Fluorescence resonance energy transfer from tryptophan to a chromium(III)complex accompanied by non-specific cleavage of albumin:a step forward towards the development of a novel
photoprotease
H.Yamini Shrivastava,Balachandran Unni Nair
*
Chemical Laboratory,Central Leather Research Institute,Adyar,Chennai 600020,India Received 13November 2003;received in revised form 10February 2004;accepted 18February 2004
Available online 19March 2004
Abstract
A chromium(III)complex,transdiaqua [N ,N 0-propylenebis(salicylideneimino)chromium(III)]perchlorate ([Cr(salprn)(H 2O)2]ClO 4)in the presence of sodium azide and upon photoexcitation was found to bring about non-selective cleavage of bovine serum albumin (BSA).Electron paramagnetic resonance (EPR)evidence has been obtained for the formation of a Cr(V)species upon photolysis of a solution cont
aining the chromium(III)complex and sodium azide.This Cr(V)species non-selectively cleaves BSA.The fluorescence excitation spectrum of BSA-[Cr(salprn)(H 2O)2]þadduct showed a band at k max ex ¼370nm due to charge transfer transition of the chromium(III)complex as well as a prominent band at 290nm attributable to tryptophan absorption.This in-dicated an efficient Forster type fluorescence energy transfer (FRET)from the tryptophan residues to the chromium(III)complex indicating that the Cr(III)complex binds in the vicinity of the tryptophan residue.Ó2004Elsevier Inc.All rights reserved.
Keywords:Chromium(III)complex;BSA;Tryptophan absorption;Forster type fluorescence energy transfer
1.Introduction
Endo-proteolytic cleavage is a subject of current in-terest with diverse applications in biochemistry [1–3].Proliferation of chemical nucleases in DNA-cleavage studies is an example of how reagents based on small molecules can be of use in molecular biology [4].Prote-ases are useful biochemical tools in correlating protein structure with activity,in the design of new therapeutic agents and for converting large proteins into smaller fragments,which are amenable for sequencing [5].The reagents that effect the degradation of proteins usually are proteolytic enzymes.However,the application of these enzymes are limited due to the fact that their activity
and hence usefulness is restricted over a narrow pH range [6].In this respect,protein cleaving agents based on transition metal ions have several advantages.Metallo-proteases can bind to the side chains of amino acid like methionine,histidine,tryptophan,aspartic and gluta-mine acid and can cleave the peptide bond either hydro-lytically,photochemically or through the involvement of reactive oxygen species [7–9].Eventhough site-specific cleavage of proteins has attracted much attention [10–14];non-specific cleavage of proteins has not received much of attention.Site-specific cleavage of protein always gives rise to protein fragments of larger molecular weight [15];whereas non-specific cleavage has the potential of pro-ducing much smaller protein fragments which are difficult to visualize through electrophoretic studies.Many pro-teins contain large number of side chain carboxylic groups due to the presence of aspartic and glutamic acid residues in their primary structure.Chromium(III)is known to have greater affinity for carboxyl group
*
Corresponding author.Tel.:+91-044-4430273/2441-1630;fax:+91-44-2491-1589.
E-mail addresses:bcun@rediffmail,nairbu@rediffmail (B.U.Nair).
0162-0134/$-see front matter Ó2004Elsevier Inc.All rights reserved.
doi:10.1016/j.jinorgbio.2004.02.014
Journal of Inorganic Biochemistry 98(2004)991–994
www.elsevier/locate/jinorgbio
JOURNAL OF
Inorganic
Biochemistry
compared to other side chain groups present in protein [16].As a result,chromium(III)based metalloproteases are expected to bind to large number of sites in protein non-selectively and an activation will lead to non-specific cleavage of protein.This communication describes the non-specific photocleavage of bovine serum albumin (BSA)in the presence of a chromium(III)complex.
2.Materials and methods
2.1.Materials
Bovine serum albumin(fraction V powder)was sourced from Sigma and was used as such without any further purification.The chromium(III)complex, transdiaqua[N,N0-propylenebis(salicylideneimino)chro-mium(III)]perchlorate,([Cr(salprn)(H2O)2]ClO4)was prepared as described previously[17].
2.2.Spectroscopic measurements
All absorption spectra were measured using Shima-dzu160A-UV–Visible double beam spectrophotometer in1Â10À3M Tris–HCl buffer.The binding constant was obtained on the basis of the variation in the ab-sorbance of[Cr(salprn)(H2O)2]þ(3.3Â10À5M)at366 nm(characteristic k max of the metal complex)with varying concentration of BSA(4.6Â10À6–5Â10À5M). The concentration of BSA solution was varied until saturation point in the spectra was obtained.From the binding plot derived from the absorption data,the binding constant was determined according to the fol-lowing equation.
ð½BSA =D e apÞ¼ð½BSA =D eÞþð1=D e KÞ;
where D e ap¼ðe aÀe fÞ;D e¼ðe bÀe fÞ,e a is the absor-bance at366nm/[metal complex],e b is the e
xtinction coefficient of the metal complex bound to protein,e f is the extinction coefficient of the free metal complex and K is the binding constant.
2.3.Fluorescence measurements
Fluorescence spectra were measured using Hitachi-model-650-40Spectrofluorimeter.Samples were excited at280nm and the emission was monitored at345nm (characteristic of tryptophan residue).BSA(1Â10À4M) was incubated with various concentration of[Cr(sal-prn)(H2O)2]þ(2Â10À5–2Â10À4M)The emission spec-tra were monitored for varying concentration of metal complexes at25°C in the presence of Tris–HCl buffer (pH7.0,I¼5Â10À3M).The quenching constant,K q was calculated from the plot of F o=F vs.[chromium(III)] (F o and F are thefluorescence intensity of BSA in the absence and presence of the metal complex).The exci-tation spectrum of[Cr(salprn)(H2O)2]þ(1Â10À5M)was
recorded in the presence of BSA(1Â10À4M)by keeping
the excitation and emission slit widths at5nm.
2.4.Electron paramagnetic resonance measurements
An aqueous solution of[Cr(salprn)(H2O)2]þ
(5Â10À5M)was incubated with sodium azide(5Â10À5
M)for0.5h and photolysed in a quartz cuvette using
Applied Photophysics photolytic setup at$290nm for
15min.The epr spectrum of the photolysed solution was
recorded on a Bruker EMX6/1spectrometer equipped
with an ER4103TM cavity.
2.5.Determination of metalloprotease activity of[Cr(sal-prn)(H2O)2]þby SDS–PAGE
BSA(1Â10À5M)was incubated with[Cr(sal-
prn)(H2O)2]þ(1Â10À4M)and sodium azide(1Â10À4
M)overnight and photoexcited($290nm)in an Ap-
plied Photophysics photolytic set up Model406/61,
consisting of water cooled xenon lamp for30,40and60
min.The progress in protein modification was moni-
tored by10%SDS–PAGE.The gel was stained with
Coomassie blue.
3.Results and discussion
Chromium(III)complex,[Cr(salprn)(H2O)2]þis a
crystographically and spectroscopically well character-
ized compound[17].This chromium(III)complex is
known to show emission at497nm from its charge
transfer excited state[17].Unlike,normal chro-
mium(III)complexes,salen type Schiffbase compounds
of Cr(III)undergo aqua-ligand substitution with relative
ease[18].Binding of[Cr(salprn)(H2O)2]þcomplex with
BSA has been investigated through spectroscopic titra-
tion and a binding constant of(7.72Æ0.25)Â105MÀ1
has been estimated from the binding isotherm.The
standard Gibbs free energy change for the binding of
[Cr(salprn)(H2O)2]þto BSA has been estimated to be )33.58kJ/mol at25°C,which indicates the spontaneity of the binding of chromium(III)complex to BSA.Be-
sides binding to the side chain carboxyl group,this
compound because of the presence of the hydrophobic
ligand around the metal ion is also expected to interact
with tryptophan residues in the protein.Such an inter-
action has been confirmed throughfluorescence
quenching studies.BSA on excitation at280nm showsreactive materials studies
emission at345nm due to the presence of two trypto-
phans in its amino acid sequence[19].Fig.1shows the
quenching of the tryptophanfluorescence on subsequent
addition of increasing concentration of[Cr(sal-
prn)(H2O)2]þ.The quenching has been found to follow
Stern–Volmer equation(K q¼1:9Â104MÀ1).Such a
992H.Y.Shrivastava,B.U.Nair/Journal of Inorganic Biochemistry98(2004)991–994
behavior indicates the interaction of the chromium(III)complex with the hydrophobic pockets in the prot
ein structure (harboring the tryptophan residues),thus leading to the energy transfer quenching of tryptophan fluorescence.The fluorescence excitation spectrum of BSA-[Cr(salprn)(H 2O)2]þadduct (Fig.2)not only shows a band at k max ex ¼370nm due to charge transfer transition of the chromium(III)complex but also a prominent band at 290nm.Appearance of the band due to tryptophan absorbance as well as charge transfer transition of the chromium(III)complex in the excita-tion spectrum of the Cr(III)complex-BSA adduct is indicative of efficient Forster type fluorescence energy
transfer (FRET)from the tryptophan residues to the chromium(III)complex.
The modifications brought about in the structural integrity and electrophoretic mobility of BSA upon binding to [Cr(salprn)(H 2O)2]þwas monitored by SDS–PAGE.Binding of this chromium(III)complex did not lead to any cleavage of protein (Fig.3,lane 2).Photo-excitation of the chromium(III)complex incubated with BSA also did not show any evidence of protein cleavage as the bands were as intact as the control BSA band (Fig.3,lane 4).When BSA incubated with [Cr(sal-prn)(H 2O)2]þand equivalent amount of sodium azide was photolysed (k ex ¼290nm),the evidence for protein cleavage was apparent from the diffused protein staining in the gel.The gel electrophoretogram (Fig.3,lanes 5–7)doesn Õt show any distinct protein band except the parent band with much reduced intensity.The lanes only showed a smeared pattern,indicating that the protein is cleaved at multiple sites,non-specifi
cally into smaller peptide fragments,which are not retained in a 10%gel.This type of protein modification is a clear indication of the non-selective cleavage of protein by the chromium species.The variation in the time period of photolysis from 30–60min was found to have only a marginal difference in affecting the extent of cleavage (lanes 5–7).The analogous Cr(III)complex,[Cr(salen)(H 2O)2]þin the presence of sodium azide gives rise to [Cr(sa-len)(N 3)(H 2O)].This azido complex of chromium(III)on photoexcitation has been known to give rise to chromium(V)nitrido complex [20].Hence,it is possible that in the case of [Cr(salprn)(H 2O)2]þalso photolysis in the presence of azide leads to the formation of chro-mium(V)azido complex.Room temperature EPR spec-trum of the solution obtained after photolysing [Cr(salprn)(H 2O)2]þin the presence of sodium azide shows a signal at g ¼1:97(Fig.4),which confirms the formation of chromium(V)species.
Chromium(V)
Fig.2.Excitation spectrum of [Cr(salprn)(H 2O)2]þ(1Â10À5M)in the presence of BSA (1Â10À4M)(excitation and emission slit widths were 5nm).Emission wavelength ¼497
nm.
Fig.3.SDS–PAGE of 1Â10À5M BSA in 1Â10À3M Tris–HCl buffer with [Cr(salprn)(H 2O)2]þunder differ
ent experimental conditions (lane 1¼control BSA,lane 2¼BSA +1Â10À4M [Cr(salprn)(H 2O)2]þ,lane 3¼BSA +1Â10À4M sodium azide,lane 4¼photoexcited sample of BSA +1Â10À4M [Cr(salprn)(H 2O)2]þ,lanes 5–7¼photoexcited BSA +1Â10À4M [Cr(salprn)(H 2O)2]þ+1Â10À4M sodium azide at different time intervals of 30,40and 60min,respectively;lane 8¼protein markers).The gel has been stained with Coomassie
blue.
Fig.1.Fluorescence emission spectra of 1Â10À4M BSA (0)and BSA in the presence of 2Â10À5M (1);4Â10À5M (2);6Â10À5M (3);7Â10À5M (4);8Â10À5M (5);1Â10À4M (6);2Â10À4M (7)[Cr(salprn)(H 2O)2]þ(k ex ¼280nm,E max ¼345nm,excitation and emission slit widths were 5nm).
H.Y.Shrivastava,B.U.Nair /Journal of Inorganic Biochemistry 98(2004)991–994993
species has previously been shown to cleave alpha 1-acid glycoproteins [21].Hence,it is clear that the chro-mium(V)nitrido species oxidatively cleaves the protein.Since,BSA has a large number of carboxyl side chains and two tryptophan residues,binding of [Cr(sal-prn)(N 3)(H 2O)]at these sites leads to non-specific cleavage of BSA.In conclusion fluorescence resonance energy transfer from tryptophan to [Cr(salprn)(H 2O)2]þin the presence of azide ion can be exploited for non-selective cleavage of BSA.
4.Abbreviations BSA Bovine serum albumin
FRET Fluorescence resonance energy transfer EPR
Electron paramagnetic resonance
Salprn N ,N 0-propylenebis(salicylideneimine)Salen
N ,N 0-ethylenebis(salicylideneimine)
SDS–PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis
Tris–HCl
Tris(hydroxymethyl)aminomethane hy-drochloride
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Fig.4.EPR spectrum of an aqueous solution containing 1Â10À4M [Cr(salprn)(H 2O)2]þand 1Â10À4M sodium azide after photolysing at $290nm for 30min.Microwave frequency ¼9.81GHz,power ¼5.06mW,modulation frequency ¼100kHz,modulation amplitude ¼1.5G.
994H.Y.Shrivastava,B.U.Nair /Journal of Inorganic Biochemistry 98(2004)991–994

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