Purification and Identification of Ne w Isoenzymes of Nereid Proteinase

ISSN 100727626 CN 1123870ΠQ Chinese Journal of Biochemistry and Molecular Biology 2008 4 24 (4) :359 365 1), 2), 1) 3 ( 1), 130021 ; 2), 154000),., ( PAGE) (HPLC) ( IEF) (MS), 3,, 315 415, 29 248175 29 007166 28 954117,,, 2,, 1., 3 2, 1 2. 3, 2. 4,, 40 50 ph 8 9.. ; ; Q55 Purification and Identification of Ne w Isoenzymes of Nereid Proteinase LI Qi 1), WANG Zhao 2), HONG Min 1) 3 ( 1) Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Jilin University, Changchun 130021, Jilin, China ; 2) Department of Biochemistry, School of Medical Sciences, Jiamusi University, Jiamusi 154000, Heilongjiang, China) Abstract The native Nereid proteinase is a serine proteinase with two known isoenzymes, which have the optimal temperature 40 50 and the optimal ph 8 9. We have identified several new Nereid proteinase isoenzymes following a combination of purification procedures of ammonium sulfate salting out, column filtration and hydrophobic chromatography. The major three proteinase identification possess fibrinolytic activities and are responsive to four narrow spectrum inhibitors in enzymological activities, and have the biochemical properties of isoelectric points between 3. 5 and 415 determined by isoelectric focusing electrophoresis. The molecular weights are 2912 kd, 2910 kd and 2819 kd, respectively, as determined and confirmed by SDS2 PAGE, high performance liquid chromatogram, and mass spectrum. The peptide finger printing revealed that the three isoenzymes appeared to be novel proteins, two of which have fairly high homologies. Although all three new enzymes have the same antigen determinant group, only the highly homologous two have the identical immunogenicity determined by antigen2antibody tittering reactions. Key words isoenzyme of nereid proteinase ; purification ; identification,,,.,, [1 ],., [2 ].,,,. 1, Swiss prot, P83433. :2007209205 ; :2007212225 (No. 20030442) 3 Tel :0431285619474,E2mail :hongmin1945 @yahoo. com Received : September 5, 2007 ; Accepted : December 25,2007 Supported by Science and Technology Foundation of Jilin Province, China (No. 20030442) 3 Corresponding author Tel :0431285619474 E2mail :hongmin1945 @yahoo. com

360 24, 0214482810,, 1500873., [3 ],,,.,,,,. 1 1. 1 215 L (10 g) A, A, : (90 cm 3 cm 20 cm 5 cm, 1, B (ph 710,012 molπl ),8823B2 ( PB), 2,, ),3057 PORTABLE RECORDER (,. ),YC21 ( 280 nm, 4 ) ; :DYY2 4. ( ) ; : TSK G2000SW, ( TOSOH ) ; : Astrup [4 ] Ultimate 3000( ) ; IPGphor. ( Amersham Biosciences ) ; Bruker 1. 4 Autoflox MALDI2TOF ( Bruker ) ; ( Beckman ) ; Virtis Advantage ( Virtis ) ; PALL ULTRAPUMP II ( ) ;DNM29602G. Sephacryl S2200 Phenyl Sepharose Fast Flow ( Pharmacia ) ; ( Sigma ) ; pi Marker ( ph 3 10 ) Ampholine PAGplate ( ph 315 915 ) Amersham Biosciences ;S2251 (250 gπl, Fluka ) ; PMSF ( 100 mmolπl, DMSO ) E64 ( 100 mmolπl) (100 gπml, DMSO ) Sigma ;1,102 (100 mmolπl, DMSO, ) ; ( ) ;. 1. 2 (10 11 ),,,, 1 (0102 molπl, ph 710,PB),,,, - 20,,. 1. 3 10 % 50 %,,,, (0102 molπl,ph 710,PB).. 50 % Sephacryl 200 ( 90 cm 3 cm), 0102 molπl NaCl,,, 5 kd 10. Phenyl Sepharose, Phenyl Sepharose (20 cm 5 cm) A [ ph 710,012 molπl PB + 018 molπl (NH 4 ) 2 SO 4 ], 1. 4. 1 [5 ] : (22 cm 12 cm), 10 %, 2 mm. 4 mg, 30 % ( ), 60 ma.,,, 1 cm, 0105 % R2250 10 30 min,,, 3,,,, 160 ma,4 h.,,, 3 a b c., a b c,. : TSK G2000SW ( 715 mm 30 cm, 10 m), 50 mmolπl ( ph 710 ), 018 mlπmin,, 214 nm. 11412 ph ( T = 5 %,C = 3 %,ph 315 915, 110 mm 110 mm 1 mm),. : 1 500 V, 50 ma, 30 W, 115 h. 10.

4 : 361 pi Marker, 1 gπ l, 20 l,. 1. 4. 3, PAGE R250 a b c,, 2, (SA),,,. : N2 ( 337 nm), 20 KV. : Matrix Science (http :ΠΠwww. matrixscience. com) Mascot NCBI, : 100 ppm, 1, [M + H] +, Gerner [6 ]. 1. 4. 4 : b, [7 ], ( 1 32). ( ) a b c. 115 11511,a b c (2 gπ l,20 l) 80 l 011 molπl, ph 714, PB, 014 gπ l. 30 l, 20 30 37 50 60 70 1 h, 5 l,. 11512 ph,a b c (2 gπ l,20 l), 80 l 011 molπl ph(ph 410 1210) PB, 5 l. 11513 9, 1 91 0198 ml Tris2HCl (ph 714) 10 l (a b c ),37 5 min. 1 6 10 l Tris2HCl (ph 714) DMSO PMSF E64 1,102,7 8 3 4 495 l 5 l [8 ],9 10 l.,37 1 h. 9 10 l. S2251 [9 ] : 96 1 10, 9 100 l 1 9,10 100 l Tris2HCl (ph 714), 100 l S2251,,37 1 h. 30 l. 405 nm 10, A. 3. : SPSS1310, gx s,. 2. 1 10 % 50 %,., 50 %,,,, 50 %. Sephacryl 200,,,, Fig. 1,. Fig. 1 Chromatorgram of Nereid proteinase on a Seph2acryl2200 column The bound proteins were eluted with 0. 02 molπl NaCl. The shadow are active ingredients Fig. 2 Chromatorgram of Nereid proteinase on a Phenyl2 Sepharose column Fractions after Sephacryl 200 were applied to a pre2equilibrated Phenyl Sepharose column, and eluted with buffer A (ph 710, 012 molπl PB + 018 molπl (NH 4 ) 2 SO 4 ). The bound proteins were eluted with buffer B (ph 710, 012 molπl PB). The peak 2 were active ingredients

362 24 Sephacryl S2200 Phenyl Sepharose, Fig12, 2, 2, 1, 2, Phenyl Sepharose,.,,, Phenyl Table 1 Purification of Nereid proteinase Sepharose,. Sephacryl 200 Phenyl Sepharose Table 1.,, 75 %, 60,. Step VΠml Total Specific activityπ Total Yield Purity proteinπmg U mg - 1 activityπu ( %) Raw material 200 2 000 500 10 00 000 1. 00 100 Ammonium sulfate precipitation 200 1 500 600 900 000 1. 2 90 Sephacryl 200 600 1 200 700 840 000 1. 4 84 Phenyl Sepharose 100 25 30 000 750 000 60 75 2. 2 PAGE,, 3, Fig13A, b,, a 30 %,b 50 %,c 20 %, b,. PAGE,,, 3,. Fig. 3B, a b c PAGE. :a b c 3,,,., 3,,. 3, HPLC. 2. 3 HPLC HPLC 3, Fig. 4, : 1 81708 min, 34102 % ; 2 101210 min, 47 % ; 3 121943 min, 14122 % ; 95124 %. 3,, 3, HPLC, N2V, 95 %. Fig. 4 Analysis of the Nereid proteinase by HPLC TSK G2000SW (7. 5 mm 30 cm, 10 m) gel filtration column with mobile phase 50 mmolπl phosphate buffer (ph 710). The conditions were 018 mlπmin flow rate, at room temperature and monitored at 214 nm. The total peak area was 95124 % Fig. 3 PAGE analysis of the Nereid proteinase The Nereid proteinase was separated on a 10 % polyacrylamide gel and stained with Coomassie brilliant blue R2250. ( A ) Electrophoresis showed 3 clear bands : a, b, c strip. (B) Three electrophoresis bands were cut off, then each band and the Nereid proteinase were determined by a PAGE. Sample a (lane 1), b (lane 2) and c (lane 3) had the same migration rate as a, b and c components in the Nereid proteinase (lane 4), respectively. It demonstrated the precise cut and reliable recovery from the gel of PAGE 2. 4, 3, Fig. 5. 315 415,. 2. 5 2. 5. 1 2 (LCΠMS) 2 (LCΠMS). :a 29 248175,b

4 : 363 Fig. 5 pi analysis of the Nereid proteinase Precast polyacrylamide gels(t = 5 %,C = 3 %,ph 315 915,110 mm 110 mm 1 mm) was utilized to determine the pi of the Nereid proteinase under the condition of 1 500 V,50 ma,30 W and condensed at 10 for 115 hours. The loading volume was 20 l (1 gπ l). The gel was stained with silver nitrate. Lane1 : pi marker ;Lane 2 : Nereid proteinase 29 007166,c 28 954117. 3,, b c 53149,a b 240109. 120, 3 1 2. 2. 5. 2 MALDI2TOF2MS (peptide mass finger printing, PMF), Fig16. : a b c 3,. a b c, 3 mπ z 762 1 073 1 649 2 764 4, mπz 1 073 1 649 2 764,. b c mπz 2 070 2 494 3 220 3, a,a b c mπz 1 629 2 094 2 734 3 428 4. b c 2, a,., 3,b c,,a. 2. 6,,a b c. :a b c, 1, 3,,. Fig. 7,,a1,b1,c1 Fig. 6 The peptide mass fingerprinting of the proteinase Nereid Strip a, b and c stained with coomassie brilliant blue R250 were cut off from PAGE. Samples were identified by mass spectrometry. (A) The PMF of a strip ; (B) The PMF of b strip ; (C) The PMF of c strip. The PMF of strip b and c suggested they could be the homogeneous component, strip a is the different. The result indicated that the Nereid proteinase was composed of two individual components with different structures 1 gπ l,a2,b2,c2 2 gπ l. 2, Fig. 8,, 4 (1 2 3 4 gπ l), 3, 2,1,., a b c,b c,,a,3, :a b c,,.

364 24 Fig. 7 The antigen2antibody reaction of a, b, c strip and antiserum The serum antibodies was in the middle well, the concentration of sample a1,b1 and c1 were 1 gπ l, a2,b2 and c2 were 2 gπ l. There was one precipitation line between the a, b, c strip and serum antibodies. indicated that three samples have identical antigen determinant group Fig. 8 The antigen2antibody reaction of the Nereid proteinase and antiserum The serum antibody was in the middle well, the samples around were 1 gπ l, 2 gπ l, 3 gπ l and 4 gπ l, respectively. There were two precipitation lines between the Nereid proteinase and serum antibody. It indicated that two compositions had identical immunogenicity, the other had different Table 3 The optimum reaction ph of the Nereid proteinase It 2. 7 2. 7. 1 Table 2. Table 2 The optimum reaction temperature of Nereid proteinase tπ 20 30 37 50 60 70 D N Πmm 3. 0 3. 6 4. 0 4. 5 2. 0 0. 1 D a Πmm 3. 0 3. 5 4. 2 4. 5 1. 9 0. 2 D b Πmm 2. 8 3. 6 4. 1 4. 4 2. 1 0. 3 D c Πmm 3. 1 3. 4 4. 0 4. 4 2. 2 0. 1 Fibrinolytic activity of the enzyme was determined by fibrin plate method. D N, D a, D b and D c represent diameters of fibrinolytic spots of Nereid proteinase, component a, b and c, respectively,a b c 50, 37,70, 40 50,a b c. 2. 7. 2 ph ph Table 3. Table 3,,a b c ph 510 1010, ph 810 910, ph 8 9,a b c ph. 2. 7. 3, Fig. 9, b. 1 2 4 5 6,3, 7 8 9. S2251 :, F = 801457. 1 3, q = 01001,. ph 4. 0 5. 0 6. 0 7. 0 7. 4 8. 0 9. 0 10. 0 D N Πmm 2. 0 2. 5 3. 0 3. 0 3. 0 4. 0 4. 0 3. 5 D a Πmm 2. 0 2. 6 3. 1 2. 9 2. 9 4. 0 3. 9 3. 6 D b Πmm 1. 9 2. 4 3. 0 3. 0 3. 1 4. 2 4. 0 3. 5 D c Πmm 2. 1 2. 5 3. 1 3. 1 3. 0 4. 1 4. 1 3. 4 Fibrinolytic activity of the enzyme was determined by fibrin plate method. D N, D a, D b and D c represent diameters of fibrinolytic spots of Nereid proteinase, component a, b and c, respectively PMSF b, DMSO E64 1,102 b.,a c, b, a b c 3,.

4 : 365,. S2251,.,. :. ( References) Fig. 9 Fibrinolytic activity of b strip on fibrin plates (A) 1 :no inhibitor ; 2 :PMSF as inhibitor ; 3 :DMSO as inhibitor. (B) 4 : E64 as inhibitor ; 5 :zymofren as inhibitor ; 6 : 1,102phenanthroline as inhibitor. (C) 7 : E64 + 2 Mercapto2ethanol as inhibitor ; 8 : PMSF + 2 Mercapto2ethanolas inhibitor ; 9 : beta2 mercaptoethanol as inhibitor. showed similar fibrinolytic activity ( # 1, # 2, # 4, # 5, and # 6), no activity ( # 3) and less activity ( # 7, # 8 and # 9). The results demonstrated that the fibrinolytic activity of component b was inhibited effectively by PMSF, but not by DMSO, E64, zymofren, 1, 102 phenanthroline and beta2mercaptoethanol [10 ] [11 ] [12 ] [13 ] 4. 4 PMSF E64 [14 ] 1,102 4 [8 ]., DMSO, PMSF,,. DMSO PMSF,.,PMSF, 3.,7 8 9,, It [ 1 ] Takahashi T, Furukawa Y, Muneoka Y, et al. Isolation and characterization of four novel bioactive peptides from a polychaete annelid, Perinereis vancaurica [ J ]. Comp Biochem Physiol C Pharmacol Toxicol Endocrinol,1995, 110 (3) :2972304 [ 2 ] Matsubara K, Yamaki M, Nagayama K, et al. Wheat germ agglutinin2reactive chains of giant hemoglobin from the polychaete Perinereis aibuhitensis[j ]. Biochim Biophys Acta, 1996, 1290 ( 3) : 2152223 [ 3 ],,,. N2V [J ]. (Li Qi, Chi Xiu2Mei, Shi Li2Hong, et al. The experimental study on fibrinolytic activity of N2 V proteinase in body and vitro [J ]. Chin Pharmacol Bull),2006,22 (8) :976-979 [ 4 ] Astrup T, Mullertz S. The fibrin plate method for estimating fibrinolytic activity [J ]. Arch Biochem Biophys,1952,40 (2) : 3462 351 [ 5 ]. [ M]. : ( Guo Yao2Jun ed. Experimental Technique of Protein Electrophoresis [M]. Beijing : Science Press), 1999 :54-115 [ 6 ] Gerner C, Frohwein U, Gotzmann J, et al. The Fas2induced apoptosis analyzed by high throughput proteome analysis [J ]. J Biol Chem,2000,275(50) :39018239026 [ 7 ],,. [M], : ( Wang Yan2Hua, Li Guan2Cheng, Zhou Xin2Fu. Antibody theory and technique [ M]. Beijing : Science Press), 2005 : 298-300 [ 8 ] Beynon R, Bond J S. Proteolytic Enzymes : A practical approach, 2nd ed[m]. Oxford University Press, 2001 : 1221 [ 9 ] Nieuwenhuizen W, Voskuilen M, Vermond A. The influence of fibrin (ogen ) fragments on the kinetic parameters of the tissue2type plasminogen2activator mediated activation of different forms of plasminogen[j ]. Eur J Biochem, 1988,174(1) : 1632169 [10 ] Lu W, Qasim M A, Laskowski M Jr, et al. Probing intermolecular main chain hydrogen bonding in serine proteinase2protein inhibitor complexes: chemical synthesis of backbone2engineered turkey ovomucoid third domain [J ]. Biochemistry, 1997,36 (4) :6732679 [11 ] Drenth J, Kalk K H, Swen H M. Binding of chloromethyl ketone substrate analogues to crystalline papain [J ]. Biochemistry, 1976,15 (17) :373123738 [12 ] Hartsuck J A, Tang J. The carboxylate ion in the active center of pepsin [J ]. J Biol Chem,1972,247(8) :257522580 [13 ] Hangauer D G, Monzingo A F, Matthews B W. An interactive computer graphics study of thermolysin2catalyzed peptide cleavage and inhibition by N2carboxymethyl dipeptides [J ]. Biochemistry,1984,23 (24) :573025741 [14 ] Stone S R, Rennex D, Wikstrom P, et al. Peptidyldiazomethanes. A novel mechanism of interaction with proly lendopeptidase[j ]. Biochem J,1992,283 (Pt 3) :8712876