supports to attach proteins to amine-activated supports by using
the glutaraldehyde32,33 or N-hydroxysuccinimide (NHS) meth-
ods.32,33,38,40 In addition, glycoproteins can be immobilized after
oxidation of their carbohydrate residues by reacting such proteins
with hydrazide- or amine-containing supports.32,33,36
polystyrene microspheres,52 and Sepharose.46 However, this ap-
proach has not yet been used with any HPLC supports. This study
will explore the use of these coupling methods with HSA and
HPLC-grade silica. The supports used in this work will be prepared
by reacting aminopropyl silica with succinimidyl 4-(N-maleimi-
domethyl)cyclohexane-1-carboxylate (SMCC) or succinimidyl io-
doacetate (SIA), as shown in Figure 1. This should give activated
materials with good stability toward hydrolysis.33,45-49 After these
supports have been developed, they will be compared in terms of
their specificity for sulfhydryl groups and their ability to im-
mobilize HSA in an active form for use in drug-binding studies
and chiral separations. The results will then be compared with
those noted for the Schiff base method, a common amine-based
coupling technique employed for HSA and other proteins.20-26,53
Human serum albumin (HSA) is one protein that is often used
in HPAC columns. HSA is the most abundant protein in blood
and is known to bind a variety of drugs and biological com-
pounds.41-43 When immobilized in an HPAC column, this protein
can be used under isocratic conditions as a weak-to-moderate
ligand for the study of drug-protein interactions44 or as a
stationary phase for separating a variety of chiral solutes.19 Most
previous work with HSA has involved its immobilization to HPLC
supports through its amine groups; however, HSA has a large
number of primary amines in its structure (i.e., 58 lysines plus
the N-terminal amine)42 which might lead to such undesirable
effects as multipoint attachment, improper orientation, or inactiva-
tion of this protein during the immobilization process.32
EXPERIMENTAL SECTION
Reagents. The SMCC (>99% pure; toxic), succinimidyl io-
doacetate (>97% pure; toxic) and Slide-A-Lyzer dialysis cassettes
(7 kDa MW cutoff, 0.1-0.5 mL or 0.5-3 mL capacity) were from
An alternative route that is appealing for the immobilization
of HSA and other proteins is to use free thiol groups in their
structures. For instance, normal HSA has only a single free thiol
(Cys-34) which could, in theory, be used for the single-point
attachment of this protein to a support. This study will explore
two approaches for such work based on maleimide- and iodoacetyl-
activated materials.32,33,45-49 Both maleimide and iodoacetyl groups
are thought to react selectively with the sulfhydryl group of
cysteine.32,33,45-49 In addition, these reactions are known to be fast
for small thiol-containing compounds,45-49 making this a possible
route for the rapid immobilization of proteins like HSA to HPLC
supports.
Pierce (Rockford, IL). The D-tryptophan (>99% pure), dimethyl
formamide (DMF, 99% pure), acetonitrile (HPLC-grade, >99.93%
pure; flammable), formic acid (96% pure; corrosive), trifluoroacetic
acid (TFA, >99% pure; corrosive), acetic anhydride (>99% pure;
flammable), and pyridine (>99% pure; flammable and corrosive)
were from Aldrich (Milwaukee, WI). HSA (Cohn fraction V,
essentially fatty acid free), 3-(R-acetonylbenzyl)-4-hydroxy-cou-
marin (racemic warfarin, >98% pure; toxic and a possible terato-
gen), racemic tryptophan (>99% pure), L-tryptophan (>98% pure),
ibuprofen (>98% pure), carbamazepine (>98% pure), 4-dimethyl-
aminopyridine (DMAP; toxic), trypsin (sequencing grade), guani-
dine hydrochloride (>99% pure), dithiothreitol (DTT, >99% pure),
Immobilization based on maleimide and iodoacetyl groups has
been used in the past for coupling proteins and peptides to quartz
disks,50 polished silicon wafers,50 poly(allylamine) polymers,51
iodoacetamide (99% pure; light sensitive), and L-cysteine hydro-
chloride (anhydrous, >98% pure; toxic) were from Sigma (St.
Louis, MO). Ethylenediamine tetraacetic acid (EDTA, disodium
salt and dihydrate, essentially 100% pure) was purchased from
J. T. Baker (Phillipsburg, NJ). The separate forms of R- and
S-warfarin (>98% pure) were from DuPont Pharmaceuticals
(Wilmington, DE).
(34) Berruex, L. G.; Freitag, R.; Tennikova, T. B. J. Pharm. Biomed. Anal. 2000,
24, 95-104.
(35) Gupalova, T. V.; Lojkina, O. V.; Palagnuk, V. G.; Totolian, A. A.; Tennikova,
T. B. J. Chromatogr., A 2002, 949, 185-193.
(36) Jiang, T.; Mallik, R.; Hage, D. S. Anal. Chem. 2005, 77, 2362-2372.
(37) Luo, Q.; Zou, H.; Zhang, Q.; Xiao, X.; Ni, J. Biotechnol. Bioeng. 2002, 80,
481-489.
(38) Mallik, R.; Jiang, T.; Hage, D. S. Anal. Chem. 2004, 76, 7013-7022.
(39) Ostryanina, N. D.; Vlasov, G. P.; Tennikova, T. B. J. Chromatogr., A 2002,
949, 163-171.
The thiol and sulfide quantitation kit (T-6060) was purchased
from Molecular Probes (Eugene, OR); this kit contained the
N-benzoyl-
CH3, and
L
-
ARGININE
,
P
-
NITROANILIDE
(
L
-BAPNA), papain-S-S-
L
-cysteine referred to later in this section. The amino-
propyl and bare silica Nucleosil Si-300 supports (both 7 µm particle
diameter, 300 Å pore size) were from Macherey Nagel (Du¨ren,
Germany). All aqueous solutions were prepared using water from
a Nanopure system (Barnstead, Dubuque, IA) and filtered using
Osmonics 0.22 µm nylon filters from Fisher (Pittsburgh, PA).
Apparatus. The system used in the chromatographic studies
consisted of a P4000 gradient pump and a UV100 absorbance
detector from Thermoseparations (Riviera Beach, FL). Samples
were injected using a Rheodyne LabPro valve (Cotati, CA)
equipped with a 20 µL sample loop. Chromatographic data were
collected and processed using in-house programs written in
LabView 5.1 (National Instruments, Austin, TX). Assays for
determining the stability of the activated silica were performed
(40) Kim, H. S.; Kye, Y. S.; Hage, D. S. J. Chromatogr., A 2004, 1049, 51-61.
(41) Kragh-Hansen, U. Pharmacol. Rev. 1981, 33, 17-53.
(42) Peters, T. J. All About Albumin: Biochemistry, Genetics and Medical
Applications; Academic Press: San Diego, CA, 1995.
(43) Sengupta, A.; Hage, D. S. Anal. Chem. 1999, 71, 3821-3827.
(44) Yang, J.; Hage, D. S. J. Chromatogr., A 1997, 766, 15-25.
(45) Hashida, S.; Imagawa, M.; Inoue, S.; Ruan, K. H.; Ishikawa, E. J. Appl.
Biochem. 1984, 6, 56-63.
(46) Partis, M. D.; Griffiths, D. G.; Roberts, G. C.; Beechey, R. B. J. Protein Chem.
1983, 2, 263-277.
(47) Rector, E. S.; Schwenk, R. J.; Tse, K. S.; Sehon, A. H.; Chan, H. J. Immunol.
Methods 1978, 24, 321-336.
(48) Thorpe, P. E.; Ross, W. C. J.; Brown, A. N. F.; Myers, C. D.; Cumber, A. J.;
Foxwell, B. M. J.; Forrester, J. T. Eur. J. Biochem. 1984, 140, 63-71.
(49) Yoshitake, S.; Imagawa, M.; Ishikawa, E.; Niitsu, Y.; Urushizaki, I.; Nishiura,
M.; Kanazawa, R.; Kurosaki, H.; Tachibana, S.; Nakazawa, N.; Ogawa, H.
J. Biochem. 1982, 92, 1413-1424.
(50) Rezania, A.; Johnson, R.; Lefkow, A. R.; Healy, K. E. Langmuir 1999, 15,
6931-6939.
(51) Tada, T.; Mano, K.; Yoshida, E.; Tanaka, N.; Kunugi, S. Bull. Chem. Soc.
Jpn. 2002, 75, 2247-2251.
(52) Tournier, E. J. M.; Wallach, J.; Blond, P. Anal. Chim. Acta 1998, 361,
33-44.
(53) Larsson, P. O. Methods Enzymol. 1984, 104, 212-223.
1412 Analytical Chemistry, Vol. 79, No. 4, February 15, 2007