INTERACTIONS OF PHOTOSENSITIZED TETRACYCLINE WITH SERUM ALBUMIN. Mateen A. Khan 1, Salman Muzammil 1 and Javed Musarrat 2 *

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1 Vl. 46, N. 5, December 1998 BCHEMSTRY and MLECULAR BLGY NTERNATNAL Pages NTERACTNS F PHTSENSTZED TETRACYCLNE WTH SERUM ALBUMN Mateen A. Khan 1, Salman Muzammil 1 and Javed Musarrat 2 * 1 nterdisciplinary Bitechnlgy Unit and 2 Department f Micrbilgy, nstitute f Agriculture, Aligarh Muslim University, Aligarh 222, ndia Received July 1, 1998 Received after revisin, August 18, 1998 Summary. nteractins f tetracycline with bvine serum albumin (BSA) were studied by flurescence quenching and circular dichrism (CD) analysis. The binding istherm exhibited at least 13 tetracycline binding sites n the albumin mlecule, Amngst these, fur were fund t be high affinity sites and the remainder were lse sites. The Scatchard analysis demnstrated the binding cnstant and capacity f BSA t be 4.6 x 16 liters/mle and 3.6, respectively. The CD data revealed a significant decrease in the mean residue ellipticity (MRE), indicating alteratins in the prtein helicity. A reductin f 2% in the Gt - helical cntent f the albumin was nted at higher levels f tetracycline in the presence f Cu () ins. Thus the strng in vitr interactins f tetracycline with albumin resulted in cnfrmatinal changes in its glbular structure and insinuate ptential health risk due t pssible macrmlecular damage, under physilgical cnditins, frm the frmatin f tetracycline/cu() cmplexes. Key wrds: Tetracycline, Serum albumin, Flurescence quenching, Circular dichrism NTRDUCTN Tetracyclines (TC) are the brad spectrum antibitics, cmmnly used in the treatment f varius bacterial infectins and acne. n additin t their pharmaclgical actin spectra, tetracyclines are knwn t exhibit phttxicity causing skin lesins, nchlysis and papular eruptins (1-3). Als the accumulatin f tetracycline in the bld due t its impaired renal clearance induces hepattxicity (4,5). ndeed, after its slubilizatin and absrptin in the gut, the tetracycline is transprted bund t the serum prteins. As estimated, apprximately 36-5% f tetracycline remains bund t plasma prteins after its absrptin frm the stmach and the small intestinal cells. n situ bth the free and albumin bund tetracycline mlecules are able t interact with Cu () ins. At neutral ph, the Cu () ins react with the xygen atms bund at C3 and amide grups and/r at C and C11 psitins f the tetracycline (6). Free tetracycline in the presence f Cu () ins prduced reactive xygen species * T whm crrespndence shuld be addressed /98/ / Cpyright by Academic P~ess Australia. All rights f reprductin in any frm reserved.

2 Vl. 46, N. 5, 1998 BCHEMSTRY and MLECULAR BLGY NTERNATNAL and the Cu ()/tetracycline cmplexatin induced single and duble strand breaks in DNA (6).The tetracycline induced free radicals have path-physilgical implicatins, resulting in macrmlecular damage t cartilage and synvial fluid, which may ultimately lead t rheumatid arthritis (7-9). The prductin f xidant-induced free radicals is als reprted t enhance the permeability f albumin int endthelial cells (1) which may lead t the frmatin f senile cataract (11). n rder t determine the extent and investigate aspects f the mechanism f txicity and/r pathlgy caused by intraceuular tetracycline, it is necessary t assess the degree f its interactins with the carrier prtein (serum albumin). What precisely is the affinity f binding and hw many mlecules f tetracycline specifically interact with the serum albumin are questins t be answered. n this initial study, sensitive techniques such as flurescence quenching and circular dichrism are used t determine (i) stichimetry and affinity f tetracycline twards the carder prtein (ii) cnfrmatinal changes induced in albumin by tetracycline binding, and (iii) the rle f Cu () ins in tetracycline-albumin interactins. MATERALS AND METHDS Bvine serum albumin (grade V) was purchased frm Sigma (St. Luis, USA). Tetracycline and NBT were purchased frm SRL, ndia. All experiments were carded ut in presence f white light emitted frm a 4 W Philips flurescent tube. The prtein cncentratin was determined by methd f Lwry et.al. (12). Flurescence Quench Titratin: Binding f tetracycline t albumin was studied by flurescence quench titratin accrding t the methd f Levine (13). nitially, the titratin was perfrmed by adding aliquts f tetracycline in the cncentratin range f ( - 8 p_m) t a fixed cncentratin (6. lam) f albumin slutin. Thus the mlar rati range f t 13.3 was btained and the resultant decrease in prtein flurescence was measured at each mlar rati. Frm a knwledge f the number f high affinity tetracycline binding sites n albumin, anther emissin spectra was recrded in the range f t 4 tetracycline/albumin mlar ratis. The relative flurescence was determined at a difference f.25 mlar rati in the range f t 24 gm tetracycline. n each case, a parallel cntrl cnsisting f prtein slutin withut tetracycline was always run. All the slutins were filtered befre use thrugh Millipre filters t minimize the inner filter effect (14). Flurescence measurements were made n RF-54, equipped with data recrder DR-3, in the wave length range f 3-4 using 28 nm as excitatin wave length. The slit width (excitatin/emissin) was.5 nm and sensitivity was high. Circular Dichric Measurements: CD measurements were carded ut using a Jasc spectrplarimeter, Mdel J-72. Data were expressed as mean residue eiiiptieity [], which is defined as [] = /1 x n x 1 x cp, Where, = ellipticity in millidegree, n = number f amin acid residues in the prtein, 1 = path length f the cell in cm, cp = mlar cncentratin f the prtein. The euipticity was measured at the prtein cncentratin range f 4.5 t 5.5 gm with a lmm path length cell in the wave length range f 2 t 25 nm. 944

3 Vl. 46, N. 5, 1998 BCHEMSTRY and MLECULAR BLGY NTERNATNAL RESULTS AND DSCUSSN n this study, the interactins f tetracycline with serum albumin were assessed by quantitative determinatin f changes in the intrinsic flurescence f albumin at varying cncentratins f tetracycline. Fig. 1 shws the emissin spectra at different tetracycline/albumin mlar ratis in the wave length range f 3-4 nm The data shws a tetracycline cncentratin dependent reductin in the flurescence intensity, suggesting significant interactins between the tetracycline and albumin. Assigning the intrinsic flurescence f the tetracycline free prtein as 1, the relative flurescence c P Ll. > l 1 =~ 8 ~ 6 a ~~ \ \ \ \ B 5 1 m l ~ ~ " ~ Wavelength (nm) Fig. 1 ~ TC/Albumin mlar rati Binding f tetracycline with serum albumin at varying TC/albumin mlar ratis. The relative flurescence was btained frm the spectra shwn in the inset- (A). The curves shw the spectra f bvine serum albumin (BSA) in the absence (uppermst) and presence f increasing amunts f tetracycline (TC) using a Shimadzu Spectrflurmeter RF-54 equipped with data recrder DR-3. The inset- (B) shws the % quenching as a functin f tetracycline/albumin mlar rati. 945

4 Vl. 46, N. 5, 1998 BCHEMSTRYnd MLECULAR BLGY NTERNATNAL was determined at a emissin maxima f 34 nm. A least square analysis f the initial linear pints n the plt f relative flurescence versus tetracycline/albumin mlar rati yielded the maximal quench (m), fllwing the relatinship: F = F - mr, where, F, is the flurescence intensity at tetracycline t albumin mlar rati and F is the flurescence intensity f the prtein at zer cncentratin f tetracycline. The plt f relative flurescence vs tetracycline/albumin mlar rati (Fig. 1) shws a biphasic characteristic curve. The flurescence data revealed the presence f at least fur high affinity binding sites fr tetracycline n albumin, while the remaining sites exhibit nn specific binding. n rder t determine the binding cnstant f tetracycline with albumin, an additinal emissin spectra was scanned at a regular difference between t 4 tetracycline/albumin mlar rati (Fig. 2). At each mlar rati, the fractinal quench Q was determined and a Scatchard plt f Q/[Tet] vs Q was pltted (Fig. 3). Using least square analysis, a straight line was btained frm which the values f the slpe and intercept prvided the binding cnstant and the binding capacity f the albumin. The binding cnstant, (Ka) and capacity (n) f albumin with the tetracycline was determined t be 4.6 x 16 1/mle and 3.5, respectively. The binding was substantially increased in the presence f Cu () ins. At a fixed cncentratin f tetracycline, the additin f 1 ~M Cu () ins induced a tw-fld higher quenching f the flurescent grups n the prteins (data nt shwn). Als the excessive binding f tetracycline t albumin in the presence f the Cu () ins lead t the destabilizatin f the carrier prtein. n rder t prbe the tetracycline/cu() cmplex-induced alteratins in prtein cnfrmatin, the tetracycline/albumin interactins were analyzed by circular dichrism (CD) at higher tetracycline cncentratins. A significant decrease in the mean residue ellipticity (MRE) frm the CD analysis cnfirmed there may be alteratins in the prtein helicity. The CD spectra f the albumin, shwn in Fig. 4 revealed a cnsiderable change (2 % reductin) in the the ~ - helical cntent f the prtein. The spectra exhibited tw CD peaks fr untreated serum albumin at 28 and 222 nm, respectively. The intensity f these peaks was decreased by increasing cncentratins f tetracycline in the range frm.2 t 1 mm in presence f.2 mm Cu () ins. N change in the ellipticity was nted in the absence f Cu () ins even at the highest test cncentratins f tetracycline. This suggests that the cpper ins enhanced the binding f tetracycline t the serum albumin pssibly by the frmatin f the metal in bridges. This bservatin crrbrates the flurescent data, exhibiting significant quenching f prtein flurescence (66.7%) in the presence f tetracycline and Cu () ins cmpared t 34.5% with tetracycline alne. Earlier studies demnstrated the frmatin f such metal bridges with 946

5 Vl. 46, N. 5, 1998 BCHEMSTRYand MLECULAR BLGY NTERNATNAL 1^,~ t-. i r J c U la. ~3 4 Wavelength (n m) Fig. 2 Emissin spectra f BSA (6. ~tm) in Tris-HCl buffer, ph 8. in the absence (uppermst curve) and presence f increasing amunts f tetracycline btained in.5 M Tris-HC1 buffer, ph 8., =.15 at 28 ~ C. The mlar ratis f tetracycline t BSA were: (tp t bttm).,.28,.56,.83, 1.1, 1.4, 1.7, 1.9, 2.2, 2.5, 2.8, 3., 3.3, 3.6, 3.9, 4.1. The excitatin wavelength was 28 nm and the slit width was 5nm. 947

6 Vl. 46, N. 5, 1998 BCHEMSTRYnd MLECULAR BLGY NTERNATNAL 6 2.'~ ~ ~N~ N ~ = 1)/ r c +1 ~ 9 e6 ~ 4 c -~.~ ~ _ ~ C '~ eq g _, x [lej.]/ t'm E._= E.n,~ ~ ~ -, ~.~ ~ ~.~ ~ ~ 9 J C euesejnu eap, ele~l,.~ ~ '3 948

7 Vi. 46, N. 5, 1998 BCHEMSTRYand MLECULAR BLGY NTERNATNAL ~ N D < - '\ \ U -d ~ Ji' i i! r \\ 1 d~ ~ ~.~ ~-~ c) + Ll. (t-luu PzUU ~ 5eP) c] tl ~. L= r..) 949

8 Vl. 46, N. 5, 1998 BCHEMSTRY and MLECULAR BLGY NTERNATNAL macrmlecules harburing binding sites fr divalent catins (15). ndeed, the ptential Cu () binding sites n bvine serum albumin have been mapped (16) and the C gruping n the tetracycline mlecules has been implicated as the binding sites fr metal ins (17). The spectrscpic studies revealed that the residues at the N-terminus with a His residue at psitin 3 and cysteine-34 in bvine serum albumin may play a critical rle in metal binding (18,19). t is, therefre, implied that intracellular Cu () ins may prmte an excessive binding f tetracycline with albumin thrugh metal bridges. Such metal in bridges with the albumin have been reprted t inhibit the phtxidatin f tetracycline (17). Als binding t serum albumin may assist in the maintenance f the bilgical activity f tetracycline by prtecting the antibitic frm degradatin by bld enzymes and ensure its delivery t the target site. ACKNWLEDGEMENTS: The authrs thank Dr. Saad Tayyab and Prf. M Saleemuddin fr helpful discussins and the prvisin f labratry facilities. This wrk was supprted in part by a grant-in-aid frm the Aligarh Muslim University, Aligarh. M.A.K is a Junir Research Fellw, S. M. is a Senir Research Fellw f the University Grant Cmmissin, New Delhi, ndia. REFERENCES Cullen, S.., Catalan, P.M. and Helfman, R,J, (1966) Arch. Dermatl., 93, 77 rentreich, N., Harber, L.C., Trmvitch, T.A. (1961) Arch. Dermatl., 83, Frst, P., Weinstein, G.D., Gmez, E.C. (197) JAMA, 216, Farhat, S.M., Schelhart, D.L. and Musselman, M.M. (1958) A.M.A. Arch. Surg., 76, Shils, M.E (1963) Ann. ntern. Med., 58, Buschfrt, C. and Witte,. (1994) Carcingenesis, 15, Mnbisse, J.C., Braquet, P., Randux, A. and Brel, J.P. (1983) Bichem Pharmacl., 32, Dean, R.T., Rberts, CR. and Fmi, L.G. (1984) Bisci. Rep., 4, McCrd, J.M., (1974), Science, 185, Shasby, D.M., Lind, S.E., Shasby, S.S., Gldsmith, J.C. and Hunninghake, G.W. (1985) Bld, 65, elrichs, B.A., Kratzing, C.C. and Winzr, D.J. (1984) nt. J. Vitam Nutr. Res., 54, Lwry,.H., Rsebrugh, N.J., Farr, A.C. and Randall, R.J. (1951) J. Bil. Chem., 193, Levine, R.L. (1977) Clinical Chemistry, 23, Chignell, C.F. (1972) Methd Pharmacl., 2, Khn. K.W. (1961)Nature, 191, Marx, G. and Chevin, M. (1986) Bichem. J., 236, Wiebe, J.A. and Mre, D.E. (1977) J. Pharmaceut. Sci., 66, Peters, T. Jr. and Blumenstck, F.A. (1967) J. Bil. Chem., 242, Lau, S.-J., Kruck, T.P.A. and Sarker, B. (1974) J. Bil. Chem., 249,

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