BIOCHIMICA ET BIOPHYSICA ACTA
BBA
503
25o93
CHARACTERIZATION OF A TRYPTIC FRAGMENT ISOLATED FROM THE
INSOLUBLE
TROPOMYOSIN
O F PI N N A NOBILIS* KENNETH
B A I L E Y * * , C E L I A P. D E M I L S T E I N * * * ,
C Y R I L M. K A Y AND L A W R E N C E B. S M I L L I E
Department of Biochemistry, Cambridge University, Cambridge (GreatBritain) and Department of Biochemistry, University of Alberta, Edmonton, Alberta (Canada) (Received D e c e m b e r I6th, 1963)
SUMMARY
The tryptic digestion of Pinna paramyosin yields, in addition to soluble products, a high-molecular-weight fragment which precipitates with 3 % trichloroacetic acid. This non-dialysable fraction has been isolated by a combination of isoelectric precipitation and ammonium sulfate fractionation, and has been subjected to chemical and physico-chemical studies. The amino acid composition of the fragment is very similar to that of the native protein. Despite the fact that it still contains a high proportion of lysine and arginine, the fragment is resistant to the further action of trypsin (EC 3.4.4.4), even when the molecule is initially exposed to a denaturing treatment. Unlike the native protein, the fragment possesses an N-terminal amino acid, glutamic acid. The physico-chemical properties of the tryptic resistant core have been established. The molecule is characterized by an S°o,~ of 2.88 S and a (,/) of o. 5 dl/g, as compared with corresponding values of 3.1 S. and 2.5 dl/g for the native molecule. The molecular weight of the fragment is IOOOOO, by both light scattering and Archibald ultracentrifugation.The shape of the molecule, on the basis of viscosity and lightscattering dissymmetry measurements, is best represented in terms of a coil model of length 525-55 ° A. Optical rotatory dispersion measurements suggest that the fragment is about 38 % helical, considerably less than the native molecule. Despite this lower helical content, the fragment is not attacked by trypsin, which may also be an indication of the globular character of the fragment as compared with the native protein which seems to be more exposed to enzyme action. This suggests that the tertiary structure must play a very important role in protecting the tryptic fragment from digestion. * The physico-chemical aspects of this w o r k were p r e s e n t e d b y C.M. KAY a t the S y m p o s i u m on the B i o c h e m i s t r y of Muscle Contraction, D e d h a m , Mass., May, 1962. ** Deceased May 22, 1963. * * * P r e s e n t address : I n s t i t u t e of A n i m a l Physiology, Agricultural Research Council, B a b r a h a m , Cambridge (Great Britain).
Biochim. Biophys. Acta, 9o (1964) 5o3-52o
504
K. BAILEY et al. INTRODUCTION
Several cases are known in the literature of proteins which are split by proteolytic enzymes giving fractions of halves or quarters of the original molecule. That is the case with certain antibodies, i.e. diphtheria 1, human y-globulin 2 and rabbit antiovalbumin y-globulin 3.Generally in the case of antitoxins these fragments are further split b y the same enzyme to smaller fractions. However, the case of y-globulin seems to be different. Papain-digested human y-globulin produces a particle of a quarter size of the original molecule which remains after 6 days of digestion indicating its stability 2. The three fragments obtained b y PORTERa from rabbit antiovalbumin y-globulin are exceptionally resistant to further digestion b y papain (EC 3.4.4.1o). LOCKERANDSCHMITT4 found b y following a short trypsin (EC 3.4.4.4) treatment of paramyosin from Venus mercenaria in the ultracentrifuge t h a t a profound change occurred in the protein. They thought that this change was identical with that found by MACFARLANE5 in the decomposition of rabbit tropomyosin by an enzyme from Clostridium oedematiens and which was interpreted by KEKWICK as a general breakdown of the molecule. However, we have found 6 that prolonged tryptic action on paramyosin from Pinna nobilis split only about one-half the total number of available bonds leaving beside soluble products a fraction which precipitates with 3 % trichloreacetic acid. This non-dialysable fraction has been isolated under neutral conditions and shown to be highly resistant to further tryptic action in spite of its amino acid composition which closely resembles that of the native protein. This isolated and purified fraction appears to be a well-defined fragment of molecular weight about iooooo and which still contains a high proportion of arginine and lysine. Chemical and physical properties were studied in order to characterize and differentiate this fragment from the native protein. A preliminary report has been published v.
MATERIALS AND METHODS Isolation of the fragment Proteolysis was carried out as previously described 6, which, in some experiments, included the presence of indole as a chymotryptic inhibitor in the incubation medium. After arresting hydrolysis by the addition of D F P or soybean trypsin inhibitor (2 parts inhibitor to i part enzyme), the digest was dialysed for 3 days at 4 ° against several changes of distilled water. The non-diffusible material was precipitated with 5 volumes of o.I N sodium acetate (pH 5.4) and dissolved in water b y adjusting the p H to 7-5 with NaOH. Neutral saturated ammonium sulphate was then slowly added to 38 % saturation. The precipitate obtained was discarded and more ammonium sulphate solution added to 5o % saturation. The resulting precipitate was dissolved in water at p H 7.5 and dialysed until free of ammonium ions. In all preparations the purity was checked by electrophoresis on paper with 0.06 M veronal buffer (pH 8.4) at a voltage gradient of 4-7 V/cm for 15 h. After drying, the paper was first sprayed with ninhydrin in order to detect material of low molecular weight, and then stained with bromophenol blue to reveal the protein-like component. Dialysis was continued until the ninhydrin-positive material was no longer present. At this stage the tryptic Biochim. Biophys. Acla, 90 (1964) 503 520
CHARACTERIZATION OF A TRYPTIC CORE OF PARAMYOSIN
505
fragment yielded a single peak both in the ultracentrifuge and free boundary electrophoresis (see Fig. I). Unlike native paramyosin the tryptic fragment after purification is soluble at p H 7.o-7.5 in water alone. A
B
Fig. I. A, u l t r a c e n t r i f u g e p a t t e r n of t h e t r y p t i c core in p h o s p h a t e buffer (0.035 M N a 2 H P O 4 a n d O.Ol 5 M KH2PO4) a t p H 7.0, t a k e n a t 64 m i n a f t e r r e a c h i n g t o p speed (59780 r e v . / m i n ) ; B, d e s c e n d i n g b o u n d a r y electrophoresis p a t t e r n of t h e t r y p t i c core in o.i M. Tris buffer (pH 7.9) a t 72 m i n , t e m p . 4 °.
Protein concentration This was determined by either the micro-Kjeldahl method as adapted by CHIBNALL, REES AND WILLIAMS 8, taking 18.5 % as the N content of paramyosin from Pinna nobilis9, or by absorbancy measurements at 280 m/z, using an El omOf%3.O (ref. IO).
Quantitative amino acid analysis Samples were hydrolysed in constant boiling HC1 under reflux for 24 h. The total acid hydrolysates were fractionated according to the improved column procedure of MOORE, SPACKMAN AND STEIN11. The yields of serine, threonine and methionine were corrected for destructive losses and lysine and ammonia were corrected for colour yield. The amino acid composition has been calculated by converting the yield of e-amino nitrogen into weight of anhydro-amino acid. The general method of calculation has been described by BAILEY AIN'DROEGG9.
N-terminal groups These determinations were carried out b y SANGER'S method 12 using about 0.2/,mole of the fragment. The protein content of the DNP-protein was assumed to be 70 % (ref. 13, I4), making allowance for water and D N P content. Correction factors due to losses in the hydrolysis and chromatography (20 and IO %, respectively) were applied. The DNP-amino acids were run on buffered paper in the p h t h a l a t e - t e r t i a r y amyl alcohol system of BLACKBURN AND LOWTHERla. The amount of e-DNP-lysine present in the hydrolysate of the DNP-tryptic fragment was estimated after fractionation of the water-soluble material by paper chromatography in p h t h a l a t e - t e r t i a r y amyl alcohol b y its absorption at 39 ° m/~ in I N HCI. Here again correction factors due to hydrolysis and chromatography losses were applied (5 and IO %, respectively). These correction factors were calculated by Biochim. Biophys. dcta, 9o (1964) 5o3 52o
506
K. B A I L E V
et al.
SANGER 12 for silica-gel-column chromatography and are therefore only a first approximation when applied to paper chromatography.
C-terminal groups C-terminal groups were determined by the carboxypeptidase method according to the procedure followed by LOCKER16 but using a solution of carboxypeptidase A (EC 3.4.2.1) (ref. 17) instead of a suspension. The enzyme was a three times recrystallised water suspension from Worthington Biochemical Corporation, Freehold, N.J.
(['.S.A.). Paramyosin Paramyosin was obtained from the whole adductor muscles of Pinna nobilis by the ethanol method is. Samples were recrystallised at least three times.
Trypsin Crystalline trypsin (50 % MgSO~) obtained either from Armour Laboratories or from Worthington Biochemical Corporation was used. It was dissolved in o.oi M HC1, dialyzed exhaustively in the cold against o.ooi M HC1 (pH 3.0) and lyophilized.
Physico-chemical measurements Sedimentation-velocity: These were made in a Spinco Model E ultracentrifuge operating at 59780 rev./min and at a temperature of 20 °. The position of the boundaries on the photographic plates was measured in a two-dimensional micro-eomparator and the calculated sedimentation coefficients were corrected to standard conditions of water at 20 °. Viscosity: These measurements were made in Ostwald-type viscometers of 5-ml capacity and having flow times of 300 sec for water. All the viscosity measurements were made at 20 -~ o.I °. Intrinsic viscosities were obtained by plotting ~sp/C versus concentration and extrapolating to infinite dilution. Light scattering: Light-scattering measurements were performed at a wavelength of 436 m/~ and at room temperature (25 ~ 2 °) with the Brice-Phoenix light-scattering photometer 19. Before the measurements, the protein solutions were clarified by filtration through Millipore filters of pore size 0.45/z. Turbidities on the pure solvents were determined in a similar fashion and were subtracted from the readings obtained with the protein solutions. Scattering dissymmetries at 45 ° and 135 ° were carried out concurrently with the 9°° measurements. The dissymmetry was found to be significant, thereby necessitating the use of the modified DEBEY equation 2° to calculate molecular weights, viz., Hc
i
+ 2Bc
(x)
~'eorr - - M
where H, the Debye factor is defined by the optical properties of the system, c is the protein concentration in g/Ioo ml, M is the molecular weight, B, the interaction constant is a thermodynamic parameter and ~eorr, the corrected turbidity is defined by the relation : T. . . .
=
i T. . . .
(2)
V(9o) Biochim. Biophys. Acta,
90 (1964) 503
520
CHARACTERIZATION OF A TRYPTIC CORE OF PARAMYOSIN
507
where T' is the turbidity value as obtained by measuring the scattered light at 9 °o and P(9 o) is a particle scattering factor accounting for the decrease of scattering due to intraparticle interference and derivable from the dissymmetry ratio ~. The refractive index increment for the trypsin-resistant core was assumed to have the same value as the native protein, viz., o.188 at 436 m/z (ref. 22) ; this corresponds to a Debye factor, H, in the light scattering equation of 1.o3" lO -5. A p p r o a c h to s e d i m e n t a t i o n - e q u i l i b r i u m : These runs were observed in the Spinco model E ultracentrifuge equipped with a slow-speed attachment (I : 3 speed reduction). The speed of the centrifuge was maintained at 9945 rev./min. The Schlieren optical system was used with the phase plate as the diaphragm. Throughout each run, the diaphragm was maintained at 80 °, since this angle position yielded the sharpest curve outlines at both the meniscus and cell-bottom positions. Runs were made with 12 m m cells using a 4 ° sector Kel-F centerpiece. The concentration of the original sample was determined in arbitrary units in a 12 mm, 2 ° sector synthetic boundary cell (single capillary type). To facilitate studying the molecular weight at the cell bottom, o.i ml of Dow-Corning No. 555 silicone fluid was first introduced into the cell and the protein solution then added. Nevertheless the extrapolation procedure at the solution-silicone interface was rendered somewhat difficult by the thickening of the boundary with time, suggesting some interaction of the protein with the silicone fluid; in fact, satisfactory agreement with measurements at the meniscus was obtained in only one run (see Table V). All sedimentation patterns were photographed on Kodak metallographic plates. They were subsequently enlarged and were then traced directly on to I m m graph paper. The tracings and the measurements were carried out in accordance with procedures recommended b y SCHACHMAN23. No solvent correction was applied, since the centrifugal force was sufficiently low that no redistribution of electrolyte occurred, The concentration of the protein at the cell meniscus, Cm, and at the cell bottom, cb, was calculated from the measurements using the equations24: O. I
cm = c o - - - -
nx
V'xm 2
X
n = o
(3 a)
Xn~'Zn
0.i nb Cb = C O - - - - - 2~ X n 2 " Z n F " Xb 2 no:
(3 b)
where co is the original concentration; o.1 cm is the value of the interval between tabulated readings along the x-axis; F is the enlargement factor; Xm, xb and Xn are the distances of the meniscus, bottom of the cell and nth interval, respectively, from the axis of rotation; Z n is the ordinate (proportional to the concentration gradient, d c / d x ) ; n x is the number of intervals needed to bring the ordinate to zero, and n = o and nb correspond to the meniscus and cell-bottom intervals. The weight average molecular weight at the meniscus and cell b o t t o m was calculated from the relations25: RT Mm = (I--~p)~o2
(dc/dx) m • xm'cm '
Mb =
RT (i--~p)w2
• (dc/dx) b Xb'Cb
(4)
where M and ~ are the molecular weight and partial specific volume, respectively; R is the gas constant ; T is the absolute temperature; p is the solution density; co is the B i o v h i m . B i o p h y s . A c t a , 90 (1964) 5o3-52o
508
K. BAILEY et al.
angular velocity in radians per sec; and c and dc/dx are the concentration and concentration gradient, respectively. The subscripts m and b refer to the positions of the meniscus and cell bottom, respectively. A value of o.728 ml/g was used for g in these calculations 22. Optical rotatory dispersion: The variation of optical rotation with wavelength was obtained using a model 26o Rudolph recording spectropolarimeter at the "medium" sensitivity range, where reproducibility of observed rotations is estimated to be ~ o.oi o,. The sample of the tryptic core was measured at a concentration of o.16 % in both Io-cm and I-cm cells. The optical rotatory changes were evaluated graphically using a Moffitt plot, which is based on the equation2*: 3 M0 ml . . . . . n~ + 2 Ioo
E~]
a0~02 X2--~02
bolt0 4 + --
(~--Z0~)2
(5)
where n is the refractive index, M 0 the average residue weight and m 1, the effective residue rotation. However, rather than making the assumption that ~ is 2100 /k, as has been done in the past when this equation is applied, ;to was evaluated by MARSH27 for this particular system by a modification of the graphical approach used by MOFFITT AND YANG; the best fit of the data was obtained for a ;to value of 2250 A. EXPERIMENTAL RESULTS
Chemical properties The amino acid composition of the fragment is strikingly similar to that of Pinna paramyosin (Table I). The amino acid composition of Pecten paramyosin is also shown. The latter is included in view of some similarities which the tryptic fragment possesses in common with paramyosin as discussed below. Indeed the similarity between the tryptic fragment and Pinna paramyosin is so close that from this result alone one might conclude that trypsin does not attack the protein. The ratios Lys/Arg and Glu/Asp of both proteins are also very similar (Table II). The only difference between them is the net charge, which in the tryptic fragment approaches the charge of Pecten paramyosin. Nevertheless, paramyosin is attacked by trypsin, whereas the fragment is essentially resistant (see Fig. 2). The presence of a large proportion of basic residues in the fragment is surprising in view of its resistance towards trypsin. Determination of the N-terminal group was carried out by the F D N B method 12. Whereas this method does not disclose an N-terminal residue in Pinna paramyosin 2s, the presence of glutamic acid as N-terminal residue in the tryptic fragment was clearly established by the same technique (Table III). The impurities found are probably due to traces of small peptides which seem to be adsorbed by the tryptic fragment and which were found later to be removed more efficiently by dialysis against 0.5 M KC1. The F D N B method gives a value of 75000 for the molecular weight of the fragment, somewhat lower than that deduced by physical measurements (see below). The content of lysine found by estimation of e-DNP-lysine was 5.1 g per IOO g o t protein. The lysine content (by the MOORE AND STEIN method) is 6.8 g per IOO g, so at least 75 % of the lysine present in the fragment is available to FDNB. * T h e s e m e a s u r e m e n t s were k i n d l y p e r f o r m e d b y M. M. MARSH of Lilly R e s e a r c h Laboratories, Eli Lilly a n d Co., I n d i a n a p o l i s , I n d i a n a (U.S.A.).
Biochim. Biophys. Acta, 9o (t964) 503-520
509
C H A R A C T E R I Z A T I O N OF A TRYPTIC CORE OF P A R A M Y O S I N TABLE
I
COMPARISON OF TRYPTIC FRAGMENTs PIN~NA PARAMYOSIN AND PECTEN PARAMYOSIN Residues per z o s g protein
Weight of anhydro-amino acid per zoo g protein Tryptic fragment Pinna paramyosin
Asp 14. 3 Thr 3.4 Ser 4 .6 Glu 25. 5 Gly 0.75 Ala 7-9 Val 3 .0 Met 1.9 Ileu 3.5 Leu 13.2 Tyr 2.0 Phe 0.8 Lys 6.8 His I .I (Amino-N) Arg I 1.6 Total lOO. 3 Average residue weight Calculated N
Pinna* paramyosin
Pecten* paramyosin (striated)
13. 5 2.4 4.5 23.6 o.I5 7-7 3.75 2.15 3.4 12. 4 2.45 1. 3 8.2 0.65
14.1 6.o 2.9
Pinna* paramyosin
124 33.6 52.3 197.6 12.7 11o.8 3 °.2 14.1 30. 5 i i6. 3 I2.2 5.5 53.0 8.0 (I 51) 74.0 874.9 I 14 17.8
25.6
0.7 7.4 3.7 i .5 2. 7 I I .o 1. 7 1. 9 I2.9 --
13.2 99.95
Tryptic fragment Pinna paramyosin
7.8 99.9
117. 3 23. 7 51.6 183 13 lO8 37 .8 16-4 29.8 lO9.3 15 8-7 64.1 4.7 (I 16) 84.6 866.9 i 15 18.9
Peeten* paramvosin
123 59 33 198 12.5 lO 4 37 ii 24 97 lO. 5 13 IOi -(I I 7) 5° 873 I 14.5 17.7
* R e s u l t of BAILEY AND RUEGG (ref. 9).
TABLE
II
ANALYTICAL CHARACTERISTICS OF TRYPTIC FRAGMENT, PINNA PARAMYOSIN AND PECTEN PARAMYOSIN T h e r e s u l t s a r e e x p r e s s e d as % of t o t a l r e s i d u e s . Tryptic fragment
T o t a l a c i d g r o u p s (a) T o t a l b a s e g r o u p s (b) A m i d e - N (c) N e t a n i o n s (a - - (b + c) Non-polar Lys/Arg Glu/Asp
36.8 15.4, 14.7 6.7 36.8 0.72 i .6
Pinna paramyosin
34.6 17.7 13.5 3.4 37.o o.76 1.57
Pecten paramyosin
37 17 12.9 7 .1 35.0 i .9 1.8
* T h i s v a l u e is c a l c u l a t e d f r o m t h e a m m o n i a d e r i v e d f r o m t h e c o l u m n , w h i c h is u s u a l l y g r e a t e r 9 t h a n t h e t r u e a m i d e - N l i b e r a t e d b y t h e m e t h o d of BAILEY 51. T h e t r u e a m i d e - N is p r o b a b l y 3 ° % l o w e r , b r i n g i n g t h e n e t c h a r g e t o a p p r o x . 8.2.
After 3 h of treatment of Pinna paramyosin with carboxypeptidase A at 37 ° (enzyme-substrate ratio, by weight, I : 25), a trace of leucine was observed. A longer treatment, 6 h, under the same conditions, showed traces of leucine, lysine and serine or glycine. The amount of material used was 0.02/,mole and the intensity of the ninhydrin colour obtained for each spot was always less than half that which would correspond to o.oi/,mole. The experiment was repeated taking 0.04/,mole of paraB i o c h i m . B i o p h y s . A c t a , 90 (1964) 503 520
K. BAILEY et al.
5IO
myosin, but even so the amount of liberated amino acid was much less than o.oi/~mole per mole. Prolonged action of carboxypeptidase A (8 h) on o.o6 #mole of the fragment under the same conditions as described above for paramyosin showed only traces 80-
J
60-
/
g 40
/
JD
E 2O z
b &
3
¢-
60
n
120
¢
180 240 Time (rain)
300
3~0
Fig. 2. C o m p a r a t i v e a c t i o n of t r y p s i n followed in t h e p H s t a r ; a, on P i n n a p a r a m y o s i n , p r o t e i n concn. 3.4 m g / m l , p H 8.5, 3o°; b, on t r y p t i c f r a g m e n t , p r o t e i n concn. : m g / m l , p H 8.2, 25 °. I n b o t h cases t h e e n z y m e - s u b s t r a t e r a t i o (by weight) w a s i : i o o ; c, as a, w i t h o u t t r y p s i n . TABLE lII APPLICATION OF
OF
THE
N-TERMINAL
FDNB GROUPS
METHOD IN
THE
FOR TRYPTIC
THE
DETECTION
FRAGMENT
Amount of protein containing x DNP-residue Expt. No.
N4erminal (DNP-Glu)
5
83000
12
62 ooo 85 ooo
Contaminants
DNP-Ala + D N P - P h e + (DNP-Ser or D N P - G l y ) = 320 ooo D N P - G I y or D N P - S e r = 43 ° ooo
of lysine (about o.oi #mole) and serine or glycine (less than o.oi/,mole). The inability of carboxypeptidase A to show a C-terminal group in either the native protein or its tryptic fragment could be due to the presence of C-terminal arginine or lysine, or to the presence of other unavailable C-terminal residues. The study of the action of carboxypeptidase B on both of them is in progress.
Physical properties When the tryptic fragment was examined in the ultracentrifuge and by free 'electrophoresis in the Tiselius apparatus a single symmetrical peak was obtained (Fig. I). A mixture of Pinna paramyosin and its tryptic fragment was also examined in the ultracentrifuge, each component at 0.5 % concentration. There was no separation of the two components, but an asymmetric peak was obtained suggesting the 13iochim. Biophys. ,4cta, 90 (I964) 503-520
CHARACTERIZATION OF A TRYPTIC CORE OF PARAMYOSIN
511
h e t e r o g e n e i t y of t h e s a m p l e (Fig. 3). T h e same solution, d i l u t e d to 0.5 %, was s t u d i e d in the Tiselius a p p a r a t u s . E l e c t r o p h o r e s i s showed an a s y m m e t r i c p e a k in b o t h limbs (Fig. 3) which again suggests h e t e r o g e n e i t y of t h e s a m p l e a n d a close s i m i l a r i t y to some p r o p e r t i e s of b o t h proteins. A B
Fig. 3. Mixture of Pinna paramyosin and its tryptic core in o. 5 M KC1, o.o35 M Na2HPO 4, O.Ol5 M NaH2PO 4 (pH 7.0). A, ultracentrifuge pattern taken at 224 min after reaching top speed (59 780 rev. per min) protein concn, i °/o, temp. 20°; B, ascending boundary (top), descending boundary (bottom), electropnoresis pattern taken at 480 min, temp. 4 °, protein concn. 0. 5 °/o, field strength 2. 3 V/cm. S e d i m e n t a t i o n d e t e r m i n a t i o n s were c a r r i e d o u t on t h e f r a g m e n t dissolved in p h o s p h a t e buffer (pH 7), ionic s t r e n g t h 0.067. A plot of s2o,w versus c is shown in Fig. 4, from which an e x t r a p o l a t e d s e d i m e n t a t i o n c o n s t a n t of 2.88 S was o b t a i n e d , as c o m p a r e d w i t h a value of 3.I S for t h e n a t i v e m a t e r i a l 22. The r e d u c e d viscosity of t h e t r y p s i n - r e s i s t a n t core in 0.067 M p h o s p h a t e buffer oi p H 7 as a function of c is p r e s e n t e d in Fig. 5. T h e least squares s t r a i g h t line is given b y : ~']sp/C ~ 0.5 + 0.72 c; [~i] = 0. 5 dl/g. The r e l a t i v e viscosity of t h e f r a g m e n t using p h o s p h a t e buffer a n d v a r y i n g ionic s t r e n g t h b y t h e a d d i t i o n of KC1 was also measured. No v a r i a t i o n in i n t r i n s i c v i s c o s i t y due to a different solvent was o b s e r v e d a n d If]] r e m a i n e d c o n s t a n t with respect t o salt c o n c e n t r a t i o n u p to 0.65 M (Table IV). Solutions of t h e t r y p t i c r e s i s t a n t core e x h i b i t e d some d i s s y m m e t r y of s c a t t e r i n g , a l t h o u g h t h e r e was no p e r c e p t i b l e d e p e n d e n c e of this p a r a m e t e r on p r o t e i n concent r a t i o n . The a v e r a g e Z value, 1.2, as m e a s u r e d w i t h b l u e light of t h e m e r c u r y arc, was used to calculate t h e m o l e c u l a r l e n g t h in a c c o r d a n c e w i t h t h e g r a p h i c s o l u t i o n s of DOTY AND STEINER 21. T h e effective w a v e l e n g t h of t h e b l u e light in t h e m e d i u m Biochim. Biophys. Acta, 90 (1964) 503-520
512
K. BAILEY et al.
employed is 4358 A/I.33 = 3277 A, and from the measured dissymmetry of 1.2o, a particle length of 750 A for a rod-shaped entity and 525 A for a random monodispersed coil was calculated. Because of the appreciable angular dissymmetry of scattering shown by the core it was necessary to correct the turbidity values as obtained by measuring the TABLE
IV
RELATIVE VISCOSITY OF THE TRYPTIC FRAGMENT AT DIFFERENT IONIC STRENGTHS o.o5 M phosphate buger + KC1
O.O5 O.I 0.2 0. 3 0. 4 o. 5 0.6
M M M M M M M
Ionic strength
Y]ret
o.I o.2 o. 3 o. 4 0. 5 0.6 0. 7
1.142 I.I48 1.137 1.132 1.141 1.153 1.143
3.0
2.8
2.6
t" 24
0I 1 02I 013 OJ4 01.5 Ppoteln concentpation (g/lOOml)
220
016
F i g . 4. A p l o t of sO20,w v e r s u s c for t h e P i n n a f r a g m e n t i n 0.035 M N a 2 H P O 4 , o.o15 M K H 2 P O ~ a t p H 7.0.
o9 r J 0"8 i
u 07 I O6
o
5if
o%
I o', o'~ o~ ~ Protein cone.(g/lOOml)
~
Fig. 5. R e d u c e d s p e c i f i c v i s c o s i t y of t h e P i n n a f r a g m e n t i n 0.035 M N a ~ H P O 4 , O.Ol 5 M. K H 2 P O 4 a t p H 7.0 as a f u n c t i o n of p r o t e i n c o n c e n t r a t i o n . B i o c h i m . B i o p h y s . A c t a , 9o (I964) 5 o 3 - 5 2 o
CHARACTERIZATION OF A TRYPTIC CORE OF PARAMYOSIN
513
scattered light at 9 o°, by the particle scattering factor, P(9o), in accordance with Eqn. 2. From the DOTY-STEINER graphs 21, the factor for correcting the turbidity for rods and coils of the above specified lengths is 0.82. The values of n c: * eo r r , as obtained for the core, are plotted as a function of c in Fig. 6, where the best straight line has been drawn by the method of least squares. The observed intercept value of (Hc:T)c~ o is 0.98 corresponding to a molecular weight of 102000 _-k 4000. 1.3
1.2
% ~
1.1
1.0
0.9
O.
L
I
I
I
I
0.1 0.2 0.3 0.4 0.5 Protein concentration (g/lOOml)
I
06
0.7
Fig. 6. Hc:Teorr versus c for t h e P i n n a f r a g m e n t in 0.035 IV[ Na~HPO~, O.Ol 5 M K H 2 P O 4 a t p H 7.o.
As an additional check on the molecular weight, the Archibald approach to sedimentation equilibrium method was applied. Fig. 7 shows a typical Schlieren photograph of the approach to sedimentation equilibrium and the companion photograph from the run in the synthetic boundary cell for the trypsin-resistant core. Table V summarizes the molecular weight results calculated at both the meniscus and cell b o t t o m positions for the Pinna fragment. It is to be noted that satisfactory agreement in molecular weight values between cell top and b o t t o m was obtained in only run I ; high, irregular values were recorded at the cell b o t t o m in runs 2 and 3. Since the best average value at the meniscus position, based on all the runs, agreed well with the molecular weight deduced by light scattering, and furthermore, since they did not change with time, it was felt that the high values at the cell bottom were due to aggregation phenomena resulting from interaction of the protein solution and the
Fig. 7. T y p i c a l s e d i m e n t a t i o n - e q u i l i b r i u m p h o t o g r a p h s on t h e P i n n a f r a g m e n t a t a c o n c e n t r a t i o n of 0.5 %. Speed of t h e u l t r a c e n t r i f u g e , 9945 r e v . / m i n a n d b a r angle, 8o °. T i m e s a f t e r full s pe e d a re : a, 77 rain; b, i o o m i n ; c, I47 r a i n ; a n d d, I98 min. P h o t o g r a p h e is a s y n t h e t i c b o u n d a r y r u n for t h e s a m e s o l u t i o n a n d cond i t i ons .
Bioch~m. Biophys. Acta, 90 (1964) 503-520
514
K. BAILEY et al.
silicone, and these values were, therefore, disregarded. Similar problems have been encountered by other investigators when working with muscle proteins29, 3° and in these cases, measurements at the cell bottom have been similarly disregarded. It is significant to note that a molecular weight of Io2ooo for the fragment corresponds to a size reduction of about 25 % from the value of 135ooo for the native TABLE V M O L E C U L A R W E I G H T O F P I N N A F R A G M E N T F R O M A P P R O A C H TO S E D I M E N T A T I O N
Time Expt. No.
(rain)
r
~oo 198 60 i24 65 I20
2 3
EQUILIBRIUM
DATA
Molecular we~ht Cell top
99 94 lO 9 IO2 I10 106
650 25o 12o 80o 300 280
Cell bottom
lO6 45 ° IOO 500 24 ° 500 * 191 6oo ~ I31 800* 151 500 *
Best average value lO 3 670. * Values not included in average for reasons discussed in text.
protein*. This figure agrees fairly well with that deduced from area measurements of ultracentrifuge patterns of the tryptic digest which established a size reduction of some 33 % (ref. 6)**. A Moffitt plot of the optical rotatory dispersion, based on a 20 value of 2250 A, for the tryptic fragment is shown in Fig. 8. It should be noted that these optical rotatory measurements were carried out at the end of the slow reaction which does * A recent publication by LOWEY el a/.al cites the molecular weight of Venus mercenaria p a r a m y o s i n as 22oooo. However, the suggestion of these authors, t h a t this is the molecular weight of all paramyosins, including t h a t of P i n n a nobilis, on the basis of the virtual equivalence of the intrinsic s e d i m e n t a t i o n constant, optical r o t a t o r y dispersion and a m i n o acid analysis is a misleading a r g u m e n t , since r a b b i t t r o p o m y o s i n essentially parallels the p a r a m y o s i n s in all the aforementioned properties, and yet its molecular weight is only 53ooo (ref. 32). F u r t h e r m o r e , TSAO el al.aa have d e m o n s t r a t e d , by osmotic pressure m e a s u r e m e n t s , a wide s p e c t r u m of molecular weights (65ooo-153ooo) for t r o p o m y o s i n s isolated from different species (rabbit, duck, p r a w n , mantle and pig) a n d different muscle t y p e s (striated, s m o o t h and cardiac). The possibility, therefore, exists t h a t there m a y be a difference in molecular m a s s for p a r a m y o s i n isolated from different phylogenetic species (in this case, P i n n a nobilis and Venus merca~ia are classified in two different suborders of the true lamellibranch division, which p r o b a b l y bear the same relationship to each other as the dog and cat within the carnivore divisiona4). This idea is not w i t h o u t precedence in o t h e r protein systems, where multiple molecular weights have been d e m o n s t r a t e d to exist in different species, such as with r a b b i t muscle phosphorylase a (495 ooo) and lobster muscle phosphorylase a (25o ooo) a5 and horse liver (84 ooo) versus yeast (i 5 ° ooo) alcohol dehydrogenase a6. LowEY and colleagues offer no definitive experimental evidence t h a t the molecular weight of P i n n a p a r a m y o s i n is not x35 ooo, but, rather, choose to criticize the m e t h o d s used to establish the molecular p a r a m e t e r s of this protein 2~. Their suggestion t h a t the light-scattering p h o t o m e t e r used (designed b y D. GORING AND P. JOHNSONaT) was not subjected to the " k i n d of cross-checks for reliability reported for the Brice i n s t r u m e n t " , is also unjustified, since p r e s u m a b l y the same criticism could be levelled against a n y other non-commercial machine and furthermore, several reliable, universally accepted results have been obtained with the machine in questionaE Their criticism a b o u t the diffusion c o n s t a n t being unreliable for such an a s y m m e t r i c molecule as param y o s ! n is also to be taken lightly, since D%0.,~, values have been deduced for skeletal myosin a9 and r a b b i t t r o p o m y o s i n % b o t h of which are equally a s y m m e t r i c molecules. ** The molecular weight calculated from sedimentation-diffusion data is of the order of 92 ooo (ref. 41 }, in fairly good agreement with the above results. Biochim. IXioph3,s. Acla, 90 (t964) 5o3 520
CHARACTERIZATION
OF A T R Y P T I C
CORE OF PARAMYOSIN
515
not necessarily represent the situation when the core is first formed at the end of the fast reaction. From the slope of the plot, the helix constant, b0, was evaluated as 228 °, corresponding to a helical content of some 38 %. Evidently then, tryptic digestion oi the Pinna molecule has resulted in a core which is appreciably less helical than the native protein. 0
°t
i
~
i
i
t
i
i
i
310
:[.~
40
-tO
o x
-20 t
N< -30
L-A
-4o
-50
i
0
5
i
I0
[
J~
210 I
i
45
X I0 e
F i g . 8. P l o t of [ x ] a ( M l I o o ) ( 3 / n 2 + 2) (22 - - 220) v e r s u s (2 z - - 202) -1 f o r t h e P i n n a f r a g m e n t a t a c o n c e n t r a t i o n of o. 5 % . M is t h e a v e r a g e r e s i d u e w e i g h t , n is t h e r e f r a c t i v e i n d e x o f t h e s o l v e n t , E~] a c o r r e s p o n d s t o t h e o b s e r v e d r o t a t i o n a t t h e w a v e l e n g t h (2) i n q u e s t i o n a n d 20 is t h e d i s p e r s i o n c o n s t a n t ( z 2 5 o 3.).
TABLE
VI
ACTION OF TRYPSIN ON THE TRYPTIC FRAGMENT OBTAINED FROM PINNA PARAMYOSIN D i g e s t i o n s w e r e c a r r i e d o u t a t p H 8.2 a n d 25 °. S a m p l e s w e r e r e m o v e d a f t e r 24 h o f p r o t e o l y s i s . Expt. No.
Protein conch. (mglml)
Enzyme substrate ratio
I
2.8
1.5 : IOO
2
I.O
I : IOO
o~-amino-N as % of total N
Alkali uptake (t~mole molesprot.) alkali
0.45 --
Bonds split per mole
--
<4
0. 7
<:I
Stability of the tryptic fragment towards further digestion by trypsin The fragment is highly resistant to further tryptic digestion. This is true both during the digestion of paramyosin b y trypsin and when the fragment is previously purified and then treated with the enzyme, showing that the resistance is not due to any inhibitory effect of the dialysable material. The fact that the purified fragment is not digested by trypsin was verified by alkali uptake (Fig. 2) and by the ~-amino nitrogen content of the trichloroacetic acid-soluble fraction. The conditions used in this case were the same as those used for the digestion of paramyosin, except for the protein concentration which was lower. The results obtained by both methods showed that trypsin has no further action on the fragment (Table VI and Fig. 2), since for the amount of protein used, the values lie within the range of experimental error. Therefore in an a t t e m p t to digest the fragment with trypsin it was decided to use a more drastic method involving the denaturation of the protein. Two methods of denaturation were first tried: (a) Dialysing the protein against 6 M urea at room Biochim.
Biophys.
A c t a , 9 o (1964) 5 o 3 - 5 2 o
516
K. BAILEY gt a[.
temperature for 4 h and diluting with a trypsin solution of o.3 % to a final concentration of 1.5 ~]1 urea. Since urea gives a positive ninhydrin reaction the digestion was only followed by the alkali uptake; (b) Heating the protein in a boiling water bath for 3o min. The extent of the reaction in this case was followed by the ~-amino nitrogen content in the trichloroacetic acid-soluble medium and by the alkali uptake. No difference in alkali uptake was found when the action of trypsin was followed on the fragment previously dialysed against urea or on a control (Fig. 9). The results obtained by the ninhydrin method for the fragment previously heated suggested that some digestion took place. However when the rate of digestion was followed by alkali uptake, no difference was found between the alkali uptake of a tryptic digest of the fragment and that of a tryptic digest of the fragment previously heated (Figs. 2 and 9).
C
6I O
/~ j~-~
0 0
I
I
30
60
I___ 90 Time (rain)
B
I
__
120
Fig. 9. Action of t r y p s i n on P i n n a f r a g m e n t followed in the p H s t a r at 3 o°, p H 8.5, e n z y m e - s u b s t r a t e ratio (by weight) i : ioo, p r o t e i n concn. 2 mg/ml. A, f r a g m e n t previously dialysed against 6 M urea; B, f r a g m e n t previously heated; C, control.
As discussed previously 6 this method has been shown to be more reliable. The apparently high alkali uptake of the control is probably due to the carbon dioxide uptake from the air since no nitrogen was bubbled through in this particular experiment. Likewise, no appreciable proteolysis could be induced in the fragment isolated by addition of trichloroacetic acid, though this procedure might be expected to cause denaturation. The presence of calcium firmly bound to crystalline paramyosin was observed. It was thought that one explanation for the resistance to further tryptic digestion could be that metals might be bound to the fragment preventing further attack. An a t t e m p t was made to eliminate these ions from the paramyosin molecule by thorough dialysis against EDTA and then against glass-distilled water. An estimation of calcium in the ash ae and a 8-hydroxyquinoline test for the presence of metals 43 was carried out on paramyosin before and after dialysis against i mM EDTA. Before dialysis the 8-hydroxy-quinoline test gave a value of A25Jmg = o.161 which decreased after dialysis to A 253/mg = o.o3o (calcium, barium and magnesium ions give no response to the test or a very weak one). The estimation of calcium ions in ash was made using 95 mg of protein each time. Precipitation of Ca 2+ and Mg 2+ with ammonium oxalate gave a positive result both before and after dialysis although the amount of precipitate Biochim. Biophys. Acta, 90 (1964) 503-52o
517
CHARACTERIZATION OF A TRYPTIC CORE OF PARAMYOSIN
in the latter case was about half that found in the first case. Thus, dialysis against i mM EDTA does not completely eliminate Ca *+ and/or Mg~+ from the paramyosin molecule. Accordingly, the fragment was dialysed against o.I M EDTA and afterwards one sample was dialysed against water to a concentration of I mM EDTA and another sample was dialysed against water until free of EDTA. Trypsin in the ratio of 1:5o (by weight) was added to both samples and the alkali uptake was followed with the autotitrator (Fig. IO). Trypsin did not act on either sample nor did it after the addition of CaC12 to 9" IO-~ M. Finally, a diminution of interaction forces between solute molecules by raising the ionic strength to 0.5 or I.O did not predispose the fragment to further proteolysis (Fig. II).
-r
? Z
Addition of Co 2+
4
:E
oo z
A d d i t i o n o f KCI
~2
0 Time (h)
Fig. Io. A c t i o n of t r y p s i n on P i n n a f r a g m e n t followed in the p H star a t 3 o°, p H 8. 5, e n z y m e substrate r a t i o (by weight) 1 : 5o, p r o t e i n concn. 1.2 mg/ml. A, previously dialysed against E D T A ; B, in presence of i mM E D T A ; C, control.
1
2 Time (h)
3
Fig. I I . Action of t r y p s i n on P i n n a f r a g m e n t followed in the p H s t a t in o. 5 M and I .o M KC1, 3 o°, p H 8.5, p r o t e i n concn, i .2 m g / m l , e n z y m e s u b s t r a t e ratio (by weight) i :5 o; control.
DISCUSSION
The tryptic digestion of Pinna paramyosin yields a high molecular weight fragment whose identification has been the main object of this study. The tryptic fragment seems to be a reproducible, well defined protein. This conclusion is supported by the constancy of samples obtained in different digestions as regards: (I) Amino acid analysis; (2) Sedimentation constant; (3) N-terminal group; (4) Molecular weight; (5) Resistance to further tryptic action. Proteolysis was continued for 24 h, which allowed a safe margin for complete digestion since the reaction was generally finished after 6 h.The fragment is different from Pinna paramyosinin spite of its similar amino acid composition. Thus the two proteins differ in the following features: (I) Molecular weights; (2) N-terminal residues, as shown by the FDNB method; (3) The values for the sedimentation constants when extrapolated to zero concentrations; (4) Intrinsic viscosity; (5) Light-scattering and optical rotatory dispersion properties; (6) Behaviour shown towards trypsin; (7) Solubility; (8) Inability of the fragment to crystallise. Biochim. Biophys. Acta, 9o (1964) 5o3-52o
518
K. BAILEY et al.
Furthermore, a mixture of Pinna paramyosin and tryptic fragment was shown to be heterogeneous when examined in the ultracentrifuge and in the electrophoresis apparatus. Summarising, it can be said that the tryptic fragment is a protein which represents about two-thirds of the paramyosin molecule. This is suggested not only by its molecular weight (about IOOOOO) but also by its ultracentrifugal area, which is about two-thirds the value for the native molecule 6. As regards the shape of the Pinna fragment, the physical constants described above were substitued into the SCHERAGA--MANDELKERNequation 44 to yield a fl value of 2.7I" lO 6, corresponding to an axial ratio of 25. Another estimate of axial ratio was deduced from SIMHA'S shape equation 45 using the calculated viscosity increment of 68.5. This value, according to SCHERAGA AND MANDELKERN'S tabular analysis (see SCHACHMAN, ref. 46) corresponds to an axial ratio of 28, in good agreement with the value based on/3. From an average axial ratio of 26.5 and a molecular weight of lO2 ooo the dimensions of an ellipsoidal Pinna fragment molecule would be 546 × 20 ,~. Light scattering dissymmetry measurements suggest a length for a rod model of 725 A and 525 A for a coil one.Thus a coil model of length 525-55o, 3t, deduced by both light scattering and viscosity measurements, seems to be the most appropriate one in terms of the physical data. That the molecule is not a fully random coil can be shown by invoking the FLORV-Fox relation for !~]~ (ref. 47):
¢.m E~I -
M
(6)
where • is a universal constant, 2 . 1 " 1 0 21, R is the length in the case of a monodispersed coil, and M is the molecular weight. Substituting the appropriate quantities in the above equation yields a value of 29o A for R, which is too low when compared with the value of 525 A, obtained by light scattering for this model. Furthermore, the molecule does possess some secondary structure as revealed by the 38 % helical content, deduced from optical rotatory dispersion measurements. LOCKER AND SCHMITT4 made a short digestion of paramyosin ~a from Venus merceneria with trypsin and observed that the digestion increased the diffusion rate without appreciable change in the sedimentation velocity. They found a rapid decline in viscosity in the first few minutes and a value of 30 % of the initial relative viscosity was obtained in about 15 min. Comparing their results with those obtained in the present study, it is possible to see striking similarities, which suggest the formation of a fragment similar to the one described here. The sedimentation velocity of the tryptic fragment obtained from Pinna did not change very much compared with that of the original protein. Furthermore, the value of the intrinsic viscosity oLthe tryptic fragment was about 25 % that of paramyosin as compared with the reported 30 % in the tryptic digest of Venus paramyosin. The authors 4 suggested from their study that a profound change occurs in the protein between 2.5 and 7 min of tryptic action. This might suggest that some of the bonds which are split at the beginning of the digestion (kinetically during the fast reaction 6) are key bonds which maintain the paramvosin molecule in a folded state. Their splitting produces a change in some of its properties, charge distribution for instance, and whereas one portion retracts into a more folded structure, tile rest of the molecule remains available to the enzyme and is further attacked. According to this interpretation the slow reaction might be Biochim. Biophys. Acla, 9 ° 0964) 503 520
CHARACTERIZATION OF A TRYPTIC CORE OF PARAMYOSIN
519
due to the proteolysis of that part of the protein which is converted into smaller peptides. Correlation of the kinetic values with results obtained following the digestion in the ultracentrifuge suggest that this is the case 6. According to the ideas of LINDERSTROM-LANGAND SCHELLMAN 49, the a-helix is relatively resistant to attack by proteolytic enzymes. Since paramyosin exists largely in the helical configuration 22, it was anticipated that initially it would be only slowly degraded by trypsin. However, the results described above show that a very rapid initial reaction does occur. It is possible that the helical content of the tropomyosin molecule is in fact somewhat less than IOO % and that the non-helical region(s) m a y be confined to a small area(s) of the molecule. In this case, these non-helical or random structures m a y be attacked in the initial fast reaction producing fragments. One of these fragments, representing at least one-half of the molecule undergoes a profound structural change as the result of changes in the charge distribution or other properties of the molecule and is resistant to further tryptic attack. The other fragment(s) undergoes further slow degradation by the enzyme. It is interesting that the resistant fragment, which contains only 40 % a-helix, is not digested by trypsin whereas the original molecule of paramyosin which must be close to completely a-helical is very rapidly degraded. Thus, in the reorganization of the structure of the fragment during its formation, the potentially susceptible bonds must become masked to the action of the enzyme by the formation of a globular-type tertiary structure. These considerations would explain the differences in the rates of the fast and slow reactions 6 in a manner similar to that proposed by MIHALYI AND HARRINGTON 5° for the kinetics of the tryptic digestion of myosin. However, as previously discussed, this is not the only interpretation and participation of the primary structure in the two rates is also possible. The position occupied by the tryptic fragment in the original paramyosin molecule cannot be deduced from the present studies, but it does not seem to be derived from the N-terminal end which does not react with FDNB. Whatever the position of the fragment in the original molecule, enzyme digestion appears to involve splitting of one portion into small peptides. It does not appear very helpful at this stage to study thoroughly their amino acid composition since they are small and it is laborious to purify them. However, since the fragment appears to be formed during the first 15 rain of digestion, it might be worthwhile to stop the proteolysis at this stage in order to obtain large peptides for further study.
ACKNOWLEDGEMENTS The University of Alberta authors would like to t h a n k Dr. F. A. HILDERMAN and Dr. W.xI. A. GREEN for their competent technical assistance. The research carried out at Alberta was financed by the National Research Council of Canada, the Life Insurance Medical Research Fund, the United States Public Health Service, Grant A-4iIO, and the Canadian Muscular Dystrophy Association. The Cambridge authors are greatly indebted to Mrs. B. D. BROWN for her skilled amino acid analysis. One of the authors (C.P.M.) is grateful to the Consejo Nacional de Investigaciones Cientificas y T6cnicas for financial aid during part of this work and to Professor F. G. YOUNG for hospitality. Biochim. Biophvs. Acta, 90 (1964) 503-520
520
K. BAILEY et
al.
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Biochim. Biophys. Acta, 9o (I964) 5o3-52o