12702 J. Am. Chem. Soc., Vol. 119, No. 52, 1997
Colton et al.
perhaps to balance helix dipoles.11 An important function of
Lys in many proteins may be to make them soluble. Modifica-
tion of ꢀ-NH3 groups of Lys on the surface of the protein,
a neutral marker peak per unit of electric field strength (eq 2).
In eq 2, Ltot (m) is the total length of the capillary, Ldet (m) is
the length from the inlet of the capillary to the detector and V
+
however, should affect the stability of the protein significantly
less than modification of residues buried inside the protein. We
have recently demonstrated that each rung of the charge ladder
derived from bovine carbonic anhydrase II (BCA II) by
Ldet
Ldet
-
[
(
tx ) (tnm
)
]
µ
)
(2)
electro
V
+
acetylation of Lys ꢀ-NH3 groups binds neutral sulfonamide
(
)
L
5
tot
ligands with equal affinity. This observation suggests that in
+
this protein, Lys ꢀ-NH3 groups are not important in determining
(
V) is the voltage applied across the capillary. The parameters
the affinity of the protein for neutral ligands, and by inference
that charge on these groups does not determine the conformation
around the active site. We do not know for which other proteins
this conclusion will hold.
tx (s) and tnm (s) are the times of migration for an analyte peak
x and a neutral marker, respectively. Equating the two
expressions for electrophoretic mobility (eqs 1 and 2) and
solving for tx (eq 3) and 1/tx (eq 4), respectively, demonstrates
that plots in the 1/time domain are linearly related to ZCE/MR
Capillary electrophoresis is an analytical technique that
provides information simultaneously about the values of charge
and coefficient of friction of proteins in solution. The electro-
phoretic mobility of a protein, as measured by CE, µelectro, is
proportional to its charge at a given value of pH, ZCE, and
tnm
tx )
(3)
t VC Z
CE
nm
P
1
1
+
R
det tot MR
(
)
inversely correlated to M /CP, its coefficient of friction (eq
L L
12-16
1),
where M is the MW of the protein, CP is a proportional-
ity constant, and R is a constant that relates to the shape of the
protein under the conditions of its analysis.
VCP
Z
1
tx
CE
)
+
(4)
det tot MR
(
)
tnm
L L
ZCE
µelectro ) CP
(1)
and consequently to mobility (eq 1), while those in the time
domain are not. We plot all primary data as absorbance vs
1/time (s ) to show trends in the mobility of rungs of protein
(
MR
)
1
7
-1
-
We define Zseq as the charge of a protein calculated from its
amino acid sequence and any charged cofactors (bound metal
ions, prosthetic groups, coenzymes), and post-translationally
added residues (charged sugars, phosphate groups), using
standard values of pKa and the pH of the solution. Uncertainties
in the values of pKa (especially for His and for ionizable residues
with anomalous values of pKa) are sufficiently large that
calculations of Zseq are inexact. The value of ZCE reflects any
differences between the actual and standard values of pKa of
the charged residues of the protein and includes the contributions
of charge from tightly associated counterions.
charge ladders accurately.
Experimental Section
Materials. Acylase I (porcine kidney), aldolase (type X: rabbit
muscle), alkaline phosphatase (type VII-NL: bovine intestinal mucosa),
anti-DNP IgE (mouse), carbonic anhydrase II (bovine), carbonic
anhydrase II (human), calmodulin (bovine brain), R-chymotrypsinogen
(
type II: bovine pancreas), cytochrome c (horse heart), ferritin (type I:
horse spleen), â-galactosidase (E. coli), glucose-6-phosphate dehydro-
genase (type XII: torula yeast), growth hormone (human), hemoglobin
(bovine), insulin (bovine), R-lactalbumin (bovine milk), L-lactate
dehydrogenase (type XI: rabbit muscle), myoglobin (horse heart),
ovalbumin (chicken egg), pyruvate kinase (type III: rabbit muscle),
superoxide dismutase (bovine erythrocytes), ubiquitin (bovine eryth-
rocytes), sodium benzoate, and 1-ethyl-3-(3-dimethylaminopropyl)
carbodiimide (EDAC) were purchased from Sigma (St. Louis, MO).
Bovine pancreatic trypsin inhibitor, carboxypeptidase B (porcine
pancreas), creatine kinase (rabbit muscle), deoxyribonuclease I (bovine
pancreas), dextranase (Penicillium sp.), hexokinase (yeast), lysozyme
Electrophoretic mobilities of analytes are expressed math-
ematically as the difference in velocities of an analyte peak and
(2) We name each rung of a charge ladder by the degree of modification
of the proteins which it comprises, starting with native protein as the zeroth
rung. The nth rung of the charge ladder of a protein having N Lys ꢀ-NH2
groups may be composed of as many as (N!/(N-n)!n!) regioisomeric
derivatives. As an example, the ninth rung of the charge ladder of bovine
carbonic anhydrase II, an enzyme that has 18 modifiable Lys ꢀ-NH2 groups,
may comprise up to 48620 regioisomeric derivatives. The assumptions made
for these calculations are that all Lys ꢀ-NH2 groups have the same reactivity
and that reaction at one site does not influence the reactivity at another;
both of these assumptions are doubtless incorrect at some presently
undefined level of detail.
(
egg white), pancreatic lipase (porcine), papain (papaya latex), peroxi-
dase (horseradish), phospholipase A2 (crotalus adamanteus venom),
and ribonuclease A (bovine pancreas) were purchased from Worthington
(Freehold, NJ). Hexadimethrine bromide (Polybrene), N-hydroxy
succinimide (NHS), 1,2,4-benzenetricarboxylic anhydride, 1,2,4,5-
benzenetetracarboxylic dianhydride, and mellitic acid were purchased
from Aldrich (Milwaukee, WI). Succinic anhydride was purchased
from Eastman (Rochester, NY). Uncoated fused silica capillaries with
an internal diameter of 50 µm were purchased from Polymicro
Technologies (Phoenix, AZ). Cholic acid was purchased from Lan-
caster (Windham, NH). N,N-Dimethyl formamide (DMF) was pur-
chased from EM Science (Gibbstown, NJ). N-Hydroxysuccinimidyl
(
3) Gao, J.; Gomez, F. A.; Haerter, R.; Whitesides, G. M. Proc. Natl.
Acad. Sci. U.S.A. 1994, 91, 12027-12030.
4) Gao, J.; Mrksich, M.; Gomez, F. A.; Whitesides, G. M. Anal. Chem.
995, 67, 3093-3100.
5) Gao, J.; Mammen, M.; Whitesides, G. M. Science 1996, 272, 535-
37.
(
1
(
5
(
6) Gao, J.; Whitesides, G. M. Anal. Chem. 1997, 69, 575-580.
(7) Creighton, T. E. Proteins: Structures and Molecular Properties; W.
H. Freeman and Company: New York, 1993; p 6.
(
(
(
(
(
8) Takahashi, K. J. J. Biol. Chem. 1968, 243, 6171-6179.
18
cholate was prepared according to the procedure of Okahata et al.
9) Roberts, C.; C o´ rdova, E.; Whitesides, G. M. Unpublished results.
10) Anderson, J. R.; Colton, I. J.; Whitesides, G. M. Unpublished results.
11) Hol, W. G. J. Prog. Biophys. Mol. Biol. 1985, 45, 149-195.
12) Grossman, P. D. Capillary Electrophoresis: Theory & Practice;
The zwitterion 3-quinuclidinopropanesulfonate was synthesized as
previously described.19 NICK Spin columns containing G-50 Sephadex
gel were purchased from Pharmacia Biotech (Piscataway, NJ). The
1
Academic Press: San Diego, CA, 1992; pp 111-132.
H-NMR spectra were recorded at 400 MHz on a Bruker spectrometer.
(
(
13) Compton, B. J.; O’Grady, E. A. Anal. Chem. 1991, 63, 2597-2602.
14) Engelhardt, H.; Beck, W.; Kohr, J.; Schmitt, T. Angew. Chem., Int.
(17) Mammen, M.; Colton, I. J.; Carbeck, J.; Bradley, R.; Whitesides,
G. M. Anal. Chem. 1997, 69, 2165-2170.
Ed. Engl. 1993, 32, 629-766.
(
15) Novotny, M. V.; Cobb, K. A.; Liu, J. Electrophoresis 1990, 11,
(18) Okahata, Y.; Ando, R.; Kunitake, T. Bull. Chem. Soc. Jpn. 1979,
52, 3647-3653.
7
35-749.
(16) Karger, B. L.; Cohen, A. S.; Guttman, A. J. J. Chromatogr. 1989,
(19) Gomez, F. A.; Avila, L. Z.; Chu, Y.; Whitesides, G. M. Anal. Chem.
1994, 66, 1785-1791.
4
92, 585-614.