Biochimica et Biophysica Acta, 393 (1975) 267-273
© Elsevier Scientific Publishing Company, Amsterdam- Printed in The Netherlands BBA 37068 PHYSICAL P A R A M E T E R S A N D C H E M I C A L COMPOSITION OF PORCINE P A N C R E A T I C ELASTASE II
WOJCIECH ARDELT Department of Biochemistry, Institute of Rheumatology, 02-637 Warsaw (Poland)
(Received January 28th, 1975)
SUMMARY Some molecular properties of the elastase II preparation, homogenous in ultracentrifugation, have been determined. The molecular weight is 25 000, the sedimentation coefficient and the diffusion coefficient are 3.69.10 -la s -1 and 12.09.10 -7 cm2/s, respectively. The partial specific volume was 0.716 g/cm 3, and the axial ratio is 1.95. Elastase II exhibited a considerably lower content of arginine, tyrosine, and valine, and a higher content of proline, serine and conjugated carbohydrates than elastase I. The N-terminal amino acid of the enzyme is leucine, and its isoelectric point was 10.7.
INTRODUCTION In previous papers [1, 2], the purification as well as some enzymatic and physical properties of porcine pancreatic elastase II were described. The enzyme appeared to be different in respect to the specificity and kinetic properties from the well characterized elastase I (EC 3.4.21.11) and other pancreatic proteases. This paper presents some molecular properties of this enzyme in comparison to those of elastase I. MATERIALS AND METHODS Bovine serum albumin, crystalline, D(+)-mannose, and o(+)-galactose were purchased from BDH; D(+)-glucosamine hydrochloride and N-acetylneuraminic acid from Fluka; human 7-globulin, Cohn fraction II from Koch-Light, and dansyl chloride from Sigma Chem. Co. Porcine pancreatic elastases, II and I were prepared from a commercial pancreas powder (POLFA, Warsaw), according to the method described previously [1]. Enzyme concentrations were determined using the absorbance indexes ~AI% ~ at 282 nm of 20.5 and 18.7 for elastase II and 1, respectively [1]. •," ~ I c r n :
268
Sedimentation and diffusion studies The experiments were carried out at 20 °C in a Beckman Spinco Model E analytical ultracentrifuge equipped with a rotor temperature control unit and schlieren optics. Runs were performed using an An-D rotor and 12 mm single-sector cell (sedimentation velocity) or a double-sector, capillary synthetic boundary centerpiece (diffusion). Sedimentation coefficients were evaluated by following the migration rate of the maximum ordinate [3]. Diffusion coefficients were evaluated from the area and the maximum height of the ultracentrifugal gradient curve [4]. The results were extrapolated to infinite dilution using the method of least squares, and corrected to water in the conventional way. Partial specific volume This was determined by the density gradient column method [5] using kerosene and bromobenzene as the gradient constituents, and potassium chloride solutions as standards. Molecular weight Molecular weight was calculated from the classical Svedberg equation, using the determined values of sedimentation and diffusion coefficient as well as partial specific volume. Shape parameters Frictional ratio was calculated using the formula given by Elias [6]. The axis lengths and intrinsic viscosity were calculated according to Sheraga [7] using the values of an axial ratio and Sihma parameter, taken from the tables [8]. Isoelectric point This was determined by paper electrophoresis in 0.1 M NaHCO3/NaOH buffers (pH range: 9.6-11.0). Runs were performed on 12 × 2 cm Whatman No. l paper strips at 6 V/cm, and room temp., for 2 h. 50 #g samples of the enzyme were employed, and visualized with Amido Black. For a comparison, samples of elastase I were investigated in parallel. Amino acid analysis Samples of elastase II were hydrolysed in 6 M HCI at 110 °C for 22 h in sealed, evacuated hydrolysis tubes. Analyses were carried out using a JEOL J L C - 6 A H automatic amino acid analyser. Halfcystine was determined as cysteic acid. Tryptophan content was calculated from the value for tyrosine and the molar ratio tyrosine: tryptophan, as determined spectrophotometrically [9]. Determination of the N-terminal amino acid This was performed by the dansyl chloride method, essentially as described by Fuller et al. [10]. However, 10-nmol aliquots of the enzyme were used for a single experiment, the final concentration of trichloroacetic acid was 25 ~ , and the precipitation of the dansylated protein proceeded overnight at --10 °C. The dansylated amino acid was determined by the thin layer chromatography on silica gel [11 ].
269
Carbohydrate content Protein-bound hexoses were determined with the orcinol method [12] using equimolar solutions of galactose and mannose as standards. For a control, the conjugated carbohydrates were determined in parallel in human ~'-globulin and bovine serum albumin. For the determination of hexosamines, the enzyme samples were hydrolysed in 6 M HC1 at 100 °C for 8 h, and the aminosugars were isolated on a cation exchange resin [13]. The determination was performed using the micromethod of Antonopoulus et al. [14]. Neuraminc acid was determined with the thiobarbituric acid method [15]. RESULTS AND DISCUSSION Disc polyacrylamide gel electrophoresis of the elastase II preparation obtained as described previously [1], revealed one main band and a small, slightly slower moving diffused fraction. The latter also appeared to be active towards all substrates of the enzyme. This probably means that some aggregation processes may occur under the conditions of preparation and/or electrophoresis. The enzyme appeared homogenous in the analytical ultracentrifuge, e.g. migrated as a single symmetrical peak (Fig. I). It was therefore decided that the preparation was pure enough to perform some structural studies.
Fig. 1. Sedimentation pattern of elastase II. The sample was dissolved in 0.3 M sodium acetate buffer, pH 5.0, at a concentration of 5.8 mg/ml. A single-sector cell in an An-D rotor was used. Pictures were taken after 32 and 40 min. after a maximum speed of 59 780 rev./min was reached. Migration is from left to right.
270 Fig. 2 and 3 present the dependence of sedimentation and diffusion coefficients, respectively, on the enzyme concentration. The values extrapolated (So = 3.2810 -'3 s -L, and D°0 = I 1.3 • 10 -7 cm2/s) were corrected to water, giving the values presented in Table I.
•~
3.5
3.0
2.5
*~
2.0
i
I
I
I
I
I
2
~
4
5
6
~lastaee I I concent~ati0n /=g/=I/
Fig. 2. Elastase II sedimentation coefficient dependence o n concentration. Conditions as in Fig. 1. T h e line was fitted to the data by the m e t h o d o f least squares.
o
14.o
12,0
o o
~o
1o.o i
0
I
2
i
4
6
KJ-aatase I I c o n c e n t r a t i o n
8
I0
/~g/mL/
Fig. 3. Elastase 1I diffusion coefficient dependence o n concentration. Conditions as in Fig. 1, except that a double-sector cell was used at 20 000 rev./min. T h e line was fitted to the data by the m e t h o d of least squares.
The molecular weight evaluated (24 990) is higher than the result obtained previously [1] by the gel filtration method (21 900), and is of the same order as the values calculated for elastase I from ultracentrifugation [16] as well as from the sequence [17] studies (25 000 and 25 900, respectively). In spite of the similar molecular weight, elastase II appeared different from elastase I in respect to volume and shape of the molecule. The partial specific volume and the frictional coefficient (Table 1) were lower then the corresponding values of 0.73 g/cm 3 and 1.2 recorded for elastase I [16].
271 TABLE I PHYSICAL PARAMETERS OF PORCINE PANCREATIC ELASTASE II Parameter
Value obtained
Sedimentation coefficient (s°z0,w) Diffusion coefficient (D°20.w) Partial specific volume (v) Molecular weight Frictional ratio (f/f0) Axial ratio (a/b) Axial length (2a) (2b) Sihma parameter Intrinsic viscosity Isoelectric point
(3.69 ___0.10).10-13-s -~ (12.09 ± 0.10). 10-7 cm2/s 0.716 ± 0.007 cm3/g 24 990 1.04 1.95 47.8 A 24.6 A 2.91 2.08 cm3/g 10.7
Electrophoretic d e t e r m i n a t i o n of the isoelectric p o i n t for elastase I1 gave the value 10.7. O n the other hand, elastase I migrated to cathode even at p H 11.0. Thus, the isoelectric p o i n t for this enzyme seems to be higher t h a n the value 9.5 :~ 0.5, recorded by Lewis et al. [16], a n d even higher t h a n 11.0. A n explanation for the higher isoelectric p o i n t of elastase I as compared to that of elastase II, was provided by the a m i n o acid analysis (Table II a n d ref. 17). TABLE II AMINO ACID COMPOSITION OF PORCINE PANCREATIC ELASTASE II Amino acid residue
/~mol/100/zmol of amino acids recovered a
No. of residues per 25 000 daltons
Nearest integer per 25 000 daltons
Lysine Histidine Arginine Aspartic acid b Threonine Serine Glutamic acid b Proline Glycine Alanine Cysteine c Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Tryptophana
1.85 2.05 2.66 8.41 7.39 I I. 10 8.24 8.41 10.00 8.24 4.23 8.65 1.20 4.80 8.30 2.39 1.70 3.33
4.26 4.74 6.13 19.41 17.06 25.62 19.02 19.41 23.10 19.02 9.74 19.96 2.77 11.08 19.16 5.52 3.93 7.68
4 5 6 19 17 26 19 19 23 l9 l0 20 3 l1 19 6 4 8
a 22 h hydrolysis. b Free plus amide. c As cysteic acid. d Determined spectrophotometrically.
272 The enzymes had a similar content of lysine and histidine, but elastase I1 exhibited half the content of arginine. Elastase II had twice as much proline as elastase I. This implies a lower contribution of a-helix in the secondary structure. Nevertheless, considering the values of partial specific volume, the molecule seems to be more compact in comparison to elastase 1. Somewhat higher cysteine content might be a reflection of a higher degree of crosslinking. Another characteristic feature of elastase 1I is its low molar ratio of tyrosine : tryptophan (0.72 as compared to 1.59 for elastase 1). This can explain the differences in ultraviolet spectra of both enzymes, recorded previously [1, 18]. Amino-terminal analysis of elastase II gave leucine. However, extensive denaturation [11] was necessary to make the residue available for dansyl chloride. It is therefore probable, that the amino-terminus is blocked or buried inside the molecule. The amino-terminal amino acid of elastase I is valine [17]. Another difference between elastase II and elastase I is the amount of conjugated carbohydrates. Neither hexosamines nor neuraminic acid could be detected in the enzymes, but elastase II contained more protein-bound hexoses (0.70~ as compared to 0.25 ~ for elastase I). This seems to indicate that one molecule of a carbohydrate, presumably hexose, is firmly bound to one molecule of the enzyme protein. The molecular properties of porcine pancreatic elastase II, presented in this paper, provide further evidences that this enzyme is different from the well characterised elastase I. Elastase II combines some specificity features of elastase 1 and chymotrypsin [1]. It seems therefore reasonable to suppose that elastase II precedes those enzymes in the evolution of pancreatic serine proteases. ACKNOWLEDGEMENTS The author is greatly indebted to Professor Irena Chmielewska for her helpful suggestions during the preparation of the manuscript and to Mrs Krystyna Minc for the technical assistance. The ultracentrifugation experiments were performed by Miss M. Majewska, Inst. of Nuclear Research, Warsaw. REFERENCES 1 Ardelt, W. (1974) Biochim. Biophys. Acta 341, 318-326 2 Ardelt, W. (1974) Abstr. 9-th FEBS Meeting, Budapest, p. 63 3 Schachman, H. K. (1959) Ultracentrifugation in Biochemistry, pp. 75-102, Academic Press, New York and London 4 Ehrenberg, A. (1957) Acta Chem. Scand. 11, 1257-1270 5 Sakura, J. D. and Reithel, F. J. (1972) in Methods in Enzymology (Hirs, C. W. H. and Timasheff, S. N., eds), Vol. 26, pp. 107-119, Academic Press, New York and London 6 Elias, H. G. (1961) Ultracentrifugen Methoden, p. 126, Beckman Instruments. GmbH., Munchen 7 Sheraga, H. A. (1961) Protein Structure, pp. 1-30, Academic Press, New York and London 8 Yang, Y. T. (1961) Adv. Prot. Chem. 16, 323-396 9 Beaven, G. H. and Holliday, E. R. (1952) Adv. Prot. Chem. 7, 319-386 10 Fuller, G. M., Boughter, J. M. and Morazzoni, M. (1974)Biochemistry 13, 3036-3041 11 Morse, D. and Horecker, B. L. (1966) Anal. Biochem. 14, 429-433.
273 12 Winzler, R. J. (1958) in Methods of Biochemical Analysis (Glick, D., ed.), Vol. 2, pp. 279-311 13 Boas, N. (1953) J. Biol. Chem. 204, 553-563 14 Antonopoulus, C. A., Gardell, S. and Szirmai, J. A. and De Tyssonsk, E. R. (1964) Biochim. Biophys. Acta 83, 1-19 15 Spiro, R. G. (1966) in Methods in Enzymology (Neufeld, E. F. and Ginsburg, V., eds), Vol. 8, pp. 3-26, Academic Press, New York and London 16 Lewis, U. J., Williams, D. E. and Brink, N. G. (1956) J. Biol. Chem. 222, 705-720 17 Shotton, D. M. and Hartley, B. S. (1970) Nature 225, 802-806. 18 Ardelt, W. and Ksi~±ny, S. (1970) Acta Biochim. Polon. 17, 279-289