Purification and partial characterisation of α2-antiplasmin and plasmin(ogen) from ostrich plasma

Comparative Biochemistry and Physiology Part B 129 Ž2001. 809᎐820

Purification and partial characterisation of ␣ 2-antiplasmin and plasminž ogen/ from ostrich plasma Adele R. Thomasa , Ryno J. Naude ´U,a, Willem Oelofsen a , Takako Naganumab, Koji Muramoto b a

Department of Biochemistry and Microbiology, Uni¨ ersity of Port Elizabeth, P.O. Box 1600, Port Elizabeth 6000, South Africa b Department of Applied Biological Chemistry, Faculty of Agriculture, Uni¨ ersity of Tohoku, Sendai 981, Japan Received 18 December 2000; received in revised form 9 March 2001; accepted 12 March 2001

Abstract This study reports the isolation and partial characterisation of the ostrich serpin, ␣ 2 AP, and its target enzyme, ostrich plasmin, in its active and inactive proenzyme, namely plasminogen, forms. Ostrich ␣ 2 AP was purified using L-lysine᎐Sepharose chromatography, ammonium sulfate fractionation, and SuperQ-650S and ostrich LBSI᎐Sepharose chromatographies. It revealed a Mr of 84 K Žthousand. and had one and two N-terminal amino acids in common with 11 of those of human and bovine ␣ 2 AP, respectively. It showed the largest inhibitory effect on ostrich plasmin, followed by bovine trypsin and plasmin, respectively, and much less plasmin inhibition than bovine aprotinin, but much more so than human ␣ 2 AP, DFP and EACA. Ostrich plasminogen was highly purified after L-lysine᎐Sepharose chromatography and showed a Mr of 92 K, a total of 775 amino acids and its N-terminal sequence showed ; 53% identity with those of human, rabbit, cat, and ox plasminogens. Ostrich plasmin, obtained by the urokinase-activation of ostrich plasminogen, revealed a Mr of 78 K, a total of 638 amino acids, an N-terminal sequence showing two to four residues identical to five of those of human, cat, dog, rabbit, and ox plasmins, and pH and temperature optima of 8.0 and 40⬚C, respectively. 䊚 2001 Elsevier Science Inc. All rights reserved. Keywords: Ostrich; Plasma; Serpin; ␣ 2 -antiplasmin; Plasminogen; Plasmin

1. Introduction ␣ 2 AP, also known as ␣ 2 PI, belongs to the family of structurally related serine protease inhibitors called serpins ŽPotempa et al., 1994.. It is the primary physiological inhibitor of plasmin, the enzyme responsible for the dissolution of fibrin clots, and an efficient inhibitor of fibrinolysis U

Corresponding author. Tel.: q27-41-5042441; fax: q2741-5042814. E-mail address: [email protected] ŽR.J. Naude ´..

ŽCollen, 1976; Moroi and Aoki, 1976; Mullertz ¨ and Clemmensen, 1976; Highsmith, 1979.. A deficiency of ␣ 2 AP therefore results in severe haemorrhagic tendencies due to premature lysis of haemostatic plugs before the restoration of injured vessels ŽWiman, 1980a; Aoki, 1990.. To date, mammalian ␣ 2 AP has been well characterised, but little attention has been given to avian ␣ 2 AP. As part of the systematic investigation of the biochemistry of proteases and their inhibitors in ostrich blood undertaken in our laboratory ŽKuhn et al., 1994; Van Jaarsveld et

1096-4959r01r$ - see front matter 䊚 2001 Elsevier Science Inc. All rights reserved. PII: S 1 0 9 6 - 4 9 5 9 Ž 0 1 . 0 0 3 9 6 - 7

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A.R. Thomas et al. r Comparati¨ e Biochemistry and Physiology Part B 129 (2001) 809᎐820

al., 1994; Frost et al., 1997, 1999., and due to the rapidly increasing ostrich industry worldwide over the past few years, this study of ostrich ␣ 2 AP and its target enzyme, plasmin, in its active and inactive zymogen form, namely plasminogen, was initiated.

2. Materials and methods 2.1. Materials Human ␣ 2 AP, bovine plasmin, bovine pancreatic trypsin Žtype III., bovine pancreatic ␣chymotrypsin Žtype I-S., D-Val᎐Leu᎐Lys-pNAHCl, N-p-tosyl-Gly᎐Pro᎐Lys-pNA-acetate, Nbenzoyl-Phe᎐Val᎐Arg-pNA-HCl, BAPNA-HCl, p-NPGB, bovine fibrinogen, bovine pancreatic trypsin inhibitor Žtype I-P., bovine aprotinin, bovine trasylol, DFP, EACA, human urokinase, cyanogen bromide, Sepharose CL-4B, BCA and the high molecular weight marker mix were all obtained from Sigma ŽUSA.. DMSO and lysine monohydrochloride were obtained from Merck ŽGermany., and Toyopearl Super Q-650S was obtained from TosoHaas ŽJapan.. All other reagents were of the best analytical grade available. 2.2. Ostrich plasma Ostrich blood was obtained from the Grahamstown abattoir where the blood was collected directly from the neck of the ostrich immediately after killing. The blood was collected in 0.1 M trisodium citrate buffer, in a ratio of 9:1, and centrifuged at 3000 rev.rmin ŽBeckman benchtop. for 15 min at room temperature. The supernatant Žplasma. was pipetted off and immediately frozen in liquid nitrogen, all within 30 min after collection. 2.3. Purification of ostrich ␣ 2 AP Unless otherwise stated, all purification procedures were performed at 4⬚C. The purification of ostrich ␣ 2 AP was based on the procedure used by Wiman Ž1980b.. Trasylol Ž100 000 KIU. was immediately added to thawed ostrich plasma Ž503 ml. before stirring overnight with L-lysine᎐Sepharose resin Ž150 ml. equilibrated with 0.1 M sodium phosphate buffer ŽpH 7.4. containing 3 mM EDTA. The supernatant Žfraction A. was

removed from the plasminogen-bound resin by suction on a Buchner funnel at room temperature ¨ and brought to 30% saturation with solid ŽNH 4 . 2 SO4 . After stirring for 2 h, the supernatant was removed by centrifugation at 17 700 g for 1.5 h and brought to 67.5% saturation with solid ŽNH 4 . 2 SO4 . After stirring overnight, the pellet was removed by centrifugation at 17 700 g for 1 h, dissolved in a small volume of 0.1 M sodium phosphate buffer ŽpH 7.4. containing 3 mM EDTA, and exhaustively dialysed against running de-ionised water. The dialysed sample was cleared by centrifugation at 17 700 g for 1 h and freeze-dried Žfraction B; 6.70 g.. Fraction B Ž1 g. was stirred overnight with Toyopearl Super Q-650S resin equilibrated with 50 mM sodium phosphate buffer ŽpH 7.4., and the supernatant was removed by suction on a Buchner funnel. The ¨ resin was washed with the equilibration buffer on the Buchner funnel, packed into a column Ž1.6= ¨ 18.7 cm. and further washed to ensure an A 280nm baseline value. ␣ 2 AP was eluted with a linear salt gradient Ž0᎐0.2 M NaCl in equilibration buffer. at ; 90 mlrh and the elution was continued with the buffer containing 0.2 M NaCl until an A 280nm baseline was reached. The tubes were pooled according to SDS-PAGE patterns and ␣ 2 AP activity, and the fraction was exhaustively dialysed against running distilled water and freeze-dried Žfraction C.. Fraction C was applied to an ostrich LBSI᎐Sepharose column, prepared by cyanogen bromide coupling ŽCuatrecasas, 1970. of purified ostrich LBSI fragment ŽSottrup-Jensen et al., 1978. to Sepharose 4B Ž1.0= 7.6 cm. and equilibrated with 10 mM sodium phosphate buffer ŽpH 7.4. at 1 mlrh. The column was first washed to an A 280nm baseline with the equilibration buffer and then with 20 mM sodium phosphate buffer ŽpH 7.4.. ␣ 2 AP was eluted with 50 mM EACA in the latter buffer at 60 mlrh, exhaustively dialysed against running distilled water and freeze-dried Žfraction D.. Ostrich ␣ 2 AP was purified 16.24-fold from 503 ml of plasma, giving a 0.27% yield. 2.4. Purification of ostrich plasminogen The purification procedure of ostrich plasminogen was adapted from that of Deutsch and Mertz Ž 1970 . . The ostrich plasm inogen-bound L-lysine᎐Sepharose resin obtained from the first purification step of ostrich ␣ 2 AP was washed with 0.3 M sodium phosphate buffer ŽpH 7.4.

A.R. Thomas et al. r Comparati¨ e Biochemistry and Physiology Part B 129 (2001) 809᎐820

containing 3 mM EDTA and 5000 KIUrl Trasylol, and equilibrated with 0.1 M sodium phosphate buffer ŽpH 7.4. containing 3 mM EDTA and 10 000 KIUrl Trasylol on a Buchner funnel ¨ at room temperature. The resin was packed into a column Ž2.6= 25.5 cm. and further washed with the equilibration buffer at 4⬚C to ensure an A 280nm baseline. Ostrich plasminogen was eluted with 10 mM EACA in the equilibration buffer at 80 mlrh. The A 280nm peak was pooled omitting the ascending and descending tails, exhaustively dialysed against running distilled water and freeze-dried Ž226.16 mg.. 2.5. Acti¨ ation of ostrich plasminogen to plasmin Ostrich plasminogen was activated according to the method described by Robbins and Summaria Ž1970.. Human urokinase solution was added at ratios of 0, 5, 10 and 600 ␮grmg plasminogen and the plasminogen᎐urokinase solution was allowed to stand at room temperature for 0, 2, 4, 6, 8 and 20r22.5 h. After ŽNH 4 . 2 SO4 precipitation, the final plasmin solutions were dialysed for a few hours against a large volume of distilled water at 4⬚C and freeze-dried. 2.6. Enzyme assays The assay procedures for plasmin and trypsin were adapted from Aiach et al. Ž1983. and Skidgel and Erdos ¨ Ž1988., respectively. Therefore, to assay for plasmin inhibitory activity, 10 ␮l of the inhibitor was incubated with 10 ␮l of plasmin for 4 min at room temperature, after which 190 ␮l of 1 mM D -Val᎐ Leu ᎐ Lys-p NA in 50 mM Tris᎐acetate ŽpH 8.0. was added to start the reaction. The increase in A 410nm was followed for 5 min at 10-s intervals. The assay for trypsin inhibitory activity involved incubating 10 ␮l of the inhibitor with 25 ␮l of trypsin for 5 min at room temperature, and starting the reaction by adding 265 ␮l of 1 mM BAPNA-HCl Žfinal concentration. in 50 mM Tris᎐HCl ŽpH 8.2. containing 20 mM CaCl 2 . The increase in A 410nm was monitored for 4 min at 10-s intervals. The definition of one unit of enzyme activity is the change of 1.0 absorbance unit per minute at 410 nm, while that of one unit of inhibitory activity ŽU. is that causing 50% inhibition of the enzyme under the defined assay conditions.

811

2.7. Protein determination Protein concentrations were determined using the BCA microtiter plate assay ŽSmith et al., 1985. with BSA as standard. 2.8. Acti¨ e-site titration The concentration of active enzyme was determined according to the method described by Chase and Shaw Ž1967. using p-NPGB. The reaction was followed at 410 nm in a microtiter plate reader. 2.9. SDS-PAGE SDS-PAGE was performed in 10% gels according to the method of Laemmli Ž1970. as described in the Bio-Rad Mini-proteanTM II Dual Slab cell instruction manual. Approximately 10 ␮g of protein was loaded per lane. 2.10. Amino acid composition and N-terminal sequence determinations Amino acid composition and N-terminal sequence determinations were performed as described by Mkwetshana et al. Ž1999.. 2.11. pH optima The pH optima of both ostrich and commercial bovine plasmins were determined by performing the plasmin assay, but using different buffer systems for the different pH values, namely 50 mM Na᎐citrate buffers for pH values 4᎐6 and 50 mM Tris᎐acetate buffers for pH values 6᎐9 and 10.1. 2.12. Temperature optima To determine the temperature optima of ostrich and commercial bovine plasmins, the plasmin assay was scaled up 2.5-fold so that a 1-ml cuvette and a Perkin-Elmer Lambda 3A spectrophotometer fitted with temperature control could be used. The assays, using the Tris᎐acetate ŽpH 8.0. buffer, were performed at 20, 25, 30, 35, 40, 45, 50 and 60⬚C by incubating the enzyme and substrate, and assaying at the respective temperatures.

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A.R. Thomas et al. r Comparati¨ e Biochemistry and Physiology Part B 129 (2001) 809᎐820

2.13. Kinetic parameters The kinetic parameters of ostrich and commercial bovine plasmins were determined for three synthetic pNA substrates, namely N-p-TosylGly᎐Pro᎐Lys-pNA, D-Val᎐Leu᎐Lys-pNA, Nbenzoyl-Phe᎐Val᎐Arg-pNA, and a natural substrate, namely bovine fibrinogen, using various substrate concentrations. The plasmin assay procedure was followed for the three synthetic pNA substrates, while the assays with bovine fibrinogen employed the discontinuous TCA assay.

3. Results and discussion 3.1. Purification of ostrich ␣ 2 AP Figs. 1 and 2 depict the elution profiles of fraction B on Toyopearl Super Q-65OS and of fraction C on ostrich LBSI᎐Sepharose, respectively. Despite the low purification factor, ostrich ␣ 2 AP was purified to homogeneity ŽFig. 3.. The fact that only the plasminogen-binding form of ␣ 2 AP was purified by ostrich LBSI᎐Sepharose chromatography could have contributed to the low purification fold and yield of active ostrich ␣ 2 AP Ž7.3 mg from 530 ml.. 3.2. Purification of ostrich plasminogen

Fig. 2. Ostrich LBSI᎐Sepharose chromatography of fraction C. Tubes 34 and 60, start of elution with 20 mM sodium phosphate buffer ŽpH 7.4. and 50 mM EACA, respectively; fraction D, tubes 60᎐67.

L-lysine᎐Sepharose

chromatography, as shown in

Fig. 4. 3.3. Acti¨ ation of ostrich plasminogen to plasmin Fig. 5 shows the activation of ostrich plasminogen to plasmin using various urokinaserplasminogen concentrations at various incubation times. An optimum activator concentration of 600 ␮g urokinasermg plasminogen and incubation period of 4 h were therefore used to activate

Ostrich plasminogen was highly purified after

Fig. 1. Gradient elution profile of Toyopearl Super Q-650S chromatography of fraction B. ᎏ, A 280nm ; ---, wNaClx ŽM.; fraction C, tubes 73᎐93.

Fig. 3. SDS-PAGE patterns of ostrich and commercial human ␣ 2 AP ŽCoomassie stained.. Where not stated, samples were non-reduced. Lanes: 1, HMW markers with Mr values in K; 2, fraction D; 3, commercial human ␣ 2 AP; 4, 5 and 6, fractions in lanes 1, 2 and 3 Žreduced., respectively.

A.R. Thomas et al. r Comparati¨ e Biochemistry and Physiology Part B 129 (2001) 809᎐820

813

ostrich plasminogen to plasmin. The decline in plasmin activity under the above conditions possibly suggests autodigestion of plasmin owing to its high concentration ŽRobbins and Summaria, 1976a.. Despite the high urokinaserplasminogen concentrations, ostrich plasminogen was not completely activated to ostrich plasmin ŽFig. 6.. Robbins and Summaria Ž1970., on the other hand, used a urokinaserplasminogen concentration of

Fig. 5. Activation of ostrich plasminogen to plasmin with various urokinaserplasminogen concentrations at various incubation times. ⽧, 600 ␮grmg; ', 10 ␮grmg; I, 5 ␮grmg; `, 0 ␮grmg.

only 0.25 ␮grmg, but an incubation time of 20 h, for the activation of human plasminogen with human urokinase. Optimal activator concentration and incubation time do, however, depend on the specific activity of the activator and probably also the source of the activator and proenzyme. Interestingly, it has been reported that some species Ždog and rabbit. require much higher streptokinase concentrations to achieve complete activation of plasminogen than other species Žman, monkey and cat., but that this is generally not the case for urokinase ŽRobbins and Summaria, 1970.. Ostrich plasminogen could, however, be an exception, requiring higher concentrations of urokinase than other species. Streptokinase could also be used to activate ostrich plasminogen and it would be interesting to establish whether low or high concentrations of the activator are required. 3.4. Partial characterisation of ostrich ␣ 2 AP

Fig. 4. SDS-PAGE pattern of ostrich plasminogen ŽCoomassie stained.. Lanes: 1, HMW markers with Mr values in K; 2, ostrich plasminogen.

3.4.1. Molecular weight determination SDS-PAGE revealed a Mr of 83.6 K for reduced and non-reduced ostrich ␣ 2 AP and 67.0 K for commercial human ␣ 2 AP ŽFig. 3.. Human ␣ 2 AP showed a Mr of 70 K before reduction ŽWiman and Collen, 1977. and 67 K after reduction ŽMoroi and Aoki, 1976; Wiman and Collen, 1977., while bovine ␣ 2 AP has a Mr of 70 K after reduction ŽChristensen and Sottrup-Jensen, 1992..

A.R. Thomas et al. r Comparati¨ e Biochemistry and Physiology Part B 129 (2001) 809᎐820

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Commercial human ␣ 2 AP revealed a dimer Ž Mr 148.7 K. under non-reducing conditions ŽFig. 3., but of a larger size than that reported Ž132 K. ŽWiman and Collen, 1977.. Ostrich ␣ 2 AP, however, did not show the presence of a dimer. 3.4.2. N-terminal sequence determination Of the 11 N-terminal amino acids determined for ostrich ␣ 2 AP, only one and two residues are identical to those of human and bovine ␣ 2 APs, respectively, as compared to the seven that human and bovine ␣ 2 APs have in common ŽTable 1..

Fig. 6. SDS-PAGE patterns of ostrich and commercial bovine plasmins ŽCoomassie stained.. Lanes: 1, HMW markers with Mr values in K; 2, ostrich plasmin; 3, commercial bovine plasmin. In lane 2 the bands at Mr 92 K, 78 K and 55 K are ostrich plasminogen, ostrich plasmin and urokinase, respectively.

3.4.3. Inhibition of different serine proteases Ostrich and commercial human ␣ 2 APs have the strongest inhibitory effects on ostrich plasmin, followed by bovine trypsin and finally bovine plasmin ŽTable 2.. Enghild et al. Ž1993. showed that human ␣ 2 AP reacts most rapidly with plasmin, followed by trypsin and then chymotrypsin. They obtained an inhibitory activityrŽ IrE . value of ; 2 U for the inhibition of bovine trypsin by human ␣ 2 AP. Comparing a few plasmin inhibitors ŽTable 2., commercial bovine aprotinin appears to be the most potent inhibitor, much more so than ␣ 2 AP, which should theoretically not be the case ŽHighsmith, 1979.. Ostrich ␣ 2 AP shows a stronger inhibitory effect on ostrich plasmin than on its bovine counterpart, whereas human ␣ 2 AP has the opposite effect. The synthetic inhibitors inhibit plasmin to a much smaller degree than the natural ones, with EACA being the weakest plasmin inhibitor, not inhibiting ostrich plasmin at all. However, at high concentrations EACA showed a maximum inhibition of 34.2% on commercial bovine plasmin Žresults not shown..

Table 1 Comparison of N-terminal sequence of ostrich ␣ 2 AP with those of human and bovine ␣ 2 APs a

Ostrich Humanb Bovineb a

1

2

3

4

5

6

7

8

9

10

11

Leu Met Met

Gln Glu Glu

Val Pro Pro

Asp Leu Leu

Tyr Gly Asp

Leu Arg Leu

Val Gln Gln

Leu Leu Leu

Glu Thr Met

Val Ser Asp

Ala Gly Gly

Amino acids identical to those of ostrich ␣ 2 AP are in bold. See Christensen et al. Ž1994..

b

A.R. Thomas et al. r Comparati¨ e Biochemistry and Physiology Part B 129 (2001) 809᎐820

815

Table 2 Effect of inhibitors on ostrich plasmin, and commercial bovine plasmin and trypsina

plasminogens is to be expected since the ostrich belongs to the avian species.

Inhibitor

3.5.3. Amino acid composition The amino acid composition of ostrich plasminogen correlates well with those of human and rabbit plasminogen ŽTable 4., the only significant differences being increased values for Asx, Leu and Tyr, and decreased values for Ser, Pro and Cyh. The decreased Cyh value is, however, expected since Cyh was not protected during the acid hydrolysis procedure. From the amino acid composition data a Mmin of 89.4 K was calculated for ostrich plasminogen.

Inhibitory effects ŽU. Ostrich plasmin

Ostrich ␣2 AP Comm. human ␣2 AP Comm. bovine aprotinin DFP EACA

Comm. bovine plasmin

Comm. bovine trypsin

7.53 3.52

0.42 0.96

3.35 2.65

206.11

2.81

ND

0.06 2.40= 10y6

ND ND

0.01 0

a

Values represent inhibitory Žmolrmol.. ND, not determined.

activity

Ž U . r Ž IrE .

3.6. Partial characterisation of ostrich plasmin 3.5. Partial characterisation of ostrich plasminogen

3.6.1. Molecular weight determination The Mr of ostrich plasmin determined by SDS-PAGE is 78.1 K ŽFig. 6., which is the same as that obtained for commercial bovine plasmin, similar to that of human plasmin Ž75.4, 76.5 or 81.0 K. ŽRobbins and Summaria, 1976b., but slightly smaller than that of rabbit plasmin Ž82.0᎐86.0 K. ŽRobbins and Summaria, 1976b.. The molecular weights of human and rabbit plasmins were, however, determined by sedimentation equilibrium methods.

3.5.1. Molecular weight determination The Mr of ostrich plasminogen, as determined by SDS-PAGE, is 92.1 K ŽFig. 4.. This corresponds well with that reported for native human plasminogen Ž90᎐94 K. ŽCastellino and Powell, 1981. and the rabbit plasminogen forms Ž89᎐94 K. ŽRobbins and Summaria, 1976b., as determined by sedimentation equilibrium analysis. 3.5.2. N-terminal sequence determination The 16-residue N-terminal sequence of ostrich plasminogen shows 53% identity with that of human plasminogen Žexcluding residue 9., and similarly with those of cat and ox plasminogens Žtheir deduced sequences are just a little shorter.; it has one more residue in common with rabbit plasminogen ŽTable 3.. The N-terminus of dog plasminogen is blocked ŽRobbins and Summaria, 1976b.. This relatively low identity with mammalian

3.6.2. N-terminal sequence determination The N-terminal sequence of ostrich plasmin, aligned with those of five mammalian plasmins in Table 5, shows only 35% identity with that of human plasmin ŽLys78 ᎐plasmin.. It shows a better identity with those of cat and ox plasmins and the best with that of dog plasmin, but these sequences are too short to make significant comparisons.

Table 3 Comparison of N-terminal sequence of ostrich plasminogen with those of four mammalian plasminogens a

Ostrich Humanb Rabbitc Catb Oxb a

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

Asn Glu Glu Asp Asp

Ile Pro Pro Pro Leu

Leu Leu Leu Leu Leu

Asp Asp Asp Asp Asp

Gly Asp Asp Asp Asp

Tyr Tyr Tyr Tyr Tyr

Val Val Val Val Val

Arg Asn Asn Asn

X Thr Thr X

Glu Gln Glu Gln

Gly Gly Gly Gly

Ala Ala Ala Ala

Trp Ser

Leu Leu

Leu Phe

Ser Ser

Amino acids identical to those of ostrich plasminogen are in bold. X, unknown. See Robbins and Summaria Ž1976a.. c See Robbins and Summaria Ž1976b.. b

A.R. Thomas et al. r Comparati¨ e Biochemistry and Physiology Part B 129 (2001) 809᎐820

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Table 4 Amino acid composition Žmolar ratios. of ostrich plasminogen and plasmin compared to those of human and rabbit plasminogens and plasmins, respectively Amino acid

Ostrich plasminogen

Human plasminogena

Rabbit plasminogenb

Human plasminc

Rabbit plasmind

Form 2

Ostrich plasmin

Form 1

Form 1

Form 2

Asx Thr Ser Glx Pro Cyh Gly Ala Val Met Ile Leu Tyr Phe His Lys Trp Arg

107.9 Ž108. 53.6 Ž54. 39.4 Ž39. 87.8 Ž88. 45.5 Ž45. 15.6 Ž16. 54.3 Ž54. 45.4 Ž45. 32.0 Ž32. 7.4 Ž7. 26.8 Ž27. 54.3 Ž54. 48.4 Ž48. 20.1 Ž20. 24.6 Ž25. 54.3 Ž54. ND Ž20.e 39.4 Ž39.

76 57 51 92 73 38 58 38 44 8 23 43 28 19 22 50 21 39

80 58 58 84 58 48 56 48 32 9 22 40 30 18 21 53 19 55

78 57 58 83 58 49 57 49 34 10 23 41 32 19 20 50 19 54

71.2 Ž72. 41.6 Ž42. 49.0 Ž49. 75.2 Ž75. 58.7 Ž59. ND Ž40.e 63.8 Ž64. 43.8 Ž44. 9.1 Ž9. ND Ž7.e 8.5 Ž9. 16.5 Ž17. 28.5 Ž29. 5.7 Ž6. 12.0 Ž12. 45.0 Ž45. ND Ž20.e 41.0 Ž41.

60 50 40 59 61 37 53 29 36 8 15 36 26 14 21 38 16 36

81 53 58 76 71 45 57 39 29 9 21 38 34 13 18 42 25 46

80 53 53 73 73 45 59 40 32 10 22 38 35 16 16 41 25 46

Total

775

780

790

792

635

755

757

638

a

See Wallen ´ and Wiman Ž1972. Žisoelectric form of pI 6.23.. See Sodetz et al. Ž1972.. c See Robbins and Summaria Ž1976a.. d See Robbins and Summaria Ž1976b.. e ND, not determined Žassumed value.. b

3.6.3. Amino acid composition Most of the amino acid residues of ostrich plasmin fell within the range of those of human and rabbit plasmins ŽTable 4., but the residues

that gave significantly lower values were Thr, Val, Ile, Leu, Phe and His. The total number of amino acid residues of ostrich plasmin are similar to that of human plasmin, while the rabbit plasmin forms

Table 5 Comparison of N-terminal sequence of ostrich plasmin with those of five mammalian plasmins a

Ostrich Humanb Catc Dogc Rabbitc Oxc

Ostrich Humanb Catc Dogc Rabbitc Oxc a

1

2

3

4

5

6

7

8

9

Arg Lys Lys Arg Lys Lys

Ile Val Ile Ile Val Ile

Tyr Tyr Tyr Tyr Tyr Tyr

Leu Leu Leu Leu Leu Leu

Asp Ser Val Gly Gly Val

Glu Glu

Val Cys

Glu Lys

Gly Thr

10 Arg Gly

11 Asp Asp

12 Val Gly

13 Val Lys

14 Tyr Asn

15 Tyr Tyr

16 Arg Arg

17 Thr Gly

Amino acids identical to those of ostrich plasmin are in bold. See Castellino and Powell Ž1981.. c See Robbins and Summaria Ž1976a.. b

A.R. Thomas et al. r Comparati¨ e Biochemistry and Physiology Part B 129 (2001) 809᎐820

displayed substantially higher values than both those of ostrich and human plasmins. The amino acid residues of the rabbit plasmin forms were, however, obtained by the direct addition of the amino acid residues of the plasmin heavy and light chains, which can give slightly overestimated values, as was the case for human plasmin ŽRobbins and Summaria, 1976a.. From the amino acid composition data a Mmin of 75.5 K was calculated for ostrich plasmin. 3.6.4. pH optimum Ostrich and commercial bovine plasmins showed similar pH profiles, both having a pH optimum of 8.0 Žthe pH optimum of commercial bovine plasmin is actually between 7.0 and 8.0., but ostrich plasmin is not as quickly inactivated at high pH values Ži.e. above pH 8.0. as commercial bovine plasmin Žresults not shown.. Christensen and Ipsen Ž1979. obtained a pH optimum of 7.60 for human plasmin. 3.6.5. Temperature optimum Ostrich and commercial bovine plasmins showed temperature optima of 40 and 45⬚C, respectively Žthe temperature optimum of commercial bovine plasmin is actually between 40 and 45⬚C. Žresults not shown.. Ostrich plasmin is much more quickly inactivated at temperatures below its optimum Žbetween 20 and 35⬚C. than its commercial bovine counterpart, having only 22% activity at 20⬚C, whereas commercial bovine plasmin showed 56% activity. However, high temperatures, above the optima, inactivated both plasmins to a similar extent, with ostrich and commercial bovine plasmins showing 44 and 49% activity, respectively, at 60⬚C. 3.6.6. Thermodynamic parameters The activation energy Ž Ea . for the reaction of ostrich plasmin with D-Val᎐Leu᎐Lys-pNA is almost three times larger than that for commercial bovine plasmin ŽTable 6., implying that ostrich plasmin is less capable of lowering the Ea for the reaction with D-Val᎐Leu᎐Lys-pNA, thus facilitating a slower reaction. Ostrich plasmin is therefore less efficient as an enzyme than its commercial bovine counterpart. ⌬ H ‡ for the reaction with ostrich plasmin is also almost three times larger than for the one with commercial bovine plasmin, implying that the reaction with ostrich plasmin to form the transition state is more endothermic and

817

Table 6 Summary of thermodynamic parameters for the reaction of ostrich and commercial Žcomm.. bovine plasmins with DVal᎐Leu᎐Lys-pNA Plasmin

Ea ⌬ H‡ ⌬ S‡ ŽkJrmol. ŽkJrmol. ŽJrmol K.

Ostrich 64.95 Comm. 23.79 bovine

62.43 21.27

y51.87 y185.52

⌬G‡ r2 ŽkJrmol. 78.67 79.37

0.983 0.994

therefore less efficient. However, ⌬ S ‡ is almost four times less negative for ostrich plasmin than for commercial bovine plasmin, which is more favourable since the increase in the order of the molecules in moving from the substrate to the transition state is not so drastic for the reaction with ostrich plasmin than for the one with commercial bovine plasmin, and therefore it does not require as much energy. The combined effects of ⌬ H ‡ and ⌬ S ‡ produce a similar ⌬G ‡ value for both ostrich and commercial bovine plasmins, but ostrich plasmin has a slightly lower value and is therefore marginally more successful at lowering the free energy of the transition state. 3.6.7. Kinetic parameters From Table 7 it is evident that Gly᎐Pro᎐LyspNA is by far the best synthetic substrate for plasmin, followed by Val᎐Leu᎐Lys-pNA, whose k cat values are more than half those of Gly᎐Pro᎐Lys-pNA for the respective plasmins, and finally Phe᎐Val᎐Arg-pNA with values more than 20 tim es sm aller than those of Gly᎐Pro᎐Lys-pNA for the respective plasmins. k cat values of 13.5" 0.5 and 8.5" 0.3 sy1 were reported for hum an plasm in with D Val᎐Leu᎐Lys-pNA and benzoyl-L-Phe᎐Val᎐ArgpNA, respectively, at pH 8.00 and 25⬚C ŽChristensen and Ipsen, 1979.. The former value is higher than that for ostrich plasmin, but lower than that for commercial bovine plasmin, while the latter value is approximately 30 times and four times higher than those for ostrich and commercial bovine plasmins, respectively. Considering the K m values, however, Phe᎐ Val᎐Arg-pNA has the lowest values of all three synthetic substrates for both plasmins ŽTable 7.. The K m value for ostrich plasmin with Gly᎐Pro᎐Lys-pNA is 1.6 times lower than that for its commercial bovine counterpart, while Val᎐Leu᎐Lys-pNA has the opposite effect, but

A.R. Thomas et al. r Comparati¨ e Biochemistry and Physiology Part B 129 (2001) 809᎐820

818

Table 7 Summary of kinetic parameters of ostrich and commercial Žcomm.. bovine plasmins Substrate

Plasmin

Gly᎐Pro᎐Lys-pNA

Ostrich Comm. bovine Ostrich Comm. bovine Ostrich Comm. bovine Ostrich Comm. bovine

Val᎐Leu᎐Lys-pNA Phe᎐Val᎐Arg-pNA Bovine fibrinogen

Vma x Ž␮Mrs.

Km ŽmM.

kcat Žsy1 .

1.230 1.143 0.591 0.520 0.060 0.056 0.030a 0.018a

0.333 0.530 0.379 0.298 0.186 0.183 0.030 0.016

5.702 45.034 2.741 20.492 0.277 2.193

kcatrKm ) ŽmMy1 ⭈ sy1 . 17.100 85.037 7.239 68.678 1.486 11.971

r2

0.998 0.999 1.000 0.992 0.990 0.981 0.939 0.987

a

⌬ A 280nm r30 min.

the difference in K m values is only 1.3 times. The K m values for both plasmins with Phe᎐Val᎐ArgpNA are very similar. Christensen and Ipsen Ž1979. reported K m values of 0.22" 0.02 and 0.69 " 0.04 mM for human plasmin with DVal᎐Leu᎐Lys-pNA and benzoyl-L-Phe᎐Val᎐ArgpNA, respectively, at pH 8.00 and 25⬚C. The former K m value is lower than those for both ostrich and commercial bovine plasmins, as well as for both with Gly᎐Pro᎐Lys-pNA, but the K m value for human plasmin with benzoylL-Phe᎐Val᎐Arg-pNA is almost four times larger than those for both ostrich and commercial bovine plasmins. Bovine fibrinogen gave the lowest K m value of all the substrates used, which is to be expected since it is a natural substrate. The K m value is especially low for commercial bovine plasmin, which is its natural enzyme and which has an almost two times higher affinity compared to ostrich plasmin, as well as for human plasmin, which has the same K m value as for ostrich plasmin, i.e. 0.03 mM ŽDeutsch and Mertz, 1970.. Comparing k catrK m values for all three synthetic substrates, commercial bovine plasmin is 5᎐9.5 times more efficient than ostrich plasmin ŽTable 7.. The k catrK m values of human plasmin w ith V al ᎐ L eu ᎐ L ys-p N A a n d b en zoylL-Phe᎐Val᎐Arg-pNA at pH 8.00 and 25⬚C are 60 " 4.1 and 12.4" 0.3 mMy1 ⭈ sy1 , respectively ŽChristensen and Ipsen, 1979., which correspond more with those values obtained for commercial bovine plasmin, the former value being slightly lower and the latter slightly higher than the respective values obtained for commercial bovine plasmin. Comparing the k catrK m values for the different synthetic substrates, Gly᎐Pro᎐Lys-pNA is the synthetic substrate most favoured by both ostrich and commercial bovine plasmins, followed

b y V a l ᎐ L e u ᎐ L ys-p N A a n d la stly b y Phe᎐Val᎐Arg-pNA. In conclusion, ostrich ␣ 2 AP, plasminogen and plasmin showed many physical, chemical and kinetic properties similar to those of other known ␣ 2 APs, plasminogens and plasmins, but this study showed a few significant differences between the fibrinolytic system of the ostrich Žor birds in general. and those of mammals. It has, however, been suggested that the fibrinolytic systems of the ostrich and chicken are comparable to that of humans based on the ␣ 2-antiplasmin levels, while the clotting system of the ostrich was equated to one between those of reptilian and avian species ŽFrost et al., 1999.. This latter finding is supported by the complete amino acid sequences of ostrich proopiomelanocortin NH 2-terminal fragment ŽNaude ´ et al., 1993. and MSEL-neurophysin and its associated copeptin ŽLazure et al., 1989., which revealed a close resemblance to non-mammalian ‘lower’ species, e.g. amphibia. A study of ␣ 2 AP, plasminogen and plasmin of other nonmammalian species would therefore prove useful for comparison purposes.

4. Nomenclature ␣ 2 AP: ␣ 2 PI: BAPNA: BCA: DABS-Cl: DFP: DMSO: EACA:

␣ 2-antiplasmin ␣ 2-plasmin inhibitor Benzoyl-DL-arginine-p-nitroanilideHCl Bicinchoninic acid 4- Ždimethylamino .azobenzene-4⬘sulfonyl chloride Diisopropyl fluorophosphate Dimethyl sulfoxide ␧-aminocaproic acid

A.R. Thomas et al. r Comparati¨ e Biochemistry and Physiology Part B 129 (2001) 809᎐820

FITC᎐PITC: Fluorescein isothiocyanate᎐phenylisothiocyanate KIU: Kallikrein inhibitory unitŽs. LBSI: Lysine-binding site I pNA: p-nitroanilide p-NPGB: p-nitrophenol p⬘-guanidinobenzoate HCl PVDF: Polyvinylidene difluoride SAPNA: N-succinyl-Ala᎐Ala᎐Ala-pnitroanilide Serpin(s): Serine proteinase inhibitorŽs. TCA: Trichloroacetic acid

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