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Physical and chemical characterization of hydroxysteroid dehydrogenases from Pseudomonas testosteroni
BIOCHIMICA ET BIOPHYSICAACTA
409
BBA 65066 PHYSICAL AND CHEMICAL CHARACTERIZATION OF HYDROXYSTEROID DEHYDROGENASES FROM PSEUDOMONAS TESTOSTERONI PHIL G. SQUIRE, STAFFAN DELIN AND JERKER PORATH Institute of Biochemistry, University of Uppsala, Uppsala (Sweden) and the Hormone Research Laboratory, University of California, Berkeley, Calif. ( U.S.A .)
(Received April 21, 1964)
SUMMARY Two steroid-induced enzymes, a-hydroxysteroid dehydrogenase (3-a-hydroxysteroid: NAD(P) oxidoreductase, EC I.I.I.5O) and fl-hydroxysteroid dehydrogenase (3 (or i7)-fl-hydroxysteroid:NAD(P) oxidoreductase, EC I.I.I.5I), which we have isolated by means of a new and simplified purification procedure, have been characterized by several physical and chemical methods. Additional criteria of homogeneity have been applied; both enzymes migrate as single bands at several pH values when examined by zone electrophoresis in continuous buffer systems on films of polyacrylamide, both enzyme preparations appear to be homogeneous as judged by sedimentation velocity, and except for a trace of very high-molecular-weight material in the a-enzyme preparation, both preparations appear to be homogeneous as judged by their sedimentation equilibrium behaviour as well. The isoelectric point of the aenzyme, as estimated from mobility studies on polyacrylamide films is 6.1 while the isoelectric point of the fl-enzyme was found to be 6.5. The molecular weights of the a-enzyme and the fl-enzyme, calculated from the results of the sedimentation equilibrium measurements were 47 IOO ± 15oo and IOO ooo + 600 respectively. The frictional ratio of the fl-enzyme, calculated from the sedimentation coefficient and the molecular weight is 1.18, suggesting that the shape of the molecule is reasonably spherical. INTRODUCTION We have recently reported a purification procedure by which three steroid-induced enzymes from Pseudomonas testosteroni can be separated from each other by gel filtration on Sephadex G-Ioo. Two of these enzymes, an a-hydroxysteroid dehydrogenase (3-a-hydroxysteroid:NAD(P) oxidoreductase, EC 1.1.1.5o ) and a fl-hydroxysteroid dehydrogenase (3 (or I7)-/5-hydroxysteroid:NAD(P ) oxidoreductase, EC I.I.I.5I), have been further purified by column zone electrophoresis to the point where several criteria of purity are satisfied; The results of some studies of the physical and chemical properties of these enzymes is reported here.
Biochim. Biophys. Acta, 89 (1964) 4o9-42x
410
P . G . SQUIRE, S. DELIN, J. PORATH MATERIALS AND METHODS
Mobility determination in polyacrylamide gels In an effort to get further information about the electrophoretic homogeneity of the highly purified preparations of the a- and/3-enzymes, and to make an estimate of the isoelectric point of the two enzymes, a series of zone electrophoretic experiments were conducted in which films of polyacrylamide gels I mm in thickness were used as the supporting medium. A schematic drawing of the apparatus used in these experiments is given in Fig. I. Continuous buffer systems were used throughout, i.e., the buffer composition of the gels was the same as that in the electrode vessels. Tile experiments were run for 3 h at a field strength of 7.5-8.o V/cm. The current varied from about 25 mA (through three slides) at the start of the runs, to about 35 mA at the end. The pH in the chambers containing the supports for the slides never varied by more than o.o5 units.
$
F
t
¢
F
20 cr~ . . . .
T £0
±
~4
Fig. I. A p p a r a t u s for zone electrophoresis on films of polyacrylamide gel. A microscope slide M is placed face-down with the gel film G in contact with the filter p a p e r - w r a p p e d s u p p o r t s . These s u p p o r t s are held in place by strips of sponge, F, placed between the s u p p o r t s and the baffle B. The buffer vessels are connected to the electrode vessels by m e a n s of agar bridges A. A strip of filter p a p e r moistened with distilled w a t e r is placed on the microscope slide as a cooling agent. The entire a p p a r a t u s is covered with a plastic cover, n o t shown, to minimize evaporation.
The solutions used in the preparation of the polyacrylamide slides were made up as follows: (a) Stock buffer solution I = o.Ioo ionic strength and the desired pH; (b) Stock monomer solution containing io.oo g of acrylamide and o. ioo g of bisacrylamide dissolved in water to make a final volume of 50 ml. (c) Buffer plus TEMED*; consisting of 44 ml buffer plus 0.200 ml TEMED plus I M HC1 to restore the pH to that of the stock buffer. (d) Ammonium persulfate solution consisting of o.14 g ammonium persulfate in IOO ml of water. This solution was freshly prepared before use.
For each experiment five or six molds were prepared with a microscope slide as the one side, and a sheet of plexiglass of the same dimensions as the other. A spacer cut from a polyvinylchloride sheet i mm in thickness was placed between the slide and plaxiglass sheet forming the bottom and two edges of the mold. Leakage of the polymerizing solution is minimized if surgical tape is wrapped around the edges of the mold. The solution for polymerization was prepared by mixing 5 ml of stock monomer solution, 5 ml of buffer plus TEMED, and IO ml of ammonium persulfate solution. * Abbreviation: T E M E D , N,N,N',N'-tetramethylethylenediamine.
Biochim. Biophys. Acta, 89 (1964) 4o9-421
CHARACTERIZATION
OF DEHYDROGENASES
411
The resulting solution was deaerated and pipetted into tile molds allowing the solution to run down the inclined edges in order not to trap bubbles. After polymerization was complete, the plexiglass plate and tile polyvinylchloride spacer were removed leaving the gels attached to the microscope slides. The gels were then covered with buffer diluted with an equal volume of water, and allowed to equilibrate at least 6 h before the electrophoretic experiment. The gels were then carefully wiped dry with filter paper, and the sample was applied as a small droplet, or a narrow band. The acrylamide and N , N , methylene-bis-acrylamide were obtained from Fluka A.G. (Switzerland) and the TEMED, from Distillation Products Industries. The reagents used in the preparation of buffers were all of reagent grade. U ltr acentrif ug ation
The ultracentrifugation experiments were performed with a Spinco model E ultracentrifuge equipped with Schlieren optics, including a phase plate as the Schlieren diaphragm, and with Rayleigh optics. The Schlieren system was used for the sedimentation velocity studies, and Rayleigh optics for sedimentation equilibrium experiments. The methods used in the analysis of fringe data from the sedimentation equilibrium experiments, and the equations used in the calculation of the weight and z-average molecular weights have been described previously 2. Analysis of data is based upon a plot of the logarithm of concentration in terms of Rayleigh fringes, as a function of the square of radial distance. Since concentration dependence is negligible in the system reported here, the weight average molecular weight of the sample has been calculated from the equation : 2 RT
cb
-
-
Cm
Mw= ( I - - 5~)o) 2 (r~ 2 - - r m ~)
cO
where co is the initial concentration as determined with a double sector synthetic boundary cell and cb -- Cm is the concentration difference between the centripetal and centrifugal solution menisci at equilibrium. The weight average molecular weight at dlnc
any point in tile cell is calculated from the slope of ~d- r
at that point by means of
the equation 2 RT
dlnc
Mw(r) = (I -
~e)oJ ~
d
~
In this study, we have calculated only the weight average molecular weights at the two menisci, M~ca ) and M~(b) from which the z-average molecular weights were calculated according to the equation c~ M ~ ( b ) - - Ca M w ( a ) iz
= Cb - - Ca
RESULTS
A m i n o acid analysis
Amino acid analysis of tile hydrolyzed samples of the enzyme preparations were carried out on the Spinco amino acid analyzer according to the procedures of SPACKBiochim. Biophys. Acta, 89 (1964) 409-42I
412
P . G . SQUIRE, S. DELIN, J. PORATH
MAN, STEIN AND MOORE~. Three samples of the/~-enzyme were hydrolyzed for 24, 48, and 72 h respectively. The results giving micromoles of each of the amino acids recovered for each of the samples were converted to the basis of a sample containing IO mg of nitrogen in order to facilitate an analysis of time dependence. These results are recorded in the first three columns of Table I. In the next column, we have recorded our "best value" estimate. In most cases this is simply the mean value, but for serine and threonine, which are known to undergo destruction on prolonged hydrolysis, a "zero time" value was estimated. The content of 1/2 cystine was calculated from the ratio of cysteic acid to leucine in a hydrolyzed sample of the performic acid oxidized enzyme, and the content of tryptophan was determined from the tyrosine-tryptophan ratio according to the method of GOODWlN AND MORTON as outlined by BEAVEN AND HOLIDAY 4. TABLE I AMINO ACID ANALYSIS OF THE /~-ENZYME A miuo acid residue
i~moles amino acid]io itmoles nitrogen 24 h
AspNH2"o.588 Glu 0.773 Gly o.814 Ala 1.oo9 Val 0.588 Leu 0.863 Ileu 0.378 Ser 0.584 Thr 0.29o 1[2 Cys Met 0'322 Pro o.26o Phe o.186 Tyr °.154 His O.ll 4 Try Lys o.31o Arg 0"355
"Best" value
Wi Res. wts.
ViWi
72 h
Molecular weight . of rtsidues
Vi
48 h
Residues per zoo ooo g
Neare st integer
o.617 o.775 0.795 0.964 0.548 o.814 0.378 0.582 0.280
o.6o7 0.770 0.796 0.968 o.581 0.829 0.376 0.574 0.282
0"308 0.254 o.172 °.144 o.129
°"31° 0.238 o.187 °.149 o.12o
o.316 0"366
o.331 0'348
o.6o4 0.773 0.802 0.98o 0.572 o.835 0.378 0.590 0.290 0.065 °"313 o.251 o.182 °.I49 o.121 0.057 o.319 0"356
114.1o 129.11 57.05 71.o7 99.13 113.15 113.15 87.07 IOI.IO lO2.13 131"19 97.11 147.17 163.17 137.14 186.2o 128.17 156'18
68.91 99.80 45.75 69.65 56.7 ° 94.48 42.77 51.37 29.32 6.64 41"°6 24.38 26.79 24.31 16.59 lO.61 40.89 55 .60
o.6o 0.66 0.64 0.74 0.86 0.90 0.9o 0.63 0.70 0.63 0.75 0.76 0.77 o.71 0.67 0.74 0.82 0"7o
41.35 65.87 29.28 51.54 48.76 85.03 38.49 32.36 2o.52 4.18 3 °.80 18.53 20.63 17-26 II.ii 7"85 33.53 38.92
74.98 95.97 99.57 121.66 71.Ol lO3.66 46.93 73.25 36.00 8.o 7 38.86 31.16 22.60 18.5o 15.o2 7 .08 39.60 44 .20
75 96 IOO 122 71 lO 4 47 73 36 8 39 31 23 19 15 7 4° 44
Wi = 805.62
ViWi = 596.Ol
vp
ViWi Vi
596.Ol 805.72
0.740
* A s s i g n m e n t of amide content to aspartic acid is arbitrary, see DISCUSSION.
These data, along with the corresponding values for the apparent specific volumes of the amino acid residues taken from COliN AND EDSALL5 were used for the calculation of the apparent specific volume of the enzyme. This value was used for the calculation of the molecular weight by the sedimentation equilibrium method. Finally, the number of moles of each of the amino acids per mole of protein was calculated. These results, as well as the value obtained by rounding off to the nearest integer, are also recorded in Table I. A consideration of the isoelectric point of the enzyme suggested that about half of the carboxyl groups were in the amide form. For purposes of the calculation of the apparent specific volume, the aspartic acid residues were all taken as asparagine. Unfortunately the amount of a-enzyme available for amino acid analysis was sufficient for only a single determination. Otherwise, the data were treated in the '.;ame manner as the data for the/3-enzyme. These results are recorded in Table II. Biochim.
Biophys.
Acta,
89 (1064) 4o9-421
CHARACTERIZATION OF DEHYDROGENASES
413
T A B L E II A M I N O A C I D A N A L Y S I S OF T H E a - E N Z Y M E
Molecular weight of residue
Amino acid residue
Residues z o #moles Nitrogen
Asp Glu N H , ~ Gly Ala Val Leu Ileu Ser Thr 1/~ Cys Met Pro
0.699 0.650 0.639 0.806 0.473 o.519 0.384 0.359 0.343
115.o8 128.13 57.05 71 .o7 99.13 113.15 113-15 87.07 ioi.io
o. 139 0.287 o.193 o.138 o. I o6 0.084 o.287 0.726 Wi
131.19 97.11 147.17 163.17 137.14 186.2o 128.17 156.18 670.6
Phe
Tyr His Try Lys Arg
Wi residue weights/ IO Itmoles N
ViWi
80.5 83. 3 36.4 52.3 46.8 58.6 39.4 31.4 40.7
0.59 0.66 0.64 0.74 0.86 o.90 0.90 0.63 0.70 Not determined 18.2 0.75 27.8 o.76 28.2 0.77 22. 5 o.71 14.5 0.67 15.6 0.74 36.8 0.82 37.6 o.7o
Vp
*
Vi
ViWi _ 490.6 Wi 670.6
47.5 54.9 23.3 38.7 4o.3 51.9 35.4 19.9 28. 5
Residues per 47 IOO g
49.1 54.7 44-9 56.6 33.2 36.5 26.9 25.2 24.1
13. 7 9.8 21.1 20.2 21. 7 13.6 16.o 9.7 9.7 7.5 11. 5 5.9 30.2 2o.2 26.3 51.o ViWi = 49o.6 o.7316
Nearest integer
49 46 45 57 33 37 27 25 24 IO 2o
14 IO
8 6 2O
51
°.732
Assignment of amide content to glutamic acid is arbitrary.
+!
'~
0
-2 I
I
I
6
7
8
pH
Fig. 2. Mobility of the a-enzyme in polyacrylamide gel ( [ Z - - [ ] and in free electrophoresis ( ( 3 - - ( 3 ) . The buffer used at p H 5.2 was composed of acetate-acetic acid, t h a t at p H 6.2 a n d 6.6, was sodium cacodylate-cacodylic acid, a t p H 6.9, phosphate, and at p H 8.2, Tris. In all cases, I ~
0.050.
Biochim. Biophys. Acta. 89 (1964) 4o9-4 aT
414
P. G. S Q U I R E , S. D E L I N , J . P O R A T H
*!
~°
\
-1
i Fig. 3- M o b i l i t y o f the /~-enzyrne in the p H region of the isoelectric point. Sodium cacodylatecacodylic acid buffers o f I = 0 . o 5 o were used in all experiments.
Mobility determinations in polyacrylamide gels The results of mobility determinations on the a-enzyme in polyacrylamide gels and by free electrophoresis are recorded in Fig. 2. An isoelectric point of 6.5 may be c alculated from the results obtained either from measurements in polyacrylamide gels or by free electrophoresis. The failure of the point representing the mobility calculated from the experiment in which a phosphate buffer was used is taken as evidence of phosphate binding. This point was ignored in drawing the line representing the mobility vs. pH relationship. The results of three mobility determinations of the/f-enzyme in polyacrylamide gel are recorded in Fig. 3- An isoelectric point of 6.1 was estimated from these results. TABLE
llI
RESULTS OF SEDIMENTATION EXPERIMENTS .Sedimentation velocity
Expt. No.
Enzyme
Protein (g/too ml)
Temperalure
2 6
fl fl
O.8Ol 0.475
19.45 18-45
Sobs x 1o t~
S,.o.w × zo t8
5.39 5-43
5 .88 6'05 (6.3 ° )
(Infinite dilution) Sedimentation equilibrium
Evpt. No.
4 8 7
Enzyme
Protein (g/~oo ml)
Rev./min
fl fl fl
o.Sol 1.183 0-475
5 227 5 227 7 447
0.888 o.515 0.306
7 447 8 225 i o 589
M~(a)
99 4 ° o 99 5 ° o i o o 400
Mean 13 12 14
a a ~
Mean
47 45 47 47
9°0 6oo 880 ioo
Biochim.
M~
Mz
lO6 99 ioo ioo
6oo 5° o 400 ooo
157 3 ° o 99 5 ° o i o o 400
52 47 49 49
ooo 3°o 500 600
i i o ooo 66 ooo 61 300
Biophys.
Acta,
89 (1964) 4 o 9 - 4 2 1
CHARACTERIZATIONOF DEHYDROGENASES
A
415
B
c D Fig. 4. Analysis of the fl~enzyme by sedimentation velocity at 59 780 rev./min and a protein concentration of o.8oi2/ioo ml. Pictures were taken 24 rain, 48 rain, 72 rain and lO4 rain after the ultracentrifuge was up to speed.
Sedimentation analysis Tile results from two s e d i m e n t a t i o n velocity experiments on the/5-enzyme as well as three s e d i m e n t a t i o n e q u i l i b r i u m experiments on b o t h enzymes are recorded in T a b l e h i . Tile buffer solution which was used in all experiments h a d tile composition 0.050 M Tris, 0.05 ° M Tris hydrochloride a n d 0.050 M NaC1. The p H was 8.1 a n d I ---- o.io. I n view of the fact t h a t b o t h enzymes have a n isoelectric point a b o v e p H 6,
Biochim. Biophys. Acta, 89 (1964) 4o9-421
416
P . G . SQUIRE, S. DELIN, J. PORATH
A
C
B
D
Fig. 5. A n a l y s i s o f t h e a - e n z y m e b y s e d i m e n t a t i o n v e l o c i t y a t 59 780 r e v . / m i n a n d a p r o t e i n conc e n t r a t i o n of 0.35 g ] i o o ml. Pictures were t a k e n 24 min, 56 min, 80 m i n a n d 114 rain after the u l t r a c e n t r i f u g e w a s up to speed. The Schlieren d i a p h r a g m angle was 7 °o .
it was assumed that charge effects would be negligible in this buffer. The observed sedimentation coefficients were calculated from plots of log x vs. t i m e which were strictly linear*. * The s y m b o l x represents t h e distance of t h e p e a k center f r o m the axis of r o t a t i o n m u l t i plied b y t h e m a g n i f i c a t i o n factor.
Biochim. Biophys. Acta, 89 (I964) 4 o 9 - 4 e i
CHARACTERIZATION OF DEHYDROGENASES
417
The observed sedimentation coefficients were converted to S2o,w by means of the usual equation (I - ~)2o,~ S20,w = Sob s • _ _ ~20,w (I - - U e ) where the symbols have their usual significance, cf., for example, SCHACHMAN6. In both experiments the Schlieren photography recorded a single symmetrical peak throughout the duration of both experiments. Representative photographs from sedimentation velocity experiments on the two enzymes are presented in Figs. 4 and 5. In view of the high degree of homogeneity inferred from the sedimentation equilibrium experiments, a detailed analysis of boundary shape in the sedimentation velocity experiments was thought unnecessary. Due to a slight leakage of solution from the cell in the experiment with the a-enzyme, a calculation of the sedimentation coefficient was not attempted. Ioq
C
~50
J
1.40
LJO
1.20
/.~0
I
2tO
712
=
2/';
¢
I
I
2/G 218 220
J
I
=
r
I
2.92 224 22G 228 230 x 2
Fig. 6. S e d i m e n t a t i o n e q u i l i b r i u m of t h e a - e n z y m e . T h e l o g a r i t h m of t h e c o n c e n t r a t i o n , in fringes, is p l o t t e d as a f u n c t i o n of x 2 w h e r e x is t h e radial d i s t a n c e m u l t i p l i e d b y t h e m a g n i f i c a t i o n factor f r o m cell to p h o t o g r a p h i c plate.
Also recorded in Table III is the value of S2o,w extrapolated to infinite dilution. The hazards of a two-point extrapolation were recognized but in view of the fact that the slope of S vs. C from these two points seemed reasonable for a non-interacting system, as inferred from the sedimentation equilibrium experiments, it was presumed that the two-point extrapolation would give the best estimate of this important molecular constant. Of the three sedimentation equilibrium experiments with the /5-enzyme, two yielded plots of log C vs. x ~ which were strictly linear. The plot obtained from Expt. 7 is recorded in Fig. 7 along with a fringe deviation analysis representing tile scatter of points from the straight line relationship calculated by the method of least squares. Since the plot of log C vs. x z is linear throughout the cell, M~a) = M~ = M~ for Expts. 7 and 8. Since any possible concentration dependence lies within the limits of Biochim. Biophys. Hcta, 89 (1964) 4o9-421
418
P . G . SQUIRE, S. DELIN, j. PORATH
our precision, the mean value of Mw, IOO ooo 4- 5oo, is taken as our best estimate of the molecular weight of the enzyme. Three sedimentation equilibrium experiments with the a-enzyme all yielded plots of log C vs. x 2 that were slightly curved upward in that part of the plot representing data near the lower (centrifugal) limit of the solution (see Fig. 6). Again concentration dependence is within the limits of our precision, and for reasons to be discussed, the mean value of Mw(a) is taken as our best estimate of the molecular weight.
1.60
/
1.50
.f
d
1.40 o t~
o
130
1
1.20[
1.10 +0.002 o
o
-J +0.001
o
o
.c o
~,
g
o
o
o o
o
o
o
o
o
o
oo
o
o
o Oo
o
o
-0.001 o
-0.002 218
~2o
2~
22,
2~6
2;8
2~o
2~2
2~,
x2
Fig. 7. S e d i m e n t a t i o n e q u i l i b r i u m of t h e /3-enzyme. The l o g a r i t h m of concentration, in fringes, is p l o t t e d as a f u n c t i o n of x 2. Fringe deviations, i.e., t h e d e v i a t i o n b e t w e e n observed values and the v a l u e calculated f r o m a linear e q u a t i o n representing all the data are also s h o w n .
DISCUSSION
From the criteria of purity that we have been abel to apply it would appear that both enzymes have been isolated in a high state of homogeneity. We have shown 1 that both enzyme preparations yield a single band when examined by disc electrophoresis according to the method of ORNSTE1N AND DAVIS 7. By the electrophoretic experiments on polyacrylamide films reported here, we have also found that both enzymes migrate as a single band in all buffers studied. Tile a-enzyme was studied by this technique over a rather wide range of pH, from pH 5.2 to 8.2, although tile/3-enzyme was examined only in the neighborhood of its isoelectrie point. It was recognized that resolution of components would not be as high as it appears to be by disc electrophoresis, since the protein band, when the experiment is performed on polyacrylamide films, is always rather broad. Yet on the other hand, the use of a continuous buffer system might offer certain advantages since boundary sharpening phenomena due to Biochim.
Biophys. Acta, 89 (1964) 4o9-421
CHARACTERIZATION OF DEHYDROGENASES
419
buffer gradients, which might conceivably prevent resolution, are absent. Under the conditions of these experiments we should have been able to detect a second component, if it amounted to IO% of the sample and had a mobility which differed by o.5 mobility units from that of the remainder of the fraction. In no case was a second component observed. As a method for determining mobility vs. pH relationships, this method has distinct disadvantages as compared with free electrophoresis. Vve expect that the apparent mobility would be decreased due to the gel. Furthermore, the temperature is not controlled, or even precisely measured, and mobility is dependent upon temperature. It was thought, however, that the isoelectric point, i.e., the pH of zero mobility, might be independent of the presence of the gel, and further that the isoelectric point might have little temperature dependence except for the effect of temperature on ionization of the ionizable groups of the protein. In the pH region of 6 - 7, where these proteins have their isoelectric points, these effects should be rather small. It was, however, gratifying to find that the values calculated for the isoelectric point of the a-enzyme by free electrophoresis did in fact agree very well with those calculated from the rate of migration in polyacrylamide films. It is also pertinent to point out that electroosmosis has been reported 7 to be negligible in polyacrylamide. Furthermore, in experiments performed by ourselves and others at the Institute of Biochemistry, the rate of migration of uncharged substances, for example NAD-ethanolamine at pH 8.6, was found to be too slow to be detected. The Schlieren photographs obtained from sedimentation velocity experiments on both enzymes displayed a single symmetrical peak which appeared to be Gaussian throughout the course of all three experiments. In view of the high degree of mass homogeneity revealed by the sedimentation equilibrium experiments, a detailed examination of peak shape from the sedimentation velocity experiments was not attempted. Of the three sedimentation equilibrium experiments performed with the /5enzyme, two of them yielded plots of log C vs. x 2 which were strictly linear. The fringe deviation diagram presented as part of Fig. 3 shows appreciable scatter, but no evidence of a trend in the scatter which might be interpreted as curvature of the log C vs. x ~ plot. This linearity provides strong evidence that tile enzyme preparation is monodisperse with regard to mass and that molecular interactions are very small. In an earlier experiment, Expt. 4, an earlier preparation of the/~-enzyme was studied and some heterogeneity was detected. In the plot of log C vs. x 2, a straight line relationship was observed through two-thirds of the plot, the curvature occurring only in that part of the plot where data from tile bottom of the cell are presented. Evidently this sample contained a small amount of very high molecular weight solute. The fact that M~(a~ calculated from the linear portion of this plot is in good agreement with M~ calculated from the two experiments in which no heterogeneity was detected provides confirmatory data regarding the molecular weight of the enzyme. Evidently the molecular weight of the contaminant in Expt. 4 was so high that its contribution to the total log C vs. x 2 plot was limited to the lower o.25 of the cell. In calculating the mean value of the molecular weight of the enzyme, we have taken the mean values of the average molecular weight calculated from Expts. 7 and 8. Thus our best estimate of the molecular weight of the fl-enzyme is ioo ooo + 6oo. All three sedimentation equilibrium experiments with the a-enzyme yielded plots Biochim. Biophys. Acta, 89 (1964) 4o9-42I
420
P. G. SQUIRE, S. DELIN, J. PORATH
of log C vs. x 2 which were curved upward starting approximately three-fourths of the way from a to b. Again the presence of a second macromolecular component (or components) of very high molecular weight is evident. Since the z-average molecular weight is highly sensitive to the presence of a high molecular weight "contaminant" the heterogeneity of these preparations is further illustrated by the differences between Mw and M~ for each of the experiments. The results of the three experiments are in reasonable agreement except that M~ in Expt. 13 is exceptionally high. It may be that interaction becomes appreciable at concentrations above I °/o. (The concentration at the bottom of the cell, Cb, was approx. 1.2%). Since the presence of the high molecular weight component affects M~ but apparently not M ~ ( a ) , we have taken the mean value of M~(a) as our best estimate of the molecular weight of the a-enzyme, i.e. 47 ooo -¢- 15oo. An estimate of the amount of high molecular weight contaminant can be calculated from M~ and M'~ if we make the assumption that M ~ ( a ) gives the molecular weight of the major component. This merely requires the solution of the quadratic equation s n~(a I ) + n ( I - ~ ) + ( / ~ - a ) = o followed by a calculation of z
a
i
n - i where n is the ratio of molecular weights of M~
M~M-z
the heavy "contaminant" to the major component, and a -- M 1 ' and fl MI 2 Taking the data from Expt. 12 as typical, we calculate n = 12.5 and x = 0.0032. Thus tile experimental values of M~ and Mz can be accounted for by the assumption that 0.32% of the sample has a molecular weight of 57 ° ooo, 12.5 times that of the major component'. The nature of the high molecular weight component is not known, but we have observed 1 that the a-enzyme develops a precipitate and loses activity when stored for a period of several weeks in the refrigerator. It seems likely that this trace of high molecular weight substance is a result of an early stage of the same process. The results of the Sephadex experiments reported elsewhere 1 suggest that the molecular weight of the a-enzyme is probably less than IOO ooo, and more than 4 ° ooo, the molecular weights of the t-enzyme and of the isomerase, respectively. The calculated value of 47 IOO lies well within these limits, but it seems quite unlikely that an enzyme with a molecular weight of several hundred thousand would appear in that portion of the Sephadex ehiate where the a-enzyme activity is observed. Thus it appears unlikely that the enzymic activity is associated with the trace component of high molecular weight. In an attempt to draw some conclusions regarding the shape of the t-enzyme, we have calculated the frictional coefficient of the equivalent anhydrous sphere, fo = 6 ~
\4~N/
5.84 • io-'
and the experimentally determined frictional coefficient M(I -- ~) f 6.91 • IO-s. Ns
• It should be pointed out that the values of x and n calculated by this method become exceedingly sensitive to the difference between M w and M, when this difference is small, but the conclusion that the sample contains a very small amount of an additional component having a molecular weight much greater than the major component seems inescapable. Biochim. Biophys. Acta, 89 (1964) 4o9-42I
CHARACTERIZATION OF DEHYDROGENASES
421
I n t h e s e c a l c u l a t i o n s w e h a v e t a k e n B = O.OLOO poise, M = IOO ooo, v = o.74o, a n d s ---- 6 . 3 o . lO -13. F r o m t h e s e v a l u e s a f r i c t i o n a l r a t i o , fifo ---- i. 18 is o b t a i n e d . T h i s v a l u e is r a t h e r low, s u g g e s t i n g t h a t t h e m o l e c u l e is r e a s o n a b l y spherical. If, as is f r e q u e n t l y d o n e , w e a s s u m e 20 % h y d r a t i o n a n d e s t i m a t e t h e a x i a l r a t i o o f a n e q u i v a l e n t p r o l a t e e l i p s o i d o f r e v o l u t i o n f r o m t h e c o n t o u r d i a g r a m s p r e p a r e d b y ONCLEY 9, a n a x i a l r a t i o o f 2. 7 is o b t a i n e d . A l t e r n a t i v e l y t h i s f r i c t i o n a l r a t i o c o u l d c o r r e s p o n d to a s o m e w h a t s w o l l e n s p h e r e w i t h 50 % h y d r a t i o n .
ACKNOWLEDGEMENT T h e a u t h o r s w o u l d like to t h a n k Dr. S. HJERTEN for p e r f o r m i n g t h e free e l e c t r o p h o r e s i s e x p e r i m e n t s on t h e a e n z y m e a n d for h e l p f u l s u g g e s t i o n s r e g a r d i n g t h e a c r y l a m i d e film t e c h n i q u e . T h e s e s t u d i e s w e r e a i d e d b y a n A m e r i c a n C a n c e r S o c i e t y S c h o l a r G r a n t to P. G. S.
REFERENCES
1 S. DELIN, P. G. SQUIRE AND J. PORATH, Biochim. Biophys. Acta, 89 (1964) 398. 2 p. G. SQUIRE, B. STARMANAND C. H. LI, J. Biol. Chem., 238 (1963) 1389. 3 D. H. SPACKMAN, W. S . STEIN AND S. MOORE, Anal. Chem., 3 ° (1958) 119 o. 4 G. H. BEAVEN AND E. R. HOLIDAY, Advan. Protein Chem., 7 (1952) 319 . 5 E. J. COHN AND J. T. EDSALL, Proteins, Amino Acids and Peptides, Reinhold, New York, 1943, p. 372. 6 H. K. SCHACHMAN, Ultracentrifugation in Biochemistry, Academic Press, New York, 1959. 7 L. ORNSTEIN AND B. J. DAVIS, Disc Electrophoresis, preprinted prior to journal publication by Distillation Products Industries, (Division of Eastman Kodak Company). 8 p. G. SQUIRE AND O. H. LI, J. Am. Chem. Soc., 83 (1961) 3521. 9 j . T. EDSALL, in H. NEURATH AND J. L. BAILEY, The Proteins, Vol I B, Academic Press, New York, 1953, p. 648. Biochim. Biophys. Acta, 89 (1964) 4o9-421
409
BBA 65066 PHYSICAL AND CHEMICAL CHARACTERIZATION OF HYDROXYSTEROID DEHYDROGENASES FROM PSEUDOMONAS TESTOSTERONI PHIL G. SQUIRE, STAFFAN DELIN AND JERKER PORATH Institute of Biochemistry, University of Uppsala, Uppsala (Sweden) and the Hormone Research Laboratory, University of California, Berkeley, Calif. ( U.S.A .)
(Received April 21, 1964)
SUMMARY Two steroid-induced enzymes, a-hydroxysteroid dehydrogenase (3-a-hydroxysteroid: NAD(P) oxidoreductase, EC I.I.I.5O) and fl-hydroxysteroid dehydrogenase (3 (or i7)-fl-hydroxysteroid:NAD(P) oxidoreductase, EC I.I.I.5I), which we have isolated by means of a new and simplified purification procedure, have been characterized by several physical and chemical methods. Additional criteria of homogeneity have been applied; both enzymes migrate as single bands at several pH values when examined by zone electrophoresis in continuous buffer systems on films of polyacrylamide, both enzyme preparations appear to be homogeneous as judged by sedimentation velocity, and except for a trace of very high-molecular-weight material in the a-enzyme preparation, both preparations appear to be homogeneous as judged by their sedimentation equilibrium behaviour as well. The isoelectric point of the aenzyme, as estimated from mobility studies on polyacrylamide films is 6.1 while the isoelectric point of the fl-enzyme was found to be 6.5. The molecular weights of the a-enzyme and the fl-enzyme, calculated from the results of the sedimentation equilibrium measurements were 47 IOO ± 15oo and IOO ooo + 600 respectively. The frictional ratio of the fl-enzyme, calculated from the sedimentation coefficient and the molecular weight is 1.18, suggesting that the shape of the molecule is reasonably spherical. INTRODUCTION We have recently reported a purification procedure by which three steroid-induced enzymes from Pseudomonas testosteroni can be separated from each other by gel filtration on Sephadex G-Ioo. Two of these enzymes, an a-hydroxysteroid dehydrogenase (3-a-hydroxysteroid:NAD(P) oxidoreductase, EC 1.1.1.5o ) and a fl-hydroxysteroid dehydrogenase (3 (or I7)-/5-hydroxysteroid:NAD(P ) oxidoreductase, EC I.I.I.5I), have been further purified by column zone electrophoresis to the point where several criteria of purity are satisfied; The results of some studies of the physical and chemical properties of these enzymes is reported here.
Biochim. Biophys. Acta, 89 (1964) 4o9-42x
410
P . G . SQUIRE, S. DELIN, J. PORATH MATERIALS AND METHODS
Mobility determination in polyacrylamide gels In an effort to get further information about the electrophoretic homogeneity of the highly purified preparations of the a- and/3-enzymes, and to make an estimate of the isoelectric point of the two enzymes, a series of zone electrophoretic experiments were conducted in which films of polyacrylamide gels I mm in thickness were used as the supporting medium. A schematic drawing of the apparatus used in these experiments is given in Fig. I. Continuous buffer systems were used throughout, i.e., the buffer composition of the gels was the same as that in the electrode vessels. Tile experiments were run for 3 h at a field strength of 7.5-8.o V/cm. The current varied from about 25 mA (through three slides) at the start of the runs, to about 35 mA at the end. The pH in the chambers containing the supports for the slides never varied by more than o.o5 units.
$
F
t
¢
F
20 cr~ . . . .
T £0
±
~4
Fig. I. A p p a r a t u s for zone electrophoresis on films of polyacrylamide gel. A microscope slide M is placed face-down with the gel film G in contact with the filter p a p e r - w r a p p e d s u p p o r t s . These s u p p o r t s are held in place by strips of sponge, F, placed between the s u p p o r t s and the baffle B. The buffer vessels are connected to the electrode vessels by m e a n s of agar bridges A. A strip of filter p a p e r moistened with distilled w a t e r is placed on the microscope slide as a cooling agent. The entire a p p a r a t u s is covered with a plastic cover, n o t shown, to minimize evaporation.
The solutions used in the preparation of the polyacrylamide slides were made up as follows: (a) Stock buffer solution I = o.Ioo ionic strength and the desired pH; (b) Stock monomer solution containing io.oo g of acrylamide and o. ioo g of bisacrylamide dissolved in water to make a final volume of 50 ml. (c) Buffer plus TEMED*; consisting of 44 ml buffer plus 0.200 ml TEMED plus I M HC1 to restore the pH to that of the stock buffer. (d) Ammonium persulfate solution consisting of o.14 g ammonium persulfate in IOO ml of water. This solution was freshly prepared before use.
For each experiment five or six molds were prepared with a microscope slide as the one side, and a sheet of plexiglass of the same dimensions as the other. A spacer cut from a polyvinylchloride sheet i mm in thickness was placed between the slide and plaxiglass sheet forming the bottom and two edges of the mold. Leakage of the polymerizing solution is minimized if surgical tape is wrapped around the edges of the mold. The solution for polymerization was prepared by mixing 5 ml of stock monomer solution, 5 ml of buffer plus TEMED, and IO ml of ammonium persulfate solution. * Abbreviation: T E M E D , N,N,N',N'-tetramethylethylenediamine.
Biochim. Biophys. Acta, 89 (1964) 4o9-421
CHARACTERIZATION
OF DEHYDROGENASES
411
The resulting solution was deaerated and pipetted into tile molds allowing the solution to run down the inclined edges in order not to trap bubbles. After polymerization was complete, the plexiglass plate and tile polyvinylchloride spacer were removed leaving the gels attached to the microscope slides. The gels were then covered with buffer diluted with an equal volume of water, and allowed to equilibrate at least 6 h before the electrophoretic experiment. The gels were then carefully wiped dry with filter paper, and the sample was applied as a small droplet, or a narrow band. The acrylamide and N , N , methylene-bis-acrylamide were obtained from Fluka A.G. (Switzerland) and the TEMED, from Distillation Products Industries. The reagents used in the preparation of buffers were all of reagent grade. U ltr acentrif ug ation
The ultracentrifugation experiments were performed with a Spinco model E ultracentrifuge equipped with Schlieren optics, including a phase plate as the Schlieren diaphragm, and with Rayleigh optics. The Schlieren system was used for the sedimentation velocity studies, and Rayleigh optics for sedimentation equilibrium experiments. The methods used in the analysis of fringe data from the sedimentation equilibrium experiments, and the equations used in the calculation of the weight and z-average molecular weights have been described previously 2. Analysis of data is based upon a plot of the logarithm of concentration in terms of Rayleigh fringes, as a function of the square of radial distance. Since concentration dependence is negligible in the system reported here, the weight average molecular weight of the sample has been calculated from the equation : 2 RT
cb
-
-
Cm
Mw= ( I - - 5~)o) 2 (r~ 2 - - r m ~)
cO
where co is the initial concentration as determined with a double sector synthetic boundary cell and cb -- Cm is the concentration difference between the centripetal and centrifugal solution menisci at equilibrium. The weight average molecular weight at dlnc
any point in tile cell is calculated from the slope of ~d- r
at that point by means of
the equation 2 RT
dlnc
Mw(r) = (I -
~e)oJ ~
d
~
In this study, we have calculated only the weight average molecular weights at the two menisci, M~ca ) and M~(b) from which the z-average molecular weights were calculated according to the equation c~ M ~ ( b ) - - Ca M w ( a ) iz
= Cb - - Ca
RESULTS
A m i n o acid analysis
Amino acid analysis of tile hydrolyzed samples of the enzyme preparations were carried out on the Spinco amino acid analyzer according to the procedures of SPACKBiochim. Biophys. Acta, 89 (1964) 409-42I
412
P . G . SQUIRE, S. DELIN, J. PORATH
MAN, STEIN AND MOORE~. Three samples of the/~-enzyme were hydrolyzed for 24, 48, and 72 h respectively. The results giving micromoles of each of the amino acids recovered for each of the samples were converted to the basis of a sample containing IO mg of nitrogen in order to facilitate an analysis of time dependence. These results are recorded in the first three columns of Table I. In the next column, we have recorded our "best value" estimate. In most cases this is simply the mean value, but for serine and threonine, which are known to undergo destruction on prolonged hydrolysis, a "zero time" value was estimated. The content of 1/2 cystine was calculated from the ratio of cysteic acid to leucine in a hydrolyzed sample of the performic acid oxidized enzyme, and the content of tryptophan was determined from the tyrosine-tryptophan ratio according to the method of GOODWlN AND MORTON as outlined by BEAVEN AND HOLIDAY 4. TABLE I AMINO ACID ANALYSIS OF THE /~-ENZYME A miuo acid residue
i~moles amino acid]io itmoles nitrogen 24 h
AspNH2"o.588 Glu 0.773 Gly o.814 Ala 1.oo9 Val 0.588 Leu 0.863 Ileu 0.378 Ser 0.584 Thr 0.29o 1[2 Cys Met 0'322 Pro o.26o Phe o.186 Tyr °.154 His O.ll 4 Try Lys o.31o Arg 0"355
"Best" value
Wi Res. wts.
ViWi
72 h
Molecular weight . of rtsidues
Vi
48 h
Residues per zoo ooo g
Neare st integer
o.617 o.775 0.795 0.964 0.548 o.814 0.378 0.582 0.280
o.6o7 0.770 0.796 0.968 o.581 0.829 0.376 0.574 0.282
0"308 0.254 o.172 °.144 o.129
°"31° 0.238 o.187 °.149 o.12o
o.316 0"366
o.331 0'348
o.6o4 0.773 0.802 0.98o 0.572 o.835 0.378 0.590 0.290 0.065 °"313 o.251 o.182 °.I49 o.121 0.057 o.319 0"356
114.1o 129.11 57.05 71.o7 99.13 113.15 113.15 87.07 IOI.IO lO2.13 131"19 97.11 147.17 163.17 137.14 186.2o 128.17 156'18
68.91 99.80 45.75 69.65 56.7 ° 94.48 42.77 51.37 29.32 6.64 41"°6 24.38 26.79 24.31 16.59 lO.61 40.89 55 .60
o.6o 0.66 0.64 0.74 0.86 0.90 0.9o 0.63 0.70 0.63 0.75 0.76 0.77 o.71 0.67 0.74 0.82 0"7o
41.35 65.87 29.28 51.54 48.76 85.03 38.49 32.36 2o.52 4.18 3 °.80 18.53 20.63 17-26 II.ii 7"85 33.53 38.92
74.98 95.97 99.57 121.66 71.Ol lO3.66 46.93 73.25 36.00 8.o 7 38.86 31.16 22.60 18.5o 15.o2 7 .08 39.60 44 .20
75 96 IOO 122 71 lO 4 47 73 36 8 39 31 23 19 15 7 4° 44
Wi = 805.62
ViWi = 596.Ol
vp
ViWi Vi
596.Ol 805.72
0.740
* A s s i g n m e n t of amide content to aspartic acid is arbitrary, see DISCUSSION.
These data, along with the corresponding values for the apparent specific volumes of the amino acid residues taken from COliN AND EDSALL5 were used for the calculation of the apparent specific volume of the enzyme. This value was used for the calculation of the molecular weight by the sedimentation equilibrium method. Finally, the number of moles of each of the amino acids per mole of protein was calculated. These results, as well as the value obtained by rounding off to the nearest integer, are also recorded in Table I. A consideration of the isoelectric point of the enzyme suggested that about half of the carboxyl groups were in the amide form. For purposes of the calculation of the apparent specific volume, the aspartic acid residues were all taken as asparagine. Unfortunately the amount of a-enzyme available for amino acid analysis was sufficient for only a single determination. Otherwise, the data were treated in the '.;ame manner as the data for the/3-enzyme. These results are recorded in Table II. Biochim.
Biophys.
Acta,
89 (1064) 4o9-421
CHARACTERIZATION OF DEHYDROGENASES
413
T A B L E II A M I N O A C I D A N A L Y S I S OF T H E a - E N Z Y M E
Molecular weight of residue
Amino acid residue
Residues z o #moles Nitrogen
Asp Glu N H , ~ Gly Ala Val Leu Ileu Ser Thr 1/~ Cys Met Pro
0.699 0.650 0.639 0.806 0.473 o.519 0.384 0.359 0.343
115.o8 128.13 57.05 71 .o7 99.13 113.15 113-15 87.07 ioi.io
o. 139 0.287 o.193 o.138 o. I o6 0.084 o.287 0.726 Wi
131.19 97.11 147.17 163.17 137.14 186.2o 128.17 156.18 670.6
Phe
Tyr His Try Lys Arg
Wi residue weights/ IO Itmoles N
ViWi
80.5 83. 3 36.4 52.3 46.8 58.6 39.4 31.4 40.7
0.59 0.66 0.64 0.74 0.86 o.90 0.90 0.63 0.70 Not determined 18.2 0.75 27.8 o.76 28.2 0.77 22. 5 o.71 14.5 0.67 15.6 0.74 36.8 0.82 37.6 o.7o
Vp
*
Vi
ViWi _ 490.6 Wi 670.6
47.5 54.9 23.3 38.7 4o.3 51.9 35.4 19.9 28. 5
Residues per 47 IOO g
49.1 54.7 44-9 56.6 33.2 36.5 26.9 25.2 24.1
13. 7 9.8 21.1 20.2 21. 7 13.6 16.o 9.7 9.7 7.5 11. 5 5.9 30.2 2o.2 26.3 51.o ViWi = 49o.6 o.7316
Nearest integer
49 46 45 57 33 37 27 25 24 IO 2o
14 IO
8 6 2O
51
°.732
Assignment of amide content to glutamic acid is arbitrary.
+!
'~
0
-2 I
I
I
6
7
8
pH
Fig. 2. Mobility of the a-enzyme in polyacrylamide gel ( [ Z - - [ ] and in free electrophoresis ( ( 3 - - ( 3 ) . The buffer used at p H 5.2 was composed of acetate-acetic acid, t h a t at p H 6.2 a n d 6.6, was sodium cacodylate-cacodylic acid, a t p H 6.9, phosphate, and at p H 8.2, Tris. In all cases, I ~
0.050.
Biochim. Biophys. Acta. 89 (1964) 4o9-4 aT
414
P. G. S Q U I R E , S. D E L I N , J . P O R A T H
*!
~°
\
-1
i Fig. 3- M o b i l i t y o f the /~-enzyrne in the p H region of the isoelectric point. Sodium cacodylatecacodylic acid buffers o f I = 0 . o 5 o were used in all experiments.
Mobility determinations in polyacrylamide gels The results of mobility determinations on the a-enzyme in polyacrylamide gels and by free electrophoresis are recorded in Fig. 2. An isoelectric point of 6.5 may be c alculated from the results obtained either from measurements in polyacrylamide gels or by free electrophoresis. The failure of the point representing the mobility calculated from the experiment in which a phosphate buffer was used is taken as evidence of phosphate binding. This point was ignored in drawing the line representing the mobility vs. pH relationship. The results of three mobility determinations of the/f-enzyme in polyacrylamide gel are recorded in Fig. 3- An isoelectric point of 6.1 was estimated from these results. TABLE
llI
RESULTS OF SEDIMENTATION EXPERIMENTS .Sedimentation velocity
Expt. No.
Enzyme
Protein (g/too ml)
Temperalure
2 6
fl fl
O.8Ol 0.475
19.45 18-45
Sobs x 1o t~
S,.o.w × zo t8
5.39 5-43
5 .88 6'05 (6.3 ° )
(Infinite dilution) Sedimentation equilibrium
Evpt. No.
4 8 7
Enzyme
Protein (g/~oo ml)
Rev./min
fl fl fl
o.Sol 1.183 0-475
5 227 5 227 7 447
0.888 o.515 0.306
7 447 8 225 i o 589
M~(a)
99 4 ° o 99 5 ° o i o o 400
Mean 13 12 14
a a ~
Mean
47 45 47 47
9°0 6oo 880 ioo
Biochim.
M~
Mz
lO6 99 ioo ioo
6oo 5° o 400 ooo
157 3 ° o 99 5 ° o i o o 400
52 47 49 49
ooo 3°o 500 600
i i o ooo 66 ooo 61 300
Biophys.
Acta,
89 (1964) 4 o 9 - 4 2 1
CHARACTERIZATIONOF DEHYDROGENASES
A
415
B
c D Fig. 4. Analysis of the fl~enzyme by sedimentation velocity at 59 780 rev./min and a protein concentration of o.8oi2/ioo ml. Pictures were taken 24 rain, 48 rain, 72 rain and lO4 rain after the ultracentrifuge was up to speed.
Sedimentation analysis Tile results from two s e d i m e n t a t i o n velocity experiments on the/5-enzyme as well as three s e d i m e n t a t i o n e q u i l i b r i u m experiments on b o t h enzymes are recorded in T a b l e h i . Tile buffer solution which was used in all experiments h a d tile composition 0.050 M Tris, 0.05 ° M Tris hydrochloride a n d 0.050 M NaC1. The p H was 8.1 a n d I ---- o.io. I n view of the fact t h a t b o t h enzymes have a n isoelectric point a b o v e p H 6,
Biochim. Biophys. Acta, 89 (1964) 4o9-421
416
P . G . SQUIRE, S. DELIN, J. PORATH
A
C
B
D
Fig. 5. A n a l y s i s o f t h e a - e n z y m e b y s e d i m e n t a t i o n v e l o c i t y a t 59 780 r e v . / m i n a n d a p r o t e i n conc e n t r a t i o n of 0.35 g ] i o o ml. Pictures were t a k e n 24 min, 56 min, 80 m i n a n d 114 rain after the u l t r a c e n t r i f u g e w a s up to speed. The Schlieren d i a p h r a g m angle was 7 °o .
it was assumed that charge effects would be negligible in this buffer. The observed sedimentation coefficients were calculated from plots of log x vs. t i m e which were strictly linear*. * The s y m b o l x represents t h e distance of t h e p e a k center f r o m the axis of r o t a t i o n m u l t i plied b y t h e m a g n i f i c a t i o n factor.
Biochim. Biophys. Acta, 89 (I964) 4 o 9 - 4 e i
CHARACTERIZATION OF DEHYDROGENASES
417
The observed sedimentation coefficients were converted to S2o,w by means of the usual equation (I - ~)2o,~ S20,w = Sob s • _ _ ~20,w (I - - U e ) where the symbols have their usual significance, cf., for example, SCHACHMAN6. In both experiments the Schlieren photography recorded a single symmetrical peak throughout the duration of both experiments. Representative photographs from sedimentation velocity experiments on the two enzymes are presented in Figs. 4 and 5. In view of the high degree of homogeneity inferred from the sedimentation equilibrium experiments, a detailed analysis of boundary shape in the sedimentation velocity experiments was thought unnecessary. Due to a slight leakage of solution from the cell in the experiment with the a-enzyme, a calculation of the sedimentation coefficient was not attempted. Ioq
C
~50
J
1.40
LJO
1.20
/.~0
I
2tO
712
=
2/';
¢
I
I
2/G 218 220
J
I
=
r
I
2.92 224 22G 228 230 x 2
Fig. 6. S e d i m e n t a t i o n e q u i l i b r i u m of t h e a - e n z y m e . T h e l o g a r i t h m of t h e c o n c e n t r a t i o n , in fringes, is p l o t t e d as a f u n c t i o n of x 2 w h e r e x is t h e radial d i s t a n c e m u l t i p l i e d b y t h e m a g n i f i c a t i o n factor f r o m cell to p h o t o g r a p h i c plate.
Also recorded in Table III is the value of S2o,w extrapolated to infinite dilution. The hazards of a two-point extrapolation were recognized but in view of the fact that the slope of S vs. C from these two points seemed reasonable for a non-interacting system, as inferred from the sedimentation equilibrium experiments, it was presumed that the two-point extrapolation would give the best estimate of this important molecular constant. Of the three sedimentation equilibrium experiments with the /5-enzyme, two yielded plots of log C vs. x ~ which were strictly linear. The plot obtained from Expt. 7 is recorded in Fig. 7 along with a fringe deviation analysis representing tile scatter of points from the straight line relationship calculated by the method of least squares. Since the plot of log C vs. x z is linear throughout the cell, M~a) = M~ = M~ for Expts. 7 and 8. Since any possible concentration dependence lies within the limits of Biochim. Biophys. Hcta, 89 (1964) 4o9-421
418
P . G . SQUIRE, S. DELIN, j. PORATH
our precision, the mean value of Mw, IOO ooo 4- 5oo, is taken as our best estimate of the molecular weight of the enzyme. Three sedimentation equilibrium experiments with the a-enzyme all yielded plots of log C vs. x 2 that were slightly curved upward in that part of the plot representing data near the lower (centrifugal) limit of the solution (see Fig. 6). Again concentration dependence is within the limits of our precision, and for reasons to be discussed, the mean value of Mw(a) is taken as our best estimate of the molecular weight.
1.60
/
1.50
.f
d
1.40 o t~
o
130
1
1.20[
1.10 +0.002 o
o
-J +0.001
o
o
.c o
~,
g
o
o
o o
o
o
o
o
o
o
oo
o
o
o Oo
o
o
-0.001 o
-0.002 218
~2o
2~
22,
2~6
2;8
2~o
2~2
2~,
x2
Fig. 7. S e d i m e n t a t i o n e q u i l i b r i u m of t h e /3-enzyme. The l o g a r i t h m of concentration, in fringes, is p l o t t e d as a f u n c t i o n of x 2. Fringe deviations, i.e., t h e d e v i a t i o n b e t w e e n observed values and the v a l u e calculated f r o m a linear e q u a t i o n representing all the data are also s h o w n .
DISCUSSION
From the criteria of purity that we have been abel to apply it would appear that both enzymes have been isolated in a high state of homogeneity. We have shown 1 that both enzyme preparations yield a single band when examined by disc electrophoresis according to the method of ORNSTE1N AND DAVIS 7. By the electrophoretic experiments on polyacrylamide films reported here, we have also found that both enzymes migrate as a single band in all buffers studied. Tile a-enzyme was studied by this technique over a rather wide range of pH, from pH 5.2 to 8.2, although tile/3-enzyme was examined only in the neighborhood of its isoelectrie point. It was recognized that resolution of components would not be as high as it appears to be by disc electrophoresis, since the protein band, when the experiment is performed on polyacrylamide films, is always rather broad. Yet on the other hand, the use of a continuous buffer system might offer certain advantages since boundary sharpening phenomena due to Biochim.
Biophys. Acta, 89 (1964) 4o9-421
CHARACTERIZATION OF DEHYDROGENASES
419
buffer gradients, which might conceivably prevent resolution, are absent. Under the conditions of these experiments we should have been able to detect a second component, if it amounted to IO% of the sample and had a mobility which differed by o.5 mobility units from that of the remainder of the fraction. In no case was a second component observed. As a method for determining mobility vs. pH relationships, this method has distinct disadvantages as compared with free electrophoresis. Vve expect that the apparent mobility would be decreased due to the gel. Furthermore, the temperature is not controlled, or even precisely measured, and mobility is dependent upon temperature. It was thought, however, that the isoelectric point, i.e., the pH of zero mobility, might be independent of the presence of the gel, and further that the isoelectric point might have little temperature dependence except for the effect of temperature on ionization of the ionizable groups of the protein. In the pH region of 6 - 7, where these proteins have their isoelectric points, these effects should be rather small. It was, however, gratifying to find that the values calculated for the isoelectric point of the a-enzyme by free electrophoresis did in fact agree very well with those calculated from the rate of migration in polyacrylamide films. It is also pertinent to point out that electroosmosis has been reported 7 to be negligible in polyacrylamide. Furthermore, in experiments performed by ourselves and others at the Institute of Biochemistry, the rate of migration of uncharged substances, for example NAD-ethanolamine at pH 8.6, was found to be too slow to be detected. The Schlieren photographs obtained from sedimentation velocity experiments on both enzymes displayed a single symmetrical peak which appeared to be Gaussian throughout the course of all three experiments. In view of the high degree of mass homogeneity revealed by the sedimentation equilibrium experiments, a detailed examination of peak shape from the sedimentation velocity experiments was not attempted. Of the three sedimentation equilibrium experiments performed with the /5enzyme, two of them yielded plots of log C vs. x 2 which were strictly linear. The fringe deviation diagram presented as part of Fig. 3 shows appreciable scatter, but no evidence of a trend in the scatter which might be interpreted as curvature of the log C vs. x ~ plot. This linearity provides strong evidence that tile enzyme preparation is monodisperse with regard to mass and that molecular interactions are very small. In an earlier experiment, Expt. 4, an earlier preparation of the/~-enzyme was studied and some heterogeneity was detected. In the plot of log C vs. x 2, a straight line relationship was observed through two-thirds of the plot, the curvature occurring only in that part of the plot where data from tile bottom of the cell are presented. Evidently this sample contained a small amount of very high molecular weight solute. The fact that M~(a~ calculated from the linear portion of this plot is in good agreement with M~ calculated from the two experiments in which no heterogeneity was detected provides confirmatory data regarding the molecular weight of the enzyme. Evidently the molecular weight of the contaminant in Expt. 4 was so high that its contribution to the total log C vs. x 2 plot was limited to the lower o.25 of the cell. In calculating the mean value of the molecular weight of the enzyme, we have taken the mean values of the average molecular weight calculated from Expts. 7 and 8. Thus our best estimate of the molecular weight of the fl-enzyme is ioo ooo + 6oo. All three sedimentation equilibrium experiments with the a-enzyme yielded plots Biochim. Biophys. Acta, 89 (1964) 4o9-42I
420
P. G. SQUIRE, S. DELIN, J. PORATH
of log C vs. x 2 which were curved upward starting approximately three-fourths of the way from a to b. Again the presence of a second macromolecular component (or components) of very high molecular weight is evident. Since the z-average molecular weight is highly sensitive to the presence of a high molecular weight "contaminant" the heterogeneity of these preparations is further illustrated by the differences between Mw and M~ for each of the experiments. The results of the three experiments are in reasonable agreement except that M~ in Expt. 13 is exceptionally high. It may be that interaction becomes appreciable at concentrations above I °/o. (The concentration at the bottom of the cell, Cb, was approx. 1.2%). Since the presence of the high molecular weight component affects M~ but apparently not M ~ ( a ) , we have taken the mean value of M~(a) as our best estimate of the molecular weight of the a-enzyme, i.e. 47 ooo -¢- 15oo. An estimate of the amount of high molecular weight contaminant can be calculated from M~ and M'~ if we make the assumption that M ~ ( a ) gives the molecular weight of the major component. This merely requires the solution of the quadratic equation s n~(a I ) + n ( I - ~ ) + ( / ~ - a ) = o followed by a calculation of z
a
i
n - i where n is the ratio of molecular weights of M~
M~M-z
the heavy "contaminant" to the major component, and a -- M 1 ' and fl MI 2 Taking the data from Expt. 12 as typical, we calculate n = 12.5 and x = 0.0032. Thus tile experimental values of M~ and Mz can be accounted for by the assumption that 0.32% of the sample has a molecular weight of 57 ° ooo, 12.5 times that of the major component'. The nature of the high molecular weight component is not known, but we have observed 1 that the a-enzyme develops a precipitate and loses activity when stored for a period of several weeks in the refrigerator. It seems likely that this trace of high molecular weight substance is a result of an early stage of the same process. The results of the Sephadex experiments reported elsewhere 1 suggest that the molecular weight of the a-enzyme is probably less than IOO ooo, and more than 4 ° ooo, the molecular weights of the t-enzyme and of the isomerase, respectively. The calculated value of 47 IOO lies well within these limits, but it seems quite unlikely that an enzyme with a molecular weight of several hundred thousand would appear in that portion of the Sephadex ehiate where the a-enzyme activity is observed. Thus it appears unlikely that the enzymic activity is associated with the trace component of high molecular weight. In an attempt to draw some conclusions regarding the shape of the t-enzyme, we have calculated the frictional coefficient of the equivalent anhydrous sphere, fo = 6 ~
\4~N/
5.84 • io-'
and the experimentally determined frictional coefficient M(I -- ~) f 6.91 • IO-s. Ns
• It should be pointed out that the values of x and n calculated by this method become exceedingly sensitive to the difference between M w and M, when this difference is small, but the conclusion that the sample contains a very small amount of an additional component having a molecular weight much greater than the major component seems inescapable. Biochim. Biophys. Acta, 89 (1964) 4o9-42I
CHARACTERIZATION OF DEHYDROGENASES
421
I n t h e s e c a l c u l a t i o n s w e h a v e t a k e n B = O.OLOO poise, M = IOO ooo, v = o.74o, a n d s ---- 6 . 3 o . lO -13. F r o m t h e s e v a l u e s a f r i c t i o n a l r a t i o , fifo ---- i. 18 is o b t a i n e d . T h i s v a l u e is r a t h e r low, s u g g e s t i n g t h a t t h e m o l e c u l e is r e a s o n a b l y spherical. If, as is f r e q u e n t l y d o n e , w e a s s u m e 20 % h y d r a t i o n a n d e s t i m a t e t h e a x i a l r a t i o o f a n e q u i v a l e n t p r o l a t e e l i p s o i d o f r e v o l u t i o n f r o m t h e c o n t o u r d i a g r a m s p r e p a r e d b y ONCLEY 9, a n a x i a l r a t i o o f 2. 7 is o b t a i n e d . A l t e r n a t i v e l y t h i s f r i c t i o n a l r a t i o c o u l d c o r r e s p o n d to a s o m e w h a t s w o l l e n s p h e r e w i t h 50 % h y d r a t i o n .
ACKNOWLEDGEMENT T h e a u t h o r s w o u l d like to t h a n k Dr. S. HJERTEN for p e r f o r m i n g t h e free e l e c t r o p h o r e s i s e x p e r i m e n t s on t h e a e n z y m e a n d for h e l p f u l s u g g e s t i o n s r e g a r d i n g t h e a c r y l a m i d e film t e c h n i q u e . T h e s e s t u d i e s w e r e a i d e d b y a n A m e r i c a n C a n c e r S o c i e t y S c h o l a r G r a n t to P. G. S.
REFERENCES
1 S. DELIN, P. G. SQUIRE AND J. PORATH, Biochim. Biophys. Acta, 89 (1964) 398. 2 p. G. SQUIRE, B. STARMANAND C. H. LI, J. Biol. Chem., 238 (1963) 1389. 3 D. H. SPACKMAN, W. S . STEIN AND S. MOORE, Anal. Chem., 3 ° (1958) 119 o. 4 G. H. BEAVEN AND E. R. HOLIDAY, Advan. Protein Chem., 7 (1952) 319 . 5 E. J. COHN AND J. T. EDSALL, Proteins, Amino Acids and Peptides, Reinhold, New York, 1943, p. 372. 6 H. K. SCHACHMAN, Ultracentrifugation in Biochemistry, Academic Press, New York, 1959. 7 L. ORNSTEIN AND B. J. DAVIS, Disc Electrophoresis, preprinted prior to journal publication by Distillation Products Industries, (Division of Eastman Kodak Company). 8 p. G. SQUIRE AND O. H. LI, J. Am. Chem. Soc., 83 (1961) 3521. 9 j . T. EDSALL, in H. NEURATH AND J. L. BAILEY, The Proteins, Vol I B, Academic Press, New York, 1953, p. 648. Biochim. Biophys. Acta, 89 (1964) 4o9-421