A single-step procedure for the synthesis of photoreactiveand radioactive glycerolipids

Suman Lata, Kanchan Bhardwaj, and Ram Rajasekharan*

Lipid Laboratory, Department of Biochemistry, Indian Institute of Science, Bangalore 560012, India

Received 5 September 2002

Abstract

A single-step procedure was developed for the incorporation of iodoazide into oleic acid, triolein, and phosphatidylcholine.

Iodoazide was generated using [125I]iodomonochloride and sodium azide that was found to add stereospecifically in a variety of

olefins. Photoreactive and radiolabeled triacylglycerol and phosphatidylcholine were synthesized with a moderate yield and high

specific activity. The stability of both the radiolabel and the photoreactive group was studied under ultraviolet light under aqueous

as well as anhydrous conditions. These synthesized analogs act as substrates in the dark, and as irreversible inhibitors under ul-

traviolet irradiation for the target hydrolytic enzymes. The synthesized radiolabeled photoprobes were subsequently used to label

lipase and phospholipase A2. The results highlighted the efficiency and rapidity of the method and its potential application in the

study of lipid-metabolizing enzymes.

� 2003 Elsevier Science (USA). All rights reserved.

Keywords: Photolabeling; Protein crosslinking; Affinity labeling; Azidolipids; Nitrene; Triacylglycerol; Phosphatidylcholine

Photolabeling and crosslinking methods have been

developed to investigate the structural and functional

properties of biological molecules. Photoaffinity labeling

involves a ligand that carries a photolabile functional

group. The photochemical reagents are stable in the

absence of light and therefore can be targeted to sites

prior to activation, which accounts for their widespreaduse in many studies. Upon UV irradiation, they trans-

form into an active intermediate that is capable of

forming a covalent bond with a broad range of func-

tional groups present in the immediate target [1].

Photoprobes have been extensively used for the identi-

fication of substrate-binding sites in a number of soluble

enzymes [2,3] and membrane-bound enzymes [4] and in

the study of receptor–ligand interactions [5–7].Photoactivable groups like diazoketones, diazirines,

diazonium, and nitrophenyl azides have been used in

which azides have been found to be far superior for ef-

ficient labeling, presumably because of a longer life time

of the resulting nitrenes over carbenes. A variety of

substrate analogs bearing aliphatic and aromatic

azides as photoactivable groups have been synthesized

[1].

The strategy presented here is based on the genera-

tion of iodoazide in situ from iodine monochloride and

sodium azide [8]. The resulting iodoazide adds stereo-

specifically at a low temperature to form azidoalkyl

iodides [9]. The presence of an olefinic bond is a requisitefor this addition reaction to be carried out. By and large,

it is a single-step method that incorporates both the

photoactive and the radiolabeled groups. This method

has not been applied so far to any fatty acid-containing

lipid and its derivatives.

Materials and methods

Materials. Sodium azide, iodine monochloride,

sodium thiosulfate, sodium sulfate, Rhizopus arrhizus

lipase (Type XI), honeybee venom phospholipase A2, N ;N 0-dicyclohexylcarbodiimide (DCC),1 N ;N 0-dimethyl-

Analytical Biochemistry 313 (2003) 155–159

www.elsevier.com/locate/yabio

ANALYTICAL

BIOCHEMISTRY

* Corresponding author. Fax: +91-80-3602627.

E-mail address: lipid@biochem.iisc.ernet.in (R. Rajasekharan).

1 Abbreviations used:DCC, N ;N 0-dicyclohexylcarbodiimide; DMAP,

N ;N 0-dimethyl-4-aminopyridine; N3-iodo-PC, azidoiodophosphatidyl-

choline; N3-iodo-TAG, azidoiodotriacylglycerol; PC, phosphatidyl-

choline; TAG, triacylglycerol; SDS–PAGE, sodium dodecyl

sulfate–polyacrylamide gel electrophoresis.

0003-2697/03/$ - see front matter � 2003 Elsevier Science (USA). All rights reserved.

PII: S0003 -2697 (02 )00540-7

4-aminopyridine (DMAP), glycerophosphocholine,CdCl2 phosphatidylcholine (PC), and triacylglycerol

(TAG) were purchased from Sigma (St. Louis, MO). All

the solvents were distilled and dried prior to use. Pre-

coated silica-TLC plates (silica gel 60 F 254), CM-cel-

lulose (fine mesh), and DEAE-cellulose (fine mesh) were

from Merck. [9,10-3H(N)]triolein (10Ci/mmol), [2-pal-

mitoyl-9,10-3H]phosphatidylcholine (92.3Ci/mmol),

and [125I]NaI (17Ci/mg/0.1ml) were obtained fromPerkin–Elmer Biosystems. Ammonium persulfate and

electrophoretic reagents were purchased from Bio-Rad.

Enzyme assays. Enzyme activity was measured using

[9,10-3H]triolein as a substrate for lipase and [2-palmi-

toyl-9,10-3H]phosphatidylcholine as a substrate for

phospholipase A2. Substrate was prepared using mea-

sured amount of lipid ([3H]PC (250,000 dpm/assay) or

[3H]triolein (500,000 dpm/assay) in chloroform, driedunder a stream of nitrogen, and resuspended in 10mM

Tris–HCl (pH 7.5) buffer containing 1% Triton X-100.

The mixture was sonicated and used in the assay. The

assay buffer contained 0.01M Tris–HCl (pH 7.5), 0.1%

Triton X-100, 1mM substrate, and an appropriate

amount of enzyme in a final volume of 100 ll. The

mixture was incubated at 37 �C for 30min, and the re-

action was stopped by the addition of 50 ll of 6N HCl.Lipids were extracted according to the method of Bligh

and Dyer [10]. The organic phase was dried and the

lipids were resuspended in chloroform and resolved on

thin-layer silica gel plates using one of the following

solvent systems: chloroform:methanol:water (65:25:4, v/

v/v), chloroform:methanol:ammonia (65:30:4, v/v/v),

petroleum ether:diethyl ether:acetic acid (70:30:1, v/v/v).

The lipids were visualized either by autoradiography orwith iodine vapor. Spots corresponding to the unreacted

substrate and the products were scraped off and quan-

tified by liquid scintillation counting or gamma count-

ing. Control incubations were carried out for zero time

and in the absence of enzyme. The control value was

subtracted from the actual assay value and the specific

activity was calculated after the correction.

Synthesis of phosphatidylcholine. The synthesis wascarried out essentially as described [11]. The reaction

mixture consisting of 70mg oleic acid (0.25mmol) in

1.5ml of dry chloroform, 30mg DCC (0.15mmol), and

6mg DMAP (0.05mmol) was stirred for 2 h under an

atmosphere of argon. This was treated with glycer-

ophosphocholine:CdCl2:H2O (2:3:1) complex, which

was dried prior to the reaction by repeated evaporation

with dry benzene. Stirring was continued for 48 h atroom temperature in an argon atmosphere. The solvents

were then removed under reduced pressure, and the

residue was treated with 1ml of methanol:chloro-

form:water, 5:4:1 (v/v) and centrifuged. The clear su-

pernatant obtained was passed through a mixed resin of

CM:cellulose and DEAE:cellulose (1:1, w/w) column,

preequilibrated, and washed with chloroform. The col-

umn was then eluted with a mixture of chloro-form:methanol (1:1, v/v). After evaporation of the

solvent, the synthesized PC was purified by preparative

silica-TLC. Phosphatidylcholine was quantified by di-

gesting the organic phosphate with perchloric acid and

the liberated phosphate was determined colorimetrically

[12]. The yield was found to be 31mg (43%).

The synthesized compound was resuspended in

chloroform and the IR spectra were recorded on JASCOFT/IR-410 system. NMR spectra were recorded using a

JEOL JNM-LA 300 FT NMR system in CDCl3.

Synthesis of N3-iodo-fatty acid. Azidoiodo stearic acid

was synthesized as follows: sodium azide (60mg;

0.92mmol) was dissolved in 0.19ml of dry acetonitrile in

a methanol ice-cold bath with an equal volume of 2M

iodine monochloride in acetonitrile for a period of

15min. The reaction mixture was stirred for an addi-tional 15min. Oleic acid was added to the above mixture

for a final concentration of 0.35mmol. The reaction

mixture was allowed to reach room temperature and

stirring continued for an additional 72 h. Water (0.4ml)

was added to the red-brown slurry, and the mixture was

extracted three times with 1.5ml diethyl ether. The ether

extracts were combined, washed with 0.5ml of 5% so-

dium thiosulfate, and dried over anhydrous sodium sul-fate. The residue was chromatographed on a preparative

silica-TLC to give the pure product with the yield of

89mg. The spectral characteristics were IR(neat), a sharp

band at 2100 cm�1 (N3); NMR(CDCl3), d 3.03 (m, 1H); d4.11 (m, 1H).

Synthesis of N3-iodo-TAG. Sodium azide (0.54mmol)

was added to 0.125ml of a 2M solution of iodine

monochloride in acetonitrile on a methanol ice-coldbath. Dry acetonitrile (0.125ml) was added and the re-

action mixture was stirred for 15min. To this, triolein

(50mg; 0.056mmol) was added later, followed by stir-

ring for 72 h at 4 �C. After the reaction, 0.25ml of water

was added to the reaction mixture and extracted with

3ml of diethyl ether. The resulting solution was washed

with 1ml of 5% sodium thiosulfate and dried over an-

hydrous sodium sulfate. Solvent was removed undervacuum and the residue chromatographed on a pre-

parative TLC using petroleum ether:diethyl ether:acetic

acid, (70:30:1, v/v/v) as a solvent system. The spot ob-

served on the TLC was found to have a Rf value of 0.77

and the Rf value of triolein was around 0.90 in this

solvent system. The spot with the Rf of 0.77 was scraped

from preparative TLC and extracted with chloro-

form:methanol (1:1, v/v) and identified as azidoiodo-TAG. Spectral characteristics: IR(neat), a sharp band at

2100 cm�1 (N3); NMR(CDCl3), d 3.01 (m, 3H); d 4.02

(m, 3H); MALDI-TOF, two strong peaks at 1380 (the-

oretical, 1389) and 1254 (theoretical, 1263 after loss of

one 127I) were observed along with other peaks that re-

sult from the fragmentation of acyl chains. The overall

yield of azidoiodo-TAG was 38%.

156 S. Lata et al. / Analytical Biochemistry 313 (2003) 155–159

Synthesis of N3-iodo-PC. Azidoiodophosphatidylch-oline was synthesized by dissolving 12mg (0.184mmol)

sodium azide in 40 ll of dry acetonitrile in a methanol

ice-cold bath and an equal volume of 2M iodine

monochloride in acetonitrile was added over a period of

15min. The reaction mixture was stirred for additional

15min and then 20mg (0.025mmol) of PC was added,

allowed to attain room temperature, and stirred for an

additional 48 h. Water (0.5ml) was added to the red-brown slurry and the mixture was extracted with 4ml of

ether and washed with 0.5ml of 5% sodium thiosulfate.

The resulting clear solution was then dried over anhy-

drous sodium sulfate. For further purification, the vol-

ume was reduced to 1ml and purified by preparative

silica-TLC. Approximately, 8mg (30%) of azi-

doiodophosphatidylcholine was obtained. IR(neat), a

sharp band at 2100 cm�1 (N3) was obtained.Synthesis of 125I-labeled compounds. Iodine mono-

chloride (0.125ml of 2M solution) was added to Na125I

(0.16 lmol). Sodium iodide was neutralized and dried at

100 �C under reduced pressure for 30min prior to use.

The reaction mixture was stirred for 1 h at room tem-

perature to exchange the 127I of ICl with 125I of NaI. The

resulting 125I-labeled ICl was then treated with sodium

azide using the protocols described earlier for the syn-thesis of [125I]N3-lipids.

Photodecomposition reaction. The photodecomposi-

tion reaction was carried out in the cap of a microfuge

tube. [125I]N3-lipid in chloroform or in 10mM Tris–HCl

(pH 7.5) was taken and irradiated for various time in-

tervals with a hand-held UV lamp with the filter re-

moved (5000 lW/cm2, Model UVG-54, UV Products) at

a 7-cm distance. After each exposure, the caps were keptunder reduced pressure for 7min and the radioactivity

was measured in a gamma counter. The contents of each

cap were then spotted on a TLC plate and developed

with a neutral lipid solvent system. The starting material

was scraped off from the TLC plate and radioactivity

was measured in a gamma counter. After correcting for

the loss of label, the half-life for the decomposition of

N3 was calculated.Photoaffinity labeling. The photolabeling experiments

were carried out in a final volume of 50 ll containing 7–

8 lg of lipase and [125I]N3-TAG or phospholipase A2

(6 lg) and [125I]N3-PC in 10mM Tris–HCl (pH 7.5),

0.1mM 2-mercaptoethanol, and 0.5 lM photoprobe

(0.5 lCi). The mixture was preincubated on ice in the

dark for 5min, in a microfuge tube cap, and irradiated

for 3min with a hand-held UV lamp with the filter re-moved (5000 lW/cm2, Model UVG-54, UV products) at

a distance of 7 cm [13]. As a control, the photoprobe was

first exposed to UV light and then added to the enzyme.

No labeling was observed in the absence of UV light.

Protein was precipitated with 10% trichloroacetic acid

on ice for 15min and centrifuged in a microfuge for

15min. The pellet was washed with ice-cold acetone,

solubilized with loading buffer, and analyzed by 12%SDS–PAGE at 100V using a discontinuous buffer sys-

tem and the gel was stained with Coomassie blue R-250

[14]. The gel was dried followed by autoradiography.

Protein concentration was determined by the bicinch-

oninic acid method [15] using bovine serum albumin as

the standard.

Results and discussion

The synthesis of azido alkyliodides was based on the

addition of pseudohalogen (IN3) at an unsaturated

center [8,9]. The reaction scheme for the synthesis of

iodoazidotristearin is presented in Fig. 1. In order to

show the applicability of this method, photoactive ra-

diolabeled fatty acid was synthesized and the productwas characterized by NMR and FT-IR. The synthesized

analogs (azidoiodo-fatty acid, azidoiodo-TAG, and az-

idoiodo-PC) were analyzed by FT-IR spectroscopy. A

sharp band at 2100 cm�1, characteristic of an azido

group was observed in the spectra of fatty acid, PC, and

TAG analogs. This band was absent in the spectrum of

natural oleic acid, PC, and triolein. Additionally, the

band at 1640 cm�1, due to C@C stretching, was notobserved in the spectrum of the analogs, indicating the

conversion of double bond to a single bond by the ad-

dition reaction. Structures of the analogs were also

confirmed by NMR where disappearance of a multiplet

at d 5.34 corresponding to olefinic protons and ap-

pearance of two new peaks for the HACAI multiplet at

d 3.0 and the HACAN3 multiplet at d 4.11 were ob-

served in the spectra of the analogs.Stability of the radioactive and the photoactive groups

of the analogs. Stability of the radiolabel and the azide in

the analog is crucial for subsequent applications and its

storage. One of the synthesized analogs, (125I)iodoazido-

stearoyl-sn-glycerol was chosen to examine the stability

of these groups under anhydrous as well as aqueous

conditions. The analog was irradiated with UV light for

different time intervals under anhydrous or aqueousconditions. Aliquots of the irradiated samples were ei-

ther resolved on a silica-TLC to find the stability of the

azido group or counted in a gamma counter directly to

Fig. 1. Reaction scheme for the synthesis of iodoazido-stearoyl-sn-

glycerol.

S. Lata et al. / Analytical Biochemistry 313 (2003) 155–159 157

find the stability of the 125Iodo group. Autoradiographs

of TLC profiles of the samples irradiated for various

time points under anhydrous and aqueous conditions

were performed. The change in Rf value of the radio-

active spot was taken as an indication of the photolysis

of the azido group, which was quantified by scraping the

spots from the TLC and counting them using the gam-

ma counter. Half-lives (t0:5) for 125I and N3 were calcu-lated based on the first-order rate equation, under

anhydrous or aqueous conditions (Table 1). The radio-

label was sufficiently stable under aqueous as well as

anhydrous conditions, at the same time the azido group

was found to be equally stable under anhydrous condi-

tions. However, it readily decomposed in an aqueous

buffer (t0:5 ¼ 2:75min). Stability studies showed that the

analog is ideal for use since it is substantially stableunder anhydrous conditions and therefore can be easily

stored for a long time. The photolabel is highly sensitive

to light, resulting in photolysis under aqueous condi-

tions where the experiments were performed. However,

similar half-lives for 125I and N3 were observed for the

PC analog (data not shown).

Interaction of photoreactive TAG and PC analogs with

lipolytic enzymes. Commercially available lipase andphospholipase A2 were used as the model systems to

evaluate the effectiveness of the synthesized photoactive

reagents. Lipase and phospholipase A2 were incubated

with [125I]iodoazido-TAG and [125I]iodoazido-PC, re-

spectively, in the dark under standard assay conditions.

The reaction mixture was analyzed by TLC followed by

autoradiography, which showed a product that migrates

close to the fatty acid. These results suggested that theTAG and PC analogs act as substrates for lipase and

phospholipase A2, respectively.

The ability of azidoiodo lipids to inhibit hydrolytic

enzyme activities by cross-linking upon irradiation was

studied under different conditions (Table 2). The enzyme

activity was slightly affected by UV irradiation in the

absence of photoprobe. Lipase was photolyzed for 3min

in the presence of 50 lM azidoiodo-TAG and assayedunder standard assay conditions without removing the

photoprobe. The enzyme activity was lost up to 66%

after photolysis as compared to the unphotolyzed sam-

ples. A similar observation was made for azidoiodo-PC

with phospholipase A2. The hydrolytic activity was

protected from inhibition when a 10-fold excess of

natural substrate was included during photolysis. These

results suggested that the synthesized substrate analogs

and natural substrates interacted with the enzymes at

the same site.

Photoaffinity labeling with the synthesized analogs.

To find out the specificity of interaction with enzymes,

the synthesized [125I]iodoazido-stearoyl-sn-glycerol was

preincubated in cold with commercially availablepurified 43 kDa lipase from R. arrhizus and the [125I]io-

doazido-stearoyl-phosphatidylcholine with the com-

mercially available 20 kDa phospholipase A2 from bee

venom. Cross-linking was carried out under UV light

and protein was resolved on 12% SDS–polyacrylamide

gel electrophoresis. Photoaffinity labeling showed that

the TAG analog interacted with lipase (Fig. 2A) and

similarly the phosphatidylcholine analog interacted withphospholipase A2 (Fig. 2B). However, no labeling was

observed either in the dark or in the absence of protein.

The specificity of labeling was demonstrated by the

protection from covalent incorporation of the labeled

photoprobe with the enzyme by natural substrates.

Photolabeling of lipase with TAG analog and phos-

Table 1

Rate constants and half-lives for iodo and azido groups incorporated into lipids

Anhydrous Aqueous (pH 7.5)

K (s�1) t0:5 (min) K (s�1) t0:5 (min)

Azide 2:4� 10�4 48.00 4:27� 10�3 2.75

Iodine 1:55� 10�4 74.00 2:00� 10�4 58.00

Decomposition of azido group in [125I]iodo-N3-TAG upon exposure to UV light under anhydrous or aqueous conditions. The synthesized TAG

analog aliquots were exposed to UV light for various time points either in chloroform or in Tris–HCl (pH 7.5).

Table 2

Photoinactivation of lipolytic enzymes by azidoiodo-TAG and

azidoiodo-PC

Concentration (mM) Irradiation Activity (%)

Triolein N3-Triolein

1 0 + 100� 6:58

0 0.05 ) 92� 8:33

0 0.05 + 34� 2:33

0.5 0.05 + 81� 4:12

1.0 0.05 + 87� 5:79

PC N3-PC

1 0 + 100� 8:24

0 0.05 ) 87� 6:45

0 0.05 + 37� 2:91

0.5 0.05 + 88� 5:23

1.0 0.05 + 86� 7:65

Lipase (7lg protein) and phospholipase A2 (8lg protein) were

photolyzed for 3min in a total volume of 50ll containing 50lM of

N3-TAG and N3-PC, respectively. The photolyzed mixture was as-

sayed under standard assay conditions for respective enzyme activities.

The presence or absence of UV irradiation is indicated by + or ),respectively. One hundred percent lipase activity represents 658lmol

of fatty acid released/min/mg protein and 100% phospholipase A2

activity represents 423lmol of fatty acid released/min/mg protein.

Values are mean percentage SD of three determinations.

158 S. Lata et al. / Analytical Biochemistry 313 (2003) 155–159

pholipase A2 with PC analog was protected in a con-centration-dependent manner (data not shown). The

half-maximal protection of photolabeling of lipase and

phospholipase A2 was achieved at approximately 0.5

and 0.25mM, respectively. These results suggested that

the photoprobe interacted at the substrate-binding site

of the lipolytic enzymes.

Various methods have previously been introduced for

incorporation of 125I and N3. All these methods in-volved more than a single step and required special

functional group such as OH group on the acyl chain

[2,11,13]. Most of the methodologies involved aqueous

conditions for incorporation of N3, where the possibility

of hydrolysis of the acyl chains could not be ruled out

completely. Our results indicated that the protocol in-

volved in synthesis of azido analogs overcame all the

above-noted problems, and demonstrated the efficacy oflabeling protein in an aqueous system.

Acknowledgments

This research was supported by Department of Bio-

technology, New Delhi, India. We thank Dr. Malathi

Srinivasan for critical reading of the manuscript.

References

[1] S.A. Fleming, Chemical reagents in photoaffinity labeling, Tetra-

hedron Lett. 46 (1995) 12479–12520.

[2] P. Chakrabarthi, H.G. Khorana, A new approach to the study of

phospholipid-protein interactions in biological membranes. Syn-

thesis of fatty acids and phospholipids containing photosensitive

groups, Biochemistry 14 (1975) 5021–5033.

[3] C.M. Tran, G. Scheiner-Bobis, W. Schoner, R.A. Farley, Iden-

tification of an amino acid in the ATP binding site of Na+/

K(+)-ATPase after photochemical labeling with 8-azido-ATP,

Biochemistry 33 (1994) 4140–4147.

[4] R. Rajasekharan, V. Nachiappan, H.S. Roychowdhury, Photola-

beling of soybean membrane proteins with photoreactive acyl-

CoA analogs, Eur. J. Biochem. 220 (1994) 1013–1018.

[5] C. Shigeno, Y. Hiraki, D.P. Westerberg, J.T. Potts, G.V.

Segre, Photoaffinity labeling of parathyroid hormone receptors

in clonal rat osteosarcoma cells, J. Biol. Chem. 263 (1988)

3864–3871.

[6] S.K. Nordeen, N.C. Lan, M.O. Showers, J.D. Baxter, Photo-

affinity labeling of glucocorticoid receptors, J. Biol. Chem. 256

(1981) 10503–10508.

[7] J. Brunner, New photolabeling and crosslinking methods, Annu.

Rev. Biochem. 62 (1993) 483–514.

[8] A. Hassner, M. Lorber, C.H. Heathcock, Addition of iodine

isocyanate to olefins. Scope and synthetic utility, J. Org. Chem. 32

(1967) 540–549.

[9] A. Hassner, G.J. Matthews, F.W. Fowler, The reduction of beta-

iodo azides. A stereospecific synthesis of aziridines, J. Am. Chem.

Soc. 91 (1969) 5046–5054.

[10] E.G. Bligh, W.J. Dyer, A rapid method of total lipid extraction

and purification, Can. J. Biochem. Physiol. 37 (1959) 911–917.

[11] C.M. Gupta, R. Radhakrishnan, H.G. Khorana, Glycero-

phospholipid synthesis: Improved general method and new

analogs containing photoactivable groups, Proc. Natl. Acad.

Sci. USA 74 (1977) 4315–4319.

[12] G.R. Bartlett, Phosphorus assay in column chromatography, J.

Biol. Chem. 234 (1959) 466–468.

[13] R. Rajasekharan, R.C. Marians, J.M. Shockey, J.D. Kemp,

Photoaffinity labeling of acyl-CoA oxidase with 12-azidooleoyl-

CoA and 12-[(4-azidosalicyl)amino]-dodecanoyl-CoA, Biochemis-

try 32 (1993) 12386–12391.

[14] U.K. Laemmli, Cleavage of structural proteins during the

assembly of the head of bacteriophage T4, Nature 227 (1970)

680–685.

[15] P.K. Smith, R.I. Krohn, G.T. Hermanson, A.K. Mallia, F.H.

Gartner, M.D. Provenzano, E.K. Fujimoto, N.M. Goeke, B.J.

Olsen, D.C. Klenk, Measurement of protein using bicinchoninic

acid, Anal. Biochem. 150 (1985) 76–85.

Fig. 2. Photolabeling of lipase with the synthesized [125I]iodo-N3-TAG (A). Photolabeling of honeybee venom phospholipase A2 with the synthesized

[125I]iodo-N3-phosphatidylcholine (B). Protein was preincubated in the dark, on ice, with the analog and then cross-linked by exposure to UV light.

Cross-linked samples were TCA-precipitated and then resolved on 12% SDS–polyacrylamide gel and stained with Coomassie brilliant blue (left

panel) and the same gel was used for autoradiography (right panel). Lane 1, protein cross-linked with the analog after preincubating together. Lane 2,

analog was photolyzed prior to addition of protein.

S. Lata et al. / Analytical Biochemistry 313 (2003) 155–159 159