Stabilization of HRP Using Hsp90 in Water-miscible Organic Solvent

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1 Korean Chem. Eng. Res., Vol. 44, No. 1, February, 2006, pp Hsp90 을이용한유기용매에서의과산화효소안정화연구 om Ç l * Ç ** Çlio *, n r r, *, ** l o ne k e 56-1 (2006 1o 3p r, o 3p }ˆ) Stabilization of HRP Using Hsp90 in Water-miscible Organic Solvent Ja Hee Chung, Yoo Seong Choi*, Seung Hoon Song** and Young Je Yoo*, Interdisciplinary Program for Biochemical Engineering and Biotechnology, *School of Chemical and Biological Engineering, **Bio-MAX institute, Seoul National University, San 56-1, Shilim-dong, Gwanak-gu, Seoul , Korea (Received 3 January 2006; accepted 3 February 2006) k o n l pn k ˆr pp d pl lr rn p n v, p kr r kv v r tp kp. o n l pe p e op p kr l o l vp foldingl l molecular chaperonep psp heat-shock protein Hsp90p pn l, rp o n pedšl p HRP kr l m. Hsp90p 30Í DMSO, 30Í 50Í dioxane m nkl HRPp e v m, e p q l ˆo m. CD(circular dichroism)l p s p l Hsp90p o n l p unfolding l p e refolding l p kk. h Abstract Enzymes in organic media afford many advantages such as chiral synthesis and resolution, modification of fats and oils and production of biodegradable polymers. However, the nature of solvents influences the activity and stability of enzymes, and the presence of organic solvents always constitute a risk of enzyme inactivation. Heat-shock protein Hsp90, one of the molecular chaperone, was applied for understanding of enzyme inactivation and for increasing of enzyme stability in water-miscible organic solvent. Hsp90 showed stabilization effect on HRP in the 30 of DMSO, in the 30 and 50 of dioxane. Hsp90 also showed reactivation effect on the inactivated HRP by water-miscible organic solvent such as dioxane and DMSO. In addition, structural analysis using fluorescence spectrophotometry and circular dichroism showed that exposure of HRP in water-miscible organic solvent caused appreciable conformational changes and enzyme inactivation, and the unfolded HRP by water-miscible organic solvent was refolded by Hsp90. Key words: Hsp90, HRP, Enzyme Activity, Enzyme Stability, Reactivation, Water-miscible Organic Solvent 1. m kl n p ˆ p v }l v p. pn l r p n, p r p np, np rp kv v v p p p lrp n p qq p v p. l pp nkl v, kr p po kv v lrp p pn v p erp t p o n l r s l p kr p p l To whom correspondence should be addressed. yjyoo@snu.ac.kr p, v vp t l l p p lr p n p rr e l pv, kv v kr p vl p p lvv kp ˆp, p po lrp pn r p p [1, 2]. o n pl o n p vp p kr l m p. o n mp qn p p š p o p Ž p f vp s e p p r eˆ k r [3, 4]. vp sr pnl o n p l p p orpp kv r v p q l rr r qnp ƒr p ol (fexibility)p e p p r eˆ., orpp v 92

2 q p pka p p eˆp f o(active site) v p p eˆ [5, 6]. l l o n l pe ˆ e p o pp s, kr p v eˆ o p Hsp90p n m, p horseradish peroxidase(hrp) n m. Chaperonep ~ l vp foldingp m vp r rp 3 s vp, rp chaperonep heat shock protein Hsp60, Hsp70, Hsp90p p [7-9]. p t Hsp90p chaperone ATP lp p qrp pp, Hsp90p pn o n l p p kr l o n l p e opp s o n l p kr p o r p p p r p., l o n l p e p vp sr unfoldingl p l ˆ v, v p k q m o n mp qnl p t v s o sp tp p. ~rp, Hsp90p unfolding vp q~p refoldingl l p k rp l l vp e opp p p o n l v kr p o r p p re p. prp e p o l l l, r o n p DMSO(dimethyl sulfoxide)m dioxanel HRPp e p s m, o n pl Hsp90p rne p p kr p r m., e nk Hsp90l p l q mp, HRP p r m, vp s p o l Fluorescence spectrometrym CD (circular dichroism) pn l o n l Hsp90 } l p HRPp s p e mp, p ˆp o n l HRPp e p vp sr unfoldingl p rp m m e l n HRPm DMSO HRP assayl n pyrogallolp Sigma(St. Louis, MO)l p m. Buffer rsl n Na 2 HPO 4 m NaH 2 PO 4 n dioxanep Junsei(Tokyo, Japan) l p mp 35Í Showa Chemicall p m HRP ~o HRPp pyrogallolp v HRP assay p Š, 25 C s l ph 7.0, 10 mm phosphate buffer n l r m. Assay p light path 1 cm celll 0.5Í(w/w) H 2 O ml, 5.0Í(w/v) pyrogallol solution 0.32 ml, 0.1 mg/mlp HRP nk 0.1 ml, buffer e l n p m s p n 2.4 mlp l r~ p 3 ml pe. vp pyrogallol p pp purpurogallinp p UV-visible spectrophotometer(uvikon 930, Tegimenta Co, Switzerland) n l 420 nml r m. p Hsp90p pn o n l p kr l 93 p ˆ o 1 unitp HRP ph 7.0, 25 Cl 1 pyrogallol 1µMp purpurogallinp rp [10] Fluorescence spectrometry and CD(circular dichroism) vp tryptophanp v intrinsic fluorescencep l p vp s r m. Fluorescence rp 295 nm l excitation e p n p l excitation/ emission band-width 3 nml 10 nm p sr l r m. p 2 s CD(circular dichroism)p pn l r m. Spectrofluorometer Shimazu p RF5000p, CD rp Jovin-Yvon(France)p CD6 Dichrograph pn m Hsp90i m m m Hsp90l p r o p p l 0.1 mg/mlp HRP 0.1 ml nkp 10 mm, ph 7 phosphate buffer 1.4 mlp l 1.5 mlp HRP nkp l. p l p p HRP 0.1 ml nk 0.2 mg/mlp Hsp ml 10 mm, ph 7 phosphate buffer 1.3 mlp l 1.5 mlp HRP nkp l. p n HRPm Hsp90p kp 1:1 (mole) p. p p 25 C msl 150 rpmp shaking pre p 0.1 mlj p l e l HRP r m. 3. y 3-1. DMSOj j}khi m HRP m nk, 30Í DMSO, 50Í(v/v) DMSOl p HRPp r m. p rp n l pv 2 l r p ph 7.0, 10 mm phosphate buffer, 25 Cl r m (Fig. 1). }p 2 p l 30Í DMSOp n 37.1Í, 50Í DMSOp n 11.3Íp p k pl., HRP n m r p n wp k Fig. 1. Relative activity of HRP in 30, 50 (v/v) DMSO and buffer at ph 7.0, 25 C. HRP was incubated at 25 C waterbath with shaking at 150 rpm. HRP concentration was 0.1 mg/ml. : buffer, : 30 DMSO, : 50 DMSO. Korean Chem. Eng. Res., Vol. 44, No. 1, February, 2006

3 94 rq Ëo Ë d Ëomr Fig. 3. Effect of Hsp90 on HRP stability in 30 (v/v) DMSO at 10 C with shaking at 150 rpm. : HRP + Hsp90, : HRP only. Fig. 2. Fluorescence of Hsp90 in 30, 50, 90 concentration (v/v) of DMSO. : 90 DMSO, : 50 DMSO, : 30 DMSO. Hsp90 was excited at 295 nm. (a) Fluorescence intensity of Hsp90 in DMSO. (b) Fluorescence emission maximum wavelength of Hsp90 in DMSO. p p m p e p pl p k pl. o n l HRP s kk o l, 30Í DMSOl HRP n m r 2 l fluorescence intensity r m. 30Í DMSOl fluorescence emission λ max nmp, 50Í DMSOl fluorescence emission λ max nmpl. 295 nml excitatione p, free tryptophanp emission λ max 350 nmp native proteinp λ max 328~332 nm pp 30Í DMSOl HRPp s p v kkv, 50Í DMSOl s plpp p pl Hsp90l mk go o n l kr l Hsp90p n o l n l l n o n p DMSOl Hsp90p kr p fluorescence pn l kk k. Fig. 2(a) 30Í, 50Í, 90Í DMSOl Hsp90p s l fluorescence intensity r p, Fig. 2(b) e l emission Žqp shift ˆ p. Fig. 2(a) 30Í DMSOl Hsp90p fluorescence intensityp p llv, 50Ím 90Í DMSO o44 o k l Hsp90p k 2e v rvrp fluorescence intensity v p k 2e l n intensity v ˆ l., Fig. 2(b)l p 30Í DMSOl Hsp90p emission Žqp nm p sr l p lv, 50Ím 90Í DMSOl emission Žqp p m. p Hsp90p tryptophan residue DMSOl mr lpp p., 30Í DMSOl Hsp90p sr p lpp ˆp 30Í DMSOl HRPp kr o Hsp90p rnp v, DMSO 50Í p p nl Hsp90p sr p Hsp90p s p Ž l Ž pl 50Í p p DMSOl Hsp90p vr rn l Ž l. HRPp kr o 30Í DMSO n p p ed Šl Hsp90p rn m. Fig. 3l m p, 10 C, 30Í DMSOl k 360e incubation p Hsp90 HRP m nkl p k 60.7Í k pv, Hsp90 p l tv kp HRP m nk p k 24.5Í p k pl. v 30Í, 50Íp dioxanel e p mp, Hsp90p 30Í DMSO edšl } HRPp kr l m (Fig. 4). rp Hsp90p unfolding v k o n l HRPp kr l l ppp k pl, o n m nkl n edšl p e p opp q~p sr unfoldingp pl. v o n l p kr o l p k q m n v p qnl l pnl, v q ~p sr folding kr p v eˆ p n tn kr r tp Ž., Hsp90p pn l e HRPp p q e p m. o n l p e p vp unfoldingl p Hsp90l p p pp q p r m. Fig. 5l m p Hsp90p o n l e HRPp p rp q m. 90Í DMSOl Hsp90p l n 10Í p p p v, 59.3Í p p kp HRP Hsp90l p 94.6Í v p

4 Hsp90p pn o n l p kr l 95 Fig. 4. Effect of Hsp90 on HRP stability in 30, 50 (v/v) dioxane at 25 C with shaking at 150 rpm. : HRP with Hsp90 in 30 dioxane, : HRP without Hsp90 in 30 dioxane, : HRP with Hsp90 in 50 dioxane,g : HRP without Hsp90 in 50 dioxane (v/v). Fig. 6. Spectra of fluorescence change of HRP. (-): initial fluorescence of inactivated HRP+Hsp90, (--): fluorescence of inactivated HRP+Hsp90 after one-day of incubation, ( U ): initial fluorescence of HRP in buffer, (-): fluorescence of HRP in buffer after one-day of incubation, (UUU ): initial fluorescence of inactivated HRP without Hsp90, ( UU ): fluorescence of HRP without Hsp90 after one-day incubation. HRP concentration is 0.01 mg/ml; molar ratio of HRP:Hsp90=1:1; HRP was inactivated by stocked at 90 DMSO for one day. HRP was incubated at 25 C with shaking at 145 rpm. Fig. 5. Reactivation of HRP in DMSO: The symbols represent the HRP inactivated in each concentration of DMSO. Reactivation by Hsp90 is marked as solid line and reactivation without HRP marked as dashed line. : buffer, : inactivated at 50 DMSO/reactivated by Hsp90, : reactivated without Hsp90, : 70 DMSO with Hsp90, : 70 DMSO without Hsp90, : 90 DMSO with Hsp90, : 90 DMSO without Hsp90. l. ql p 40Í p Hsp90l p q tl ~. p, DMSO n ed Šl HRPp e p o n l p vp sr unfolding l p p p pl. v, e s l p inactivation ˆp o s eˆ š ep r p. p l, p lrp unfoldingl p e pnl lr s l p k inactivation p v k o kp l n p Ž, kp v kr r p o l n r rp ~rp l n p., 1993 v Hsp90p signal transductionl l r vp sm p sr chaperonep k r l [11] Jakob, 1996 Buchnerl p Hsp90p in vitrol non-native protein ˆrp p l aggregationp rp v p e l [12, 13]. l l Hsp90p o n l p kr p Jakob Buchnerp e m p p aggregation v ql p p p. p o Hsp90l p HRP reactivation HRPp s s m. Fig. 6p 90Í DMSOl e Hsp90l p reactivation p 3 s ˆ p. HRPp n tryptophan residuep mv sq vp l o p. Fluorescence intensityp l vp folding ˆ rrp p p [10]. ~rp intensityp v vp sr unfoldingp p v intensityp vp refoldingp t. e l e v kp m p e 24e k incubation, p p pv fluorescence intensity v m. v, p intensity v e l Hsp90p l tlp fluorescence intensity m p p m. vp fluorescence intensity o n } prp intensity m l vp o p s m tq pp p. v, p 3 s p m p p HRP Hsp90l p reactivation Korean Chem. Eng. Res., Vol. 44, No. 1, February, 2006

5 96 rq Ëo Ë d Ëomr y Fig. 7. Far-UV CD spectra of HRP. (-): spectra of HRP in buffer, (-): spectra of reactivated HRP by Hsp90, (--): spectra of inactivated HRP without Hsp90. HRP concentration was 0.2 mg/ml. HRP was incubated with Hsp90 for 24 hours with shaking at 145 rpm at 25 C. Molar ratio of HRP: Hsp90 is about 5:1. k vp srp refolding lpp k p. Fig. 7p 90Í DMSOl e reactivation lp p 2 s l CD ˆ p. 75.9Í e l Hsp90 1:5p p pe p 24e HRP p p 85.1Í reactivation lp p HRPp 2 s 100Í p native HRPp 2 sm mp, Hsp90l p reactivation native structurel n s p p m. l l water-miscible co-solvent systeml HRPp e p vp srp unfoldingl p p, v foldingl l Hsp90l p p s native ˆp s l p pl. rp, o n l p kr p eˆ o l v v n mp qnl p n tn v, v q~p sr kr p v eˆ n tn p. n p l n v, Hsp90p pn o n l p v kr l o n l vp sr k r p p l p pp, p l kp o n l p kr p } p np p Ž. 1. Laane, C., Boeren, S., Vos, K. and Veeger, C., Rules for Optimization of Biocatalysis in Organic Solvents, Biotechnol. Bioeng., 30(1), 81-87(1987). 2. Lee, S. M., Yeo, J. S., Park, K. and Yoo, Y. J., Synthesis of Lignin-phenol Copolymers Using Horseradish Peroxidase, Kor. J. Biotechnol. Bioeng., 15(1), 22-26(2000). 3. Ryu, K. and Dordick, J. S., How do Organic Solvents Affects Peroxidase Structure and Function?, Biochemistry, 31(9), (1992). 4. Park, B. J., Lee, C. H. and Koo, Y. M., Development of Novel Protein Refolding Using Simulated Moving Bed Chromatography, Kor. J. Chem. Eng. 22(3), (2005). 5. Bell, G., Halling, P. J., Moor, B. D. and Rees, D. G., Biocatalyst Behavior in Low-water System, Trends. Biotech., 13(11), (1995). 6. Schmitke, J. L., Stern, L. J. and Klibanov, A. M., Comparison of x-ray Crystal Structures of an Acyl-enzyme Intermediate of Subtilisin Carlsberg Formed in Anhydrous Acetonitrile and in Water, Proc. Natl. Acad. Sci. USA, 95(22), (1998). 7. Jaenicke, R. and Buchner, J., Protein Folding: From Unboiling an Egg to Catalysis of Folding, Chemtract: Biochem. Mol. Biol., 4, 1-30(1993). 8. Wiech, H., Buchner, J., Zimmermann, R. and Jakob, U., Hsp90 Chaperones Protein Folding in Vitro, Nature, 358(6382), (1992). 9. Freeman, B. and Morimoto, R., The Human Cytosolic Molecular Chaperones Hsp90, Hsp70, and Hdj-1 Have Distinct Roles in Recognition of a Non-native Protein and Protein Refolding, EMBO J., 15(12), (1996). 10. Kim, J. R., Choi, Y. S. and Yoo, Y. J., Reactivation of Horseradish Peroxidase in Organic Solvent Using Extraction, Biotechnol. Lett., 22(6), (2000). 11. Xu, Y. and Lindquist, S., Heat Shock Protein Hsp90 Govern the Activity ofpp60 v-src Kinase, Proc. Natl. Acad. Sci. USA, 90, (1993). 12. Jakob, U. and Buchner, J., Assisting Spontaneity-the Role of Hsp90 and Small Hsps as Molecular Chaperones, Trends in Biochem. Sci., 19(5), (1994). 13. Buchner, J., Supervising the Fold: Functional Principles of Molecular Chaperones, Faseb J., 10(1), 10-19(1996). o44 o k

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