Syntheses and transglutaminase-catalyzed incorporation of novel spin-labeled primary amines into proteins

Article abstract of DOI:10.1006/bioo.1996.0006

A series of spin-labeled primary amine derivatives, namely, 2,2,6,6-tetramethyl-piperidinyl-oxyl-4-amidoalkylamines with varying alkyl chain lengths, have been synthesized. The spin-labeled primary amine-with a tetramethylene or a pentamethylene chain covalently modifies human plasma fibronectin with a stoichiometry of 0.97-to-1.0 (probe-to-subunit) in the presence of coagulation factor XIIIa. The labels with two or one methylene chain also similarly modify fibronectin, but with a stoichiometry of only about 0.3-0.4 per subunit. The spin-labeled primary amine with a trimethylene chain does not label fibronectin. The labeling site appears to be the glutamine-3 residue at the amino-terminal region of fibronectin. Electron spin resonance studies show that the bound labels are partially immobilized with an effective rotational correlation time of 0.4-0.6 ns. The spin-labeled primary amine with tetramethylene chain also is shown to covalently incorporate into bee venom melittin in the presence of guinea-pig liver transglutaminase. The syntheses of the various spin-labeled primary amines and their applications in the study of structure and dynamics of different proteins and peptides are discussed. The observations from this study suggest that these spin-labeled primary amines have potentially wide application as structural probes.

Full text of DOI:10.1006/bioo.1996.0006

BIOORGANIC CHEMISTRY 24, 50–58 (1996)
ARTICLE NO. 0006
Syntheses and Transglutaminase-Catalyzed Incorporation
of Novel Spin-Labeled Primary Amines into Proteins1
C
HAKRAVARTHY
N
ARASIMHAN,*,2 CHING-SAN
LAI,† AND
J
OY
J
OSEPH
†
*Biochemistry Department and †National Biomedical ESR Center, Medical College of Wisconsin, 8701
Watertown Plank Road, Milwaukee, Wisconsin 53226
Received August 2, 1995
A series of spin-labeled primary amine derivatives, namely, 2,2,6,6-tetramethyl-piperidinyl-
1-oxyl-4-amidoalkylamines with varying alkyl chain lengths, have been synthesized. The spin-
labeled primary amine with a tetramethylene or a pentamethylene chain covalently modifies
human plasma fibronectin with a stoichiometry of 0.97-to-1.0 (probe-to-subunit) in the
presence of coagulation factor XIIIa. The labels with two or one methylene chain also similarly
modify fibronectin, but with a stoichiometry of only about 0.3–0.4 per subunit. The spin-
labeled primary amine with a trimethylene chain does not label fibronectin. The labeling site
appears to be the glutamine-3 residue at the amino-terminal region of fibronectin. Electron
spin resonance studies show that the bound labels are partially immobilized with an effective
rotational correlation time of 0.4–0.6 ns. The spin-labeled primary amine with tetramethylene
chain also is shown to covalently incorporate into bee venom melittin in the presence of
guinea-pig liver transglutaminase. The syntheses of the various spin-labeled primary amines
and their applications in the study of structure and dynamics of different proteins and peptides
are discussed. The observations from this study suggest that these spin-labeled primary amines
have potentially wide application as structural probes.
1996 Academic Press, Inc.
INTRODUCTION
The enzymes that belong to the transglutaminase family catalyze the calcium-
dependent cross-linking of glutaminyl residues of proteins and glycoproteins to the
epsilon-amino group of lysine residues to create inter- or intramolecular isopeptide
bonds (1, 2). This class of enzymes can also covalently link a variety of primary
amines to peptide-bound glutamine residues. Cellular transglutaminases occur
widely in differentiating cells and tissues in mammals. Coagulation factor XIII, the
proenzyme of plasma transglutaminase, is present in blood plasma at a concentration
of 10–20 Ȑg/liter. Upon activation by thrombin, factor XIII is converted into factor
XIIIa which catalyzes an acyl transfer reaction that involves glutaminyl residues in
proteins as acyl donors and a variety of primary amines as acyl acceptors, resulting
in the formation of monosubstituted Ͳ-amide bonds (3, 4). Coagulation factor XIII
is the best studied among the transglutaminases known to date.
The site-specific, transglutaminase-directed labeling of proteins with primary
amines such as the fluorescent probe, dansylcadaverine (1), has opened up a general
1 This work was supported by NIH Grants GM35719, RR01008, and GM22923.
2 To whom correspondence should be addressed. Fax: (414) 266-8497.
50
0045-2068/96 $18.00
Copyright 1996 by Academic Press, Inc.
All rights of reproduction in any form reserved.

PRIMARY AMINE SPIN LABELS AS STRUCTURAL PROBES
51
strategy for exploring cross-linking domains of proteins by fluorescence spectros-
copy (5–8). Of equal importance is the possibility of studying posttranslational
modification of proteins by small molecular weight primary amines catalyzed by
transglutaminases. Further, primary amines such as isoniazid and hydrazaline that
are used in the treatment of tuberculosis and hypertension, respectively, require
transglutaminase-catalyzed incorporation into their respective targets in order to
impart their drug effect (9). As the physiological relevance of transglutaminase
sites in a variety of proteins and peptides are identified (1), the need to develop
novel probes that will help to study their structure and function becomes important.
Plasma fibronectin (Fn),3 a glycoprotein, circulates in vertebrate blood at a con-
centration of about 0.3 mg/ml and participates in many important processes includ-
ing blood coagulation and wound healing (10). The protein is a dimer consisting
of 240–250 kD subunits linked near the carboxyl termini by two disulfide bonds.
Fn contains one plasma transglutaminase-reactive site per subunit which has been
identified as the glutamine-3 residue near the amino-terminus (11). The presence
of transglutaminase sites on Fn has made it possible to selectively label the protein
with a variety of fluorescent primary amine derivatives including dansylcadaverine
and fluorescenylcadaverine for structural analyses using fluorescence techniques
(5–7).
Melittin is a basic peptide of 26 amino acid residues which constitutes about 50%
of the dry weight of bee venom (12). The peptide has Gln residues in positions 25
and 26. Perez-Paya et al. showed that in the presence of guinea-pig liver transgluta-
minase, the fluorescent probe, dansylcadaverine, could be introduced into Gln25
(12).
Here we report the syntheses and applications of a series of new, spin-labeled
primary amine derivatives, namely, 2,2,6,6-tetramethyl-piperidinyl-1-oxyl-4-ami-
doalkylamines (TEMPO-Cn; Fig. 1).Transglutaminase-catalyzed incorporation of
these new spin labels into the Fn molecule and into melittin are examined, and
their usefulness in deriving structural and dynamic information by electron spin
resonance (ESR) spectroscopy is discussed.
MATERIAL AND METHODS
Tris(hydroxymethyl)aminomethane (Tris), phenylmethylsulfonyl fluoride,
guinea-pig liver transglutaminase, and melittin were obtained from Sigma (St. Louis,
MO). Coagulation factor XIII and thrombin were obtained from Calbiochem (San
Diego, CA). Plasma Fn was isolated from fresh frozen human plasma using gelatin-
Sepharose affinity chromatography (13). The purity of the protein was determined
by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE). Other
chemicals and solvents were obtained from Aldrich chemical Co., (Milwaukee, WI).
The TLC plates were from Whatman (Kent, England). Elemental analyses were
performed by Galbraith Laboratories, Knoxville, TN.
3 Abbreviations used: ESR, electron spin resonance; Fn, fibronectin; TEMPO, (2,2,6,6-tetramethyl-
1-piperidinyl-1-oxyl-); FMOC, fluorenylmethyloxycarbonyl; THF, tetrahydrofuran.

52
NARASIMHAN, LAI, AND JOSEPH
F
IG. 1. Schematic diagram for the synthetic routes of spin-labeled primary amines, n ϭ 1, TEMPO-
C 1; n ϭ 2, TEMPO-C2; n ϭ 3, TEMPO-C3; n ϭ 4, TEMPO-C4; n ϭ 5, TEMPO-C5. The chemical
names of compounds involved in the synthesis of TEMPO-C4 are given under Material and Methods.
Synthesis of Spin-Labeled Primary Amines
A schematic diagram of the general synthetic route is outlined in Fig. 1 in which
the major steps in the preparation of the spin-labeled primary amines are shown.
A typical procedure for the preparation of 4-(5-amino-pentaneamido)-2,2,6,6-tet-
ramethyl-piperidinyl-1-oxyl (TEMPO-C4; Fig. 1) is described below. Fluorenyl-
methyloxycarbonyl (FMOC) derivative of 5-amino pentanoic acid was synthesized
following the published protocol (14). In a typical procedure, 5-amino pentanoic
acid (4 mmol) and sodium carbonate (8 mmol) were taken in 8 ml of water and
the mixture was stirred with 7 ml of dioxane at 0ЊC. To this mixture, fluorenylmeth-
yloxycarbonylchloride (4 mmol) in 7 ml dioxane was added dropwise and stirred
for 1 h at 0ЊC and for 18 h at room temperature. The contents were poured into
100 ml of cold water and extracted with 3 ϫ 50 ml ether. The ether extract was
discarded and the aqueous phase was adjusted to pH 2–3 and extracted with 3 ϫ
50 ml ethylacetate. The organic layer was dried by rotary evaporation. The com-
pound was recrystallized from ethylacetate by adding hexane to get white solid (1.1
g, 81%). The progress of the synthesis was followed by IR (KBr) spectroscopy. The
synthesized compound showed the following characteristic IR spectral features:
1
3310(m) 2920(s) 2850(s) 1700(s) cmϪ .

PRIMARY AMINE SPIN LABELS AS STRUCTURAL PROBES
53
TABLE 1
Analytical Data for TEMPO Alkyamines
Elemental analysis
Calculated (%)
Found (%)
Sample
C
H
N
C
H
N
Rfa
TEMPO-C1
TEMPO-C2
TEMPO-C3
TEMPO-C4
TEMPO-C5
53.66
55.38
56.93
58.33
59.60
9.76
10.00
10.21
10.42
10.59
17.07
16.15
15.33
14.58
13.91
53.60
55.33
56.86
58.35
59.65
9.92
9.98
10.09
10.51
10.56
17.12
16.21
15.43
14.62
14.02
0.62
0.50
0.40
0.33
0.33
a Values measured on silica gel-60 plates using the solvent system : NH4OH (Conc.):
CH3OH : CHCl3 (1 : 10 : 26).
The spin label was introduced by reacting the FMOC aminopentanoic acid with
4-amino-TEMPO by the mixed anhydride method (15). FMOC aminopentanoic
acid (1 mmol) in 5 ml of tetrahydrofuran (THF) was kept cooled in an ice bath.
To this 1.1 mmol of triethylamine was added followed by 1 mmol of isopropylchloro-
formate in 5 ml THF and stirred at 0ЊC for 30 min. After stirring, 5 ml of 4-amino
TEMPO (1 mmol) in THF was added to the mixture, stirred for 1 h, and then
boiled under reflux for 20 min, rotary evaporated, and extracted with chloroform.
The chloroform layer was washed with 0.1
N
HCl until washings were acidic and
later washed with NaHCO3 solution to remove acid. The resulting solution was dried
over MgSO4 and then rotary evaporated. The residue was then chromatographed on
silica gel 60 and eluted with ethyl acetate. The brown band that eluted was collected
and rotary evaporated to get a brown solid (0.405 g, 82%). Rf ϭ 0.40 (ethylacetate).
1
IR (KBr) 3320(m) 3050(w), 2925(s), 2850(s), 1690(s), 1640(s), 1600(w)cmϪ
The FMOC protecting group was removed by stirring the solid in 2 ml each of
CH2Cl2 and morpholine for 6 h. The solution was rotary evaporated and the residue
was dissolved in CHCl3 and applied to a silica gel 60 column. The column was first
washed with chloroform and the bound product was eluted with CHCl3 : CH3OH
(90 : 10). Homogeneous fractions were combined and rotary evaporated to get a
brown–red semisolid (125 mg, 56%). Rf ϭ 0.33 (CHCl3 : CH30H : NH4OH
1
75 : 25 : 2.5). IR(KBr) 3400(m) 3280(m) 2920(s) 2850(s) 1650(s) 1530(m) cmϪ . The
spin-labeled amines were isolated as monohydrates. Anal. Calcd for C14H30N3O3:
C, 58.33; H, 10.42; N, 14.58. Found: C, 58.35; H, 10.51; N, 14.62. In addition, the
spin quantitation using ESR double integration technique revealed a spin-mass
ratio of 1-to-1 stoichiometry. Other spin-labeled primary amines were synthesized
and characterized similarly and their analytical data are summarized in Table 1.
Labeling of Plasma Fibronectin
Labeling of Fn with the spin-labeled primary amines was essentially the same as
the method of Wolff and Lai (6) modified from that of Mosher et al. (16) for
introducing fluorescenylcadaverine probes into Fn using thrombin-activated factor

54
NARASIMHAN, LAI, AND JOSEPH
XIII. Briefly, Fn(2 mg/ml) was incubated with the probe (0.5 m
M
) in the presence
of factor X111a at 37ЊC for 5 h. The labeled protein was purified from the reaction
mixture by gelatin–Sepharose affinity chromatography. Fn concentration was esti-
1
1
mated based on the extinction coefficient of 1.28 mg mlϪ cmϪ (17). SDS–PAGE
of the labeled protein indicated that thrombin, which was used during the labeling
procedure, did not degrade Fn.
Labeling of Melittin
Prior to labeling mellitin with the spin label, the commercially obtained peptide
was further purified by heparin–Sepharose affinity chromatography to remove phos-
pholipase A2 contamination (18). Melittin was subsequently labeled with the spin-
labeled primary amine TEMPO-C4, following the procedure of Perez-Paya et al.
(12). Melittin (1 mg/ml) was incubated with the probe (1.5 m
M
) in the presence of
dithiothreitol
guinea-pig liver transglutaminase (2 Ȑ ), 20 m CaCl2, and 20 m
M
M
M
at 37ЊC for 2 h. The labeled peptide was purified from the reaction mixture by
heparin–Sepharose affinity chromatography (18). Melittin concentration was esti-
1
mated based on the extinction coefficient of 5400 MϪ cmϪ1 (19). Phospholipase A2
contamination in melittin was checked using the rapid spin-label assay for the
enzyme developed by Lai et al. (20). The assay was performed before and after
purifying the peptide using the heparin–Sepharose affinity column.
ESR Measurements
ESR spectra were recorded with a Varian Century-Line 9-GHz spectrometer
equipped with a Varian variable temperature accessory and a digital thermometer
(Fluke 2100A). The field sweep was 100 G and the incident microwave power was
10 mW. The modulation frequency was 100 kHz and the modulation amplitude
was 1.0 G. All spectra were recorded at 22ЊC. The stoichiometry of probes incorpo-
rated into the protein was determined by double integration of the ESR spectra.
The effective rotational correlation times (␶2) were calculated from the experimental
ESR spectra using the motional narrowing formalisms (21–23). The linear and
quadratic terms for ␶ were calculated from the following equations:
2
␶2 ϭ 6.5 ϫ 10Ϫ10 ⌬H(0) [(h0/hϪ )1/2 Ϫ (h0/hϩ )1/2] (linear term)
[1]
[2]
1
1
␶2 ϭ 6.5 ϫ 10Ϫ10 ⌬H(0)[(h0/hϪ )1/2 ϩ (h0/hϩ )1/2 Ϫ 2] (quadratic term)
1
1
where ⌬H(0) is the peak-to-peak linewidth of the central field line (in gauss), and
hϪ , h0 and hϩ are the peak-to-peak amplitudes of the first derivative resonances
1
1
of the high-, central-, and low-field peaks, respectively.
RESULTS AND DISCUSSION
All of the newly synthesized spin-labeled primary amines in aqueous solution
exhibited ESR spectra consisting of three sharp and relatively symmetric lines
characteristic of a small nitroxide tumbling rapidly in solution. For example, the

PRIMARY AMINE SPIN LABELS AS STRUCTURAL PROBES
55
F
IG. 2. ESR X-band spectra of TEMPO-C4 spin label in various conditions. (A) The label alone (0.4
) in 20 m Tris, 150 m NaCl, pH 7.4. (B) The label bound to soluble Fn (6.7 Ȑ ). The labeling
was performed in the presence of coagulation factor XIIIa, as described under Material and Methods.
m
M
M
M
M
(C) The label bound to melittin (48.1 ȐM). Melittin was labeled in the presence of guinea-pig liver
transglutaminase (12).
spectrum of TEMPO-C4 in aqueous solution shown in Fig. 2A indicates that the
label undergoes an isotropic rotational diffusion with an effective rotational correla-
11
tion time of about 10Ϫ s and an isotropic hyperfine splitting constant of 16.5 G.
Fibronectin
When TEMPO-C4 was incubated with Fn in the presence of factor XIIIa, the
spectrum in Fig. 2A was converted into a partially immobilized spectrum as shown
in Fig. 2B. The results suggested that the label was attached to the protein and that
its motion (␶2 Ȃ 4.4 ϫ 10Ϫ10 s) was restricted compared to the free probe (Fig. 2A;
Table 2). No incorporation of the label was found in the absence of factor XIIIa.
In addition, the TEMPO-C4-labeled Fn did not react with fluoresceinylcadaverine
in the presence of factor XIIIa which has been shown to selectively label the
transglutaminase-reactive site located at the glutamine-3 residue of each subunit
(11). The data indicate that TEMPO-C4 was covalently incorporated onto the
amino-terminal region of Fn through transamination reaction with a stoichiometry
of 0.97-to-1 (probe-to-subunit, Table 2). A second transglutaminase-reactive site
has been suggested to be present near the carboxyl-terminal region of plasma Fn

56
NARASIMHAN, LAI, AND JOSEPH
TABLE 2
Stoichiometry of Labeling and Effective Rotational Correlation Times of Spin-Labeled Primary
Amines Bound to Fn
10
␶ (s) ϫ 10Ϫ
2
Sample
Stoichiometrya
Linear (A)
Quadratic (B)
B/A
TEMPO-C4
0.74 Ϯ 0.04
4.48 Ϯ 0.15
4.80 Ϯ 0.33
—
5.28 Ϯ 0.07
6.02 Ϯ 0.65
0.71 Ϯ 0.05
4.38 Ϯ 0.17
4.67 Ϯ 0.23
—
5.09 Ϯ 0.09
5.94 Ϯ 0.84
0.96
0.98
0.97
—
0.97
0.99
Fn ϩ TEMPO-C5
Fn ϩ TEMPO-C4
Fn ϩ TEMPO-C3
Fn ϩ TEMPO-C2
Fn ϩ TEMPO-C1
1.93 Ϯ 0.06b
1.97 Ϯ 0.11
—
0.70 Ϯ 0.05
0.57 Ϯ 0.03
a Spin labels per Fn molecule.
b The data are presented as mean Ϯ standard deviation.
(24), which may be located in the non-type-III homologous region between the
cell-binding and heparin-binding domains of the larger subunit (25). However, this
site in native Fn appears to react only with tissue transglutaminase but not plasma
transglutaminase (26), i.e., factor XIIIa.
Experiments using other spin-labeled primary amines showed that while TEMPO-
C5 is as effective as TEMPO-C4 for factor-XIIIa-mediated transamidation, other
spin-labeled primary amines with shorter methylene chains were less effective. The
results are summarized in Table 2. The TEMPO-C3 probe with three methylene
carbons was virtually unreactive while the shorter chain (TEMPO-C2 and TEMPO-
C1) probes were incorporated in significant amounts (Table 2). The reason for this
anamoly is not clear at present. Previously, in studies of structural requirements
for inhibitors of fibrin-stabilizing factor, namely, transglutaminase, Nilsson et al.,
prepared various dansyl derivatives of primary amines as potential inhibitors for
factor-XIIIa-mediated cross-linking of fibrin molecules (27). They found that the
dansyl derivative with a pentamethylene chain (dansylcadaverine) had an optimal
activity, whereas the dansyl derivative with a trimethylene chain had virtually no
activity at all. Although the mechanistic aspects of factor XIII activation by thrombin
including cleavage of the catalytic A subunits and dissociation of the A subunits
from the B subunits are well characterized, the topology of the active site in the
A subunits is not yet known (4). We speculate that the reactive sulfhydryl group
on the active center of the A subunits may be situated in a hydrophobic pocket at
˚
least 5 A in depth in order to accommodate a tetramethylene chain length.
Comparison of the rotational correlation times of the labels attached to Fn in
Table 2 shows that the labels undergo relatively fast tumbling motion. The TEMPO-
C1 probe attached to Fn undergoes somewhat slower motion compared to the other
probes that are attached to Fn. This reflects restricted rotation of the probe perhaps
because of the shorter methylene chain length (one methylene group). The motion
increases as the chain length increases. The ratios of the linear to quadratic terms
estimated from Eqs. 1 and 2 for these probes are very similar and close to unity

PRIMARY AMINE SPIN LABELS AS STRUCTURAL PROBES
57
which is indicative of isotropic motion (21–23), similar to that for the free probe
in solution (Table 2). Thus, the labels bound to the amino-terminal region of Fn
undergo a relatively fast isotropic rotational diffusion with an effective rotational
correlation time of 0.4–0.6 ns. This fast motion of the bound labels can be attributed
to long alkyl chains of the labels which are capable of exhibiting rapid segmental
flexibility.
Recently, it has been shown that the 70-kDa amino-terminal fragment of Fn
is involved in cellular matrix assembly of fibroblasts (28, 29). McKeown-Longo
and Mosher (28) postulated the existence of a matrix-assembly receptor that
recognizes the amino-terminal region of Fn. Although the exact nature of this
putative receptor has not been identified, gangliosides (30) and heparin-like
molecules (31) that are present in the cell surface have been suggested to be
potential candidates. The availability of selective labeling of the amino-terminal
region of Fn with spin-labeled primary amines reported here opens an opportunity
for studying dynamic interactions of Fn with the cell surface molecules using
ESR spectroscopy.
Melittin
Following a procedure similar to that used for Fn, melittin was labeled with the
spin-labeled primary amine, TEMPO-C4, except that guinea-pig liver transgluta-
minase was used instead of plasma transglutaminase. The resulting spectrum from
such a labeling is shown in Fig. 2C. The ESR spectral line shape of the bound
10
label indicates considerable motional freedom (␶ Ȃ 1 ϫ 10Ϫ s) compared to the
2
9
rotational dynamics of a 2800-D peptide (␶ Ȃ 1 ϫ 10Ϫ s). This opens up the
c
possibility of investigating the mechanism of interaction of melittin with red blood
cells and membranes and the dynamics of melittin aggregation in solution. The fact
that TEMPO-C4 can be enzymatically incorporated into melittin indicates the
potential use of this new class of spin labels to study the structure and dynamics
of other protein systems that contain transglutaminase sites.
Thus, the newly synthesized spin-labeled primary amines, with the exception of
the TEMPO-C3 probe, were incorporated enzymatically into the transglutaminase-
reactive site near the aminoterminal region of Fn. The TEMPO-C4 and TEMPO-
C5 probes showed very simlar reactivity while the other probes (TEMPO-C1,
TEMPO-C2, and TEMPO-C3) were less reactive. The TEMPO-C4 probe was also
incorporated into melittin (Gln25) in the presence of guinea-pig liver transglutami-
nase. The present method should be useful for the introduction of the spin-labeled
primary amines into other proteins with transglutaminase-reactive sites such as
fibrinogen (32), ͱ-casein (33), thrombospondin (34), von Willebrand factor (35),
and peptides such as substance P (36) and ͱ-endorphin (37). Use of such spin labels
would permit the investigation of the structural and dynamic properties of these
proteins and peptides using ESR spectroscopy.
ACKNOWLEDGMENTS
The authors gratefully acknowledge Drs. Jimmy Feix and Balaraman Kalyanaraman for critically
reading the manuscript and many helpful discussions.

58
NARASIMHAN, LAI, AND JOSEPH
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