9. Measure- 10-
ments were made as described in detail in the text. The data for
titration are expressed as titratable numbers of ionized residues per 8-
protein molecule. A titration curve was calculated from the numbers
of ionizable amino acid residues of each protein molecule and the 6-
corresponding pK values for the isolated amino acids. Data from
titrationmeasurements of pepsin and cytochrome c using either 4-
PDDA or MGC as cationic polyelectrolytes are given forcomparison.
2- A '
Experiments with dilution series revealed a sensitivity with I
1 1 1 1
a minimum of10"" charge equivalents detectable. The coef- 2 3 5 7 9 PH
ficient of variation at amounts between5 and 50 X lo-' charge
equivalents of polyelectrolyte is 1-2%. FIG. 6. Comparison of charge distributionof ribonuclease A
Measurements of. Surface
. Charge
- , Proteins-Fie. 3 shows
of between pH 2 and 9 as evaluated fromuolvelectrolvte titration
the titration curves for ribonuclease A preincubated with and from-potentiometry (data taken &om ref. 36).-
1668 Determination
Charge of Proteins
TABLEI
Chemical data forproteins
Terminal
Protein n(Arg)
M. n(Lys) n(His)
nWp) n(Glu) Ref.
n(NHL)"
n(CO0H)

Trypsin 23,746 1 1 5 4 3 14 2 (38)

Chymotrypsin 25,500 3 3 9 5 2 14 3 (39)
Ribonuclease A I 13,680 1 1 5 5 4 10 4 (40)
Cytochrome c 12,900 1 1 3 9 3 19 2 (41)
Pepsin 32,700 1 1 29 13 1 1 2 (42)

PK,~in amino acid 8-9 2-3 3.5 4.5 6.0 10.3 11.8 (31)
'' n, number of amino acids/molecule

a. b counterionicpolyelectrolyte KPVSresultedin a complete

recovery of basic charge equivalents of protein and PDDA.
Although this experiment indicated that direct titrationof a
protein may also be possible, we preferred to use the retitra-
tion method, since with this technique the end pointof titra-
tion could be detected with higher accuracy. In fact, the
resultsdemonstratethat electrostat.ic interaction between
protein and equally charged polyelectrolyte does not occur.

u I Incubaleon DISCUSSION

The principle of polyelectrolyte titration as appliedfor

cationically charged biopolymers is presented in Fig. 7a.
Briefly, the titration methodis based on two phenomena (28,
30): 1) the cooperative binding between oppositely charged
polymers; and 2) the metachromatic shiftof light absorption
of certain basic charged dyes interacting with chromotropic
polyanions.
During incubation a fraction of anionic sites of the chrom-
otropic polyelectrolyte equivalent to the number of positively
charged residues of the protein is neutralized. The indicator
molecules interact with the excessive amount of polyanion
resulting in a metachromatic shift of light absorption. In the
ensuing process of titration the polycationic titrant interacts
stoichiometrically withthe excessive polyanion. The end point
of this reaction is marked by the replacement of the bound
dye molecules, since the degree of cooperativity of the poly-
anion/polycation interaction exceeds significantly that of the
polymer/dye interaction.Therefore,the coupled reactions
occur consecutively.
In the case of negatively charged proteins, polycations are
used for incubation and excessive
the amount of the polycation
FIG. 7. Scheme of the principle of polyelectrolyte titration.
The scheme in a exemplifies the titration of a protein with cationic is back-titrated with a suitable polyanion as explained in the
net charge. The protein is preincubated with a defined excess of experimental part (Fig. 7 b ) . The indicator reaction is insen-
chromotropic polyanion and a metachromatic dye. The polyanion sitive to pHbetween 2 and 10 and to salt concentrations up to
binds to basic charges of the protein. The dye molecules interact with lo-" M for divalentcationsand M for monovalent ions
the excess polyanion to form a complex characterized by a metachro- (28). Therefore, the application of this method does not re-
matic shift of light absorption. A polycation added stepwise binds quire the complete removal of electrolytes of low molecular
stoichiometrically to the excess polyanion. The end point is marked
by the replacement of the dye molecules, which enables a photometric weight as a prerequisite.
end point detection. The reverse scheme of titration for measurements Previously, it was demonstrated that the interaction be-
of proteins with anionic net charge is shown in b. tween oppositely uniformly charged polyelectrolytes is deter-
mined byelectrostatic interaction resulting binding
in through
ion-pair complex formation (18-20,32,33). For syntheticionic
ation between calculated and titrated values at a given pH polymers and glycosaminoglycans the degree of ionization has
were usually not more than 3-4 charge equivalents. For pep- been measured with polyelectrolyte titration (30, 43). How-
sin, calculation reveals 4 positively charged residues within ever, proteins are ampholytes. Their electrostatic properties
the molecule at pH 3, whereas nopositive or negative charges are morecomplex. Their surface charge may notbe uniformly
could be titrated. At pH 4 and pH 5 differencesbetween distributed a t a given pH. On the other hand, intramolecular
calculated and measured numbers of charge equivalents per neutralization may occur (34). Therefore, the applicability of
molecule are even higher whereas a t p H 7 and pH 9 the this technique for protein measurement seemed to be uncer-
titratednumbercorrespondstothe calculated number of tain. In view of previous observations demonstrating that
charged residues. electrostatic interaction may dominatea specific biochemical
Experiments were also performed in which the proteinwas reaction (6-15), it seemedindispensable to obtain quantitative
incubated with polyelectrolyte equally chargedat a given pH, information about the surface charge of a protein in a given
i.e. trypsin with PDDA at pH 7. As expected, retitration with milieu.
 Charge Determination of Proteins 1669

The data obtained from globular proteins with well char- therefore, besimilar to that of a polycarboxylic acid. For
acterized amino acidcomposition shownin Figs. 5 and 6 polymethacrylate (37) and polyacrylate (32) dissociation of
demonstrate: carboxylic groups only occurs minimally near the pK of the
1. Electrostatic binding occurs betweena protein andcoun- isolated monomeric acid.
terionic polyelectrolyte whereas it does not occur between a Even at alkaline pHa considerableportion of the carboxylic
protein and equally charged polyelectrolyte, indicating that groups remains undissociated. Since experiments with pepsin,
only the net charge of a protein contributes to ionic interac- inwhich either MGC or PDDA was used as counterionic
tion. The inverse linearcorrelationbetween increasing polyelectrolyte, result in the same numberof charge equiva-
amounts of protein and retitratable charge equivalents of the lents at pH4 and 5 an erroneous estimationof charge density
oppositely charged polyelectrolyte added in excess moreover is not evident. On the contrary, it seems that pepsin behaves
indicates stoichiometryof the electrostatic interaction. like a polycarboxylic acid with high negative surface charge.
2. The complexes formed between a protein and counter- For these polymers the assumptionof a complete dissociation
polyelectrolyte are not disrupted by titrant polyelectrolyte of acidic groups slightly above the pKof the monomeric acid
charged equallyas the protein. Therefore, cooperative binding is invalid. The calculation of the theoretical curve, however,
between protein and counter polyelectrolyte appears to be was based on this assumption.
operative. Further, the experimental data obtained frompolyelectro-
3. The accuracy of charge determination can be verified at lyte titration reasonably agree with charge densityvalues
pH 2. In this rangeof pH all acidic residues withinthe protein calculated from the electrophoretic mobility-pH curve of pep-
molecule should be undissociated. The numberof measurable sin (44). Considering a specific volume of 0.76 cm"/g (48) and
charges corresponds to the numberof basic amino acid con- the known molecular weight of pepsin a spherical equivalent
stituents. Despite thecomplex pattern of charge distribution radius of 2.2 nm is calculated. From the electrophoretic mo-
at the surface of globular proteins the comparison of calcu- bility data taken from the literature (44) the net charge of
lated and titrated numbers reveals close agreement. The dif- pepsin as a function of pH is obtainedby applying the Debye-
ference is not more than one charge equivalent. This obser- Huckel-Henry-Gorin equationas describedbyBrown and
vation can easily be explained by an error of about 5% in Timasheff (45): QE = -0.54 (pH 2.47); -2.2 (pH 3.16); -5.6
quantitating small amounts of highly purifiedsalt-free protein (pH 4.15); and -8.5 (pH 4.57), respectively. As compared to
by weight. the titrimetric data plotted in Fig. 6, at pH values above 4
4. At higher pH thecalculated data must be regarded only where in the case of pepsin reliable polyelectrolyte titrations
as approximatesince the pK valuesof charged residuesin the can be conducted the charges determined electrophoretically
sequence of the polymer chain may differ from the pKvalues are about 60%of those determined titrimetrically. This result
of the isolated amino acids. Still, the conformity between is quite in accord with similar findings obtained with other
calculated and titrated numbers of charge equivalents is sur- proteins when electrophoretic and potentiometric data are
prising. Obviously, interaction between protein and counter- compared (45).In fact,in the case of egg albumin the electro-
polyelectrolyte occurs predominantly through one by one ion- phoresis charge was also found to be only 60% of the charge
pair formation irrespective of an irregular distributionof the by titration (46, 47). In order to reconcile this difference it is
ionic sites at theprotein surface. This observation agrees withgenerally understood that electrophoresis relates to the net
recent experimental results regarding the interactionof KPVS charge inside the surface of shear where part of the particle
with carboxyhemoglobin (35). surface charge may be screened by counterionic binding (47).
5. Under conditionswith dissociated carboxylic groups and In conclusion, the r.mults obtained from polyelectrolyte titra-
protonated amino groups the titratable number of charge tion are corroborated by the electrophoretic measurements.
equivalents approximates the net chargecalculable from the Our data demonstrate thatpolyelectrolyte titration offers a
number of ionized amino acidresidueswithin the protein convenient means for assessment of surface charge of biopol-
molecule. Obviously, the tertiary structure of a protein is ymers, at least of globular proteins, mucopolysaccharides, or
controlled by amaximaldegree of intramolecularcharge biological micellar systems such as serum lipoproteins. The
compensation. procedure allows quantitative determination from amounts
6. In the proximity of the isoionic point the relative impre- far less than needed for determination by conventional tech-
cision of polyelectrolyte titration increases, since cooperative niques. This confirms our belief that the methodmay provide
electrostatic interaction does not occur witha number smaller access to studies which until now were hampered because a
than 3 to 4 charge equivalents(30). The measurement of only simple methodological approach was lacking.
one or two charge equivalents may result from cluster for- Such investigations seem to be particularly interesting for
mation between protein molecules through hydrophobic in- the analysis of biopolymers participating in physiologically
teraction. In thiscase, the number of charges on anaggregate occurring colloid chemical processes.
of protein molecules could suffice for cooperative interaction
with the counterpolyelectrolyte. Acknowledgments-We are grateful to I. Fielhauer, K. Frech, and
7. Higher degrees of protonation are obtained with polye- U. Wildenberg for experienced technical assistance.
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