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Isolation and characterization of bovine, canine and ovine α-crystallins
Comp. Bioehon. Physiol., 1972, Vol. 4311,pp. 151 to 161. Pergamon Press. Printed in Great Britain
ISOLATION AND CHARACTERIZATION OF BOVINE, CANINE AND OVINE a-CRYSTALLINS* W I L L E. F E R G U S O N and V I R G I L L. K O E N I G Department of Biochemistry, The University of Texas Medical Branch, Galveston, Texas 77550 (Received 18 ffanuary 1972)
Abstract--1. a-CrystaUins were isolated from bovine, canine and ovine ocular lenses by chemical methods. 2. An alkaline-modified bovine ¢-crystallin was prepared by subjecting normal bovine ¢-crystallin to pH > 9"5 with subsequent precipitation at pH 5.1. 3. Electrophoretic mobilities of the various ¢-crystallins were determined by moving boundary electrophoresis in Na-veronal and Tris-veronal buffers. Canine ~-crystallin had the highest mobility. 4. The a-crystallins were characterized by sedimentation, viscosity and partial specific volume determinations. 5. Molecular weights were determined by means of sedimentation equilibrium. The values were: bovine, 1,035,000; alkaline-modified bovine, 499,000; ovine, 1,047,000; and canine, 924,000. 6. Molecular weights and dimensions were calculated assuming prolate and oblate ellipsoidal and spherical models from sedimentation, viscosity and partial specific volume data. INTRODUCTION SINCE Moerner (1894) first studied systematically the proteins of the crystalline lens, the larger emphasis has been on the relatively easily isolatable c~-crystallin. Francois et al. (1955) prepared ~-crystallin from bovine lenses by means of isoelectric precipitation in the presence of low concentrations of ethanol. Later, workers have used starch block electrophoresis (Bloemendal & Ten Cate, 1959), vertical column electrophoresis (Bj6rk, 1960), density gradient centrifugation (Bloemendal et al., 1964), gel filtration (Van Dam & Ten Cate, 1966) and continuous flow electrophoresis (deGroot et al., 1970) to isolate c~-crystallin. Only small amounts of material may be isolated by the above specialized procedures, consequently analytical studies are performed on a variety of samples. Bj6rk (1968) prepared ~-crystallins from eight mammalian species including the cow, sheep, pig, horse, rabbit, fox, mink and rat by zone electrophoresis. While the molecular *The data in this paper are taken from a dissertation submitted by W. E. Ferguson to the Graduate Faculty of the University of Texas in partial fulfillment for the degree, Doctor of Philosophy, 1972. This investigation was supported in part by Grant EY-00283 from the National Eye Institute, U.S. Public Health Service. 151
152
WILL E. FERGUSONAND VIRGIL L. KOENIG
w e i g h t s a n d e l e c t r o p h o r e t i c m o b i l i t i e s differ f r o m species to species, the i m m u n o logical c h a r a c t e r i s t i c s are similar. T h e v a r i o u s ~ - c r y s t a l l i n s are d i s s o c i a t e d in 7 M u r e a , t h u s s u g g e s t i n g t h a t t h e y are m a d e u p o f s u b u n i t s . R e s n i k (1957) a n d P e r r y & K o e n i g (1961) w e r e a m o n g t h e first to r e p o r t p h y s i c o c h e m i c a l studies. N i y o g i & K o e n i g (1963, 1965) r e p o r t e d a d d i t i o n a l p h y s i c o c h e m i c a l p r o p e r t i e s of b o v i n e a - c r y s t a l l i n . Still f u r t h e r p h y s i c o c h e m i c a l s t u d i e s have b e e n r e c e n t l y m a d e b y S p e c t o r et al. (1971) a n d A u g u s t e y n a n d S p e c t o r (1971). T h e p r e s e n t i n v e s t i g a t i o n is c o n c e r n e d w i t h t h e isolation b y c h e m i c a l m e a n s of b o v i n e , ovine a n d c a n i n e c~-crystallins. I n t h e p r o c e s s o f p r e p a r i n g b o v i n e c~-crystallin, an alkaline m o d i f i e d a - c r y s t a l l i n r e s u l t e d . P h y s i c o - c h e m i c a l p r o p e r t i e s o f all these c~-crystallins were d e t e r m i n e d in o r d e r to c h a r a c t e r i z e a n d o b s e r v e differences t h a t m i g h t exist. MATERIALS AND METHODS A total of 700 bovine (Bos taurus) eyes were collected at the Burton Brothers Abattoir in Houston, Texas. Approximately 200 ovine (Ovis aries) eyes were purchased from Armour and Company, San Angelo, Texas. A total of 650 canine (Canis familiaris) eyes were collected at the Houston Dog Pound. T h e eyes were collected as soon after the death of the animal as possible and stored on dry ice. T h e lenses were later removed from the frozen eyes and decapsulated. Bovine and ovine lenses were extracted in distilled water at 4°C by mechanical stirring using 160 g of lenses to 1500 ml H , O . T h e insoluble albuminoid was removed by centrifugation. T h e supernatant was diluted to a protein concentration of 0"25%. T h e p H of the diluted solution was 7"2. The p H was adjusted to 5"1 by adding 1 N HC1 slowly through a capillary with rapid stirring. After adjustment of pH, ethanol was added slowly through a capillary to a concentration of 10% (v/v). T h e a-crystallin precipitate was allowed to settle for a short time, and then removed by centrifugation at 10,000 rev/min on the Sorvall RC2-B for 20 min. The precipitate was redissolved in distilled water by adding enough 0"04 M NaHCOa to maintain the p H at 7'4. The at-crystallin in each case was reprecipitated twice. T h e final a-crystallin solution was dialyzed free of NaHCO3 and lyophilized. The lyophilized samples were stored at 1 °C. Canine a-crystallin was prepared in a similar manner. The precipitating solution was 2 per cent with respect to protein concentration. In order to remove an impurity lighter than a-crystallin, the final a-crystallin solution was dialyzed against 0"2 M NaCl-0.067 M phosphate buffer, p H 6'8 and then placed on a Sephadex G-200 column equilibrated to the same buffer. T h e a-crystallin was recovered in the void volume, dialyzed free of buffer and lyophilized. T h e lyophilized samples were stored at 1 °C. In the process of the purification of bovine a-crystallin, the solution was inadvertently exposed to p H 10'0. T h e resulting c~-crystallin although precipitating at p H 5"1 was lighter upon sedimentation than a-crystallin not exposed to p H above 7"4. A quantity of the modified a-crystallin was prepared by exposing the solution to p H 10 for 24 hr. T h e modified ct-crystallin was precipitated at p H 5"1 as described above. The final solution was dialyzed free of electrolytes against distilled water and lyophilized. The dried samples were stored at 1 °C.
Eleetrophoretic analysis Moving boundary electrophoresis was performed on the Spinco Model H electrophoresis and diffusion apparatus. T h e procedures have been described by Cobb & Koenig (1968). Veronal buffer, p H 8"6 I'/2 0"1 ( K = 0"00293-0"00300 ~-1 cm-1) and Tris-veronal buffer, p H 8"2, 0"075 M ( K = 0"001030-0"001047/Y~ -1 cm -1) were used. The preparation of these buffers has been previously described by Cobb & Koenig (1968). Protein concentrations were 0"5 % as determined with a Goldberg refractometer.
ISOLATION AND CHARACTERIZATION OF Ot-CRYSTALLINS
153
Sedimentation analysis Velocity sedimentation analyses were performed on a Spinco Model E ultracentrifuge at a speed of 50,740 rev/min. The solvent was 0"2 M NaCI for all sedimentation studies. Sedimentation values were determined at no less than ten different concentrations and were corrected to water as solvent at 20°C. Regression lines for sedimentation coefficient (Svedberg units) vs. concentration (g/100 ml) were determined by the method of least squares. Sedimentation coefficients at infinite dilution were thereby obtained. Correlation coefficients and standard errors of the slopes and intercepts were calculated. Protein concentrations were determined by means of a Goldberg refractometer. The amounts of the 3S component were determined from the composition analyses of the sedimentation patterns and corrected for radial dilution. Meniscus depletion sedimentation equilibrium studies were performed as described by Chervenka (1969). The following modifications were made: FC-43 fluorocarbon oil was not used and the temperatures were regulated at 20°C. A titanium rotor (An-H) was used to minimize errors in speed control. Fringe displacement ( Y r - Yo) for each radial interval (r) was determined by means of a Nikon microcomparator with a 50 x lens. Log ( Y r - Yo) vs. r * was plotted over the entire length of the cell. Molecular weights were calculated for the equation, M = 2R____~Tx 2-303 d log( Y r - Yo) ( 1 - ~p)w ~ dr ~ where M is the molecular weight; R, the gas constant; T, the absolute temperature; ~, the partial specific volume of solute; p, the density of solution; w, the rotational velocity in rad/sec; Yr - Yo, the fringe displacement at any point, r; and r, the distance from the center of rotation in cm.
Viscosity determination Viscosity determinations were made on a series of protein concentrations in 0"2 M NaC1 for each a-crystallin preparation. The concentrations were obtained by serial dilution. The initial solutions dialyzed against 0"2 M NaC1 were centrifuged at 13,000 r.c.f, in a Lourdes centrifuge for 30 min in order to remove insoluble material. Flow times were measured in an Ostwald viscometer at 21°C. Densities of the solutions were measured in an Ostwald pycnometer also at 21°C. The concentrations of the solutions were determined from weights of dried residues from solutions and solvents evaporated at 105°C. The reciprocals of the relative viscosities were plotted against concentrations. Equations of the resulting straight lines were determined by the method of least squares: liar = 1 - K c , where ,/r is the relative viscosity; K, the weight intrinsic viscosity; and c, the protein coneentration (g/100 ml). Volume intrinsic viscosity (~) is K(100)/~.
Partial specific volume determination The method of Drucker (1941) was used to determine the apparent partial specific volumes of the ~-crystallins. At least four different concentrations of protein were used. Details of the procedure are the same as described by Koenig (1950). Concentrations of protein were determined from the weights of dried residues from solutions and solvents evaporated at 105°C. Calculation of molecular dimensions The calculation of the molecular dimensions from viscosity data were according to the methods described by Perry & Koenig (1961) and Niyogi & Koenig (1963). Both prolate and oblate ellipsoids of revolution were assumed as models.In addition dimensions were calculated for a spherical model, and the molecular weight and dimensions were calculated
154
WILL E. FERGUSONAND VIRGIL L. KOENIG
according to the Scheraga-Mandelkern equation as was done by Niyogi & Koenig (1963). Calculations of molecular weights from the frictional coefficients of the assumed models, sedimentation coeffÉcients and partial specific volumes were made by employing the Svedberg equation according to procedures described by Perry & Koenig (1961). RESULTS All ~-crystallins were homogeneous by moving boundary electrophoresis in sodium veronal and Tris-veronal buffers. The mobilities in both buffers are reported in Table 1 and are expressed in arbitrary units of 10 -5 cm2/V sec. The values reported are the average of two determinations. The average deviations from the mean values are also listed in the parentheses in Table 1. TABLE 1--ELECTBOPHORETICMOBILITIES
Species Bovine Bovine (alkaline-modified) Ovine Canine
Na-veronal, pH 8"6,
Tris-veronal, pH 8"2
(F/2 0"10)
(0-075 M)
5'26 5'33
( _+0"03) ( _+0"07)
5.56 5"79
( _+0'00) ( _+0'01)
5"05 6"74
( _+0'18) ( _+0"15)
5"69 6"31
( + 0'06) ( _+0"01)
Typical sedimentation patterns for the intact ~-crystallins are presented in Fig. 1. T h e bovine ~-crystallin had a small amount (4 per cent) of a lighter component (3S). Ovine o~-crystallin had a similar impurity amounting to 5 per cent in the 3S component. This lighter material could be removed completely by gel filtration on a Sephadex G-200 column. The canine ~-crystallin was completely free of the 3S component by sedimentation having been subjected to purification by gel filtration. The progressive modification of bovine a-crystallin upon exposure to alkali is illustrated in Fig. 2. Apparently the modification is time dependent and the pattern for bovine ~-crystallin exposed to pH > 9.5 for approximately 1 hr at 4°C shows an intermediate stage in the modification of the ~-crystallin. There is modified o~-crystallin (shoulder on main component) in the presence of unchanged a-crystallin (main component). The final pattern shows the modified ~-crystallin after exposure to pH > 9.5 for 24 hr at 4°C. The pattern for the modified ~crystallin shows the presence of the 3S component to the extent of 4 per cent. T h e sedimentation coefficient of the modified a-crystallin is substantially reduced (14-29 S). T h e lines of regression of Sz0, w on c are given in Table 2. The standard errors of the slopes (%) and the intercepts (%) are given. The correlation coefficient, r, the number of concentrations studied, and the range of concentrations are also listed. The equations for the lines of regression of the reciprocal of relative viscosity (1/~/,) on concentration are listed in Table 3. The standard errors of the slopes
1-41%, 1-62% 29 rain, 60 °
Canine
50,740 r e v / m i n
FIe;. 1. Sedimentation patterns for the normal ~-crystallins in 0-2 M NaCI.
Ovine 1.06%, 0"91% 14min, 50 =
2-22%, 1.92% 16min, 55 °
Bovine
50°740 rev/rain
59,780 rev/min
a - Crystollins
Bovine a -Crystallin 59,780 r e v / m i n
2.22, 192 % 16 rain, 55 °
Short exposure to pH>9"5 H 6 % , ITmin, 55 °
Alkaline modified I.B2, 1-41%; 19min, 60 ° Fro. 2. S e d i m e n t a t i o n p a t t e r n s s h o w i n g tile progressive modification of b o v i n e ,~crystallin exposed to p H > 9.5.
0·003 0·003 0·001 0·003
0·076-1·43 0,51-1,76 0·05-1·68 0·21-0·54
8 8 7 8
1/'1/, = l-o'083c 1/'1/, = l-o'089c 1/'1/, = l-o'068c 1/'1/, = l-o'094c
Bovine Bovine (alkaline-modified) Ovine Canine
a,
Equation
Determinations
Range of concentrations in g/100 ml
TABLE 3-VISCOSITY
Species
0·17 0'14
0·10-1·26 0·10-0·86
14 10
S.o.w = 21·90-2·12c Sso.w = 22'38-1'27c
0'13 0·27
0·10-2·53 0·10-1·82
26 10
Sso.w = 22'16-1'33c Sao.w = 14'29-1'57c
Bovine Bovine (alkaline-modified) Ovine Canine
a,
Determinations
Equation
Species
Range of concentrations in g/100 ml
TABLE 2-SEDIMENTATION
0'11 0·15
0·04 0·13
aI
0·999 0'995
0'996 1'000
T
0·971 0·949
0·990 0·947
T
VI VI
...
rn
...z
~ ~t"
n
I
R
"'I
0
~
...~
...~
~
lI:
n
~t:;j
z
0
~
t"
0
...rn
156
WILL
E.
FERGUSON AND VIRGIL
L.
KOENIG
(as) and the correlation coefficients, r, are also listed. The number of concentrations studied, and the range of concentrations are given. The slopes of the regression lines are the weight intrinsic viscosities. TABLE 4--MOLECULAR WEIGHTS BY SEDIMENTATION EQUILIBRIUM (MENISCUS DEPLETION METHOD)
Components
Range of concentrations Determination in g/100 ml
Species
Major
Bovine Bovine (alkaline-modified) Ovine Canine
1,035,000 499,000
518,000 705,000
4 3
0·05-0·15 0·10-0·20
1,047,000 924,000
1,497,000 1,309,000
3 3
0·025-0·06 0·033-0·06
Minor
The molecular weights by the method of meniscus depletion equilibrium sedimentation are listed in Table 4. The number of concentrations studied and the range of concentrations are listed. The heterogeneity of the
The
14·02
Canine
12·28
Bovine (alkalinemodified)
9·15
11·22
Bovine
Ovine
[1]]
Species Mol. wt.
24
33 28
0·743 859,000 ( ±0'009)
0-694 773,000 ( ±0-002)
32
281
240
237
272
a (A) b (A)
0·725 490,000 (± 0'004)
0·740 923,000 ( ±0'017)
v
Prolate ellipsoid
1-55
1·40
1-56
1·47
fifo
926,000
865,000
545,000
1,065,000
Mol. wt.
9
12
8
11
169
144
137
166
a(A) b(A)
Oblate ellipsoid
517,000
1·75
849,000
1·40 862,000
1-68
62
63
53
66
Mol. wt. r (A)
1·62 994,000
flfll
Sphere
935,000
64
66
54
536,000
989,000
69
r (A)
1,097,000
Mol. wt.
ScheragaMandelkem
TABLE 5-MoLECULAR WEIGHTS AND DIMENSIONS CALCULATED FROM SEDIMENTATION AND VISCOSITY DATA
~
VI
....
'"
t""
...Zt""
~
RI
'
0
Z
0
~ ...N~ ......;0>
:z:
tl 0
~
Z
0
~
'"0t""
...
158
WILL
E.
FERGUSON AND VIRGIL
L.
KOENIG
exposure of more charged groups during the alkaline modification of the bovine ex-crystallin. The canine ex-crystallin had the highest electrophoretic mobilities in both buffers. The ovine ex-crystallin had the lowest electrophoretic mobility in the Na-veronal buffer, whereas the bovine ex-crystallin had the lowest electrophoretic mobility in the Tris-veronal buffer. The sedimentation patterns in Fig. 1 suggest some heterogeneity in addition to the 38 component of the bovine and ovine ex-crystallins. The main ex-crystallin boundary is somewhat skewed on the heavy side of the boundary in all three ex-crystallins. This skewness on the heavy side of the boundary suggests the presence of heavy material at a concentration insufficient for resolution or having a spectrum of sedimentation coefficients incapable of resolution into a single component. The presence of the 38 impurity is in agreement with the finding by Spector (1964) of 10 per cent of the 38 component in a preparation of bovine ex-crystallin. The 38 component is readily removed by gel filtration on Sephadex G-200. There appears to be no significant differences in the sedimentation coefficients at infinite dilution (intercepts of the lines of regression for 8 20 on c) for the three intact ex-crystallins (Table 2). The sedimentation coefficient at infinite dilution was significantly reduced for the alkaline-modified ex-crystallin (from 22·16 to 14·298). There was somewhat more variation in the concentration dependence of the sedimentation coefficient as manifested in the slopes of the regression lines of 8 20 on c (Table 2). Ovine ex-crystallin had the greatest concentration dependence (largest slope). Alkaline modification of the bovine excrystallin caused an increase in the slope of the regression line over that of the intact bovine ex-crystallin. In view of the standard errors of the slopes, the difference would not seem significant. The slopes of the lines of regression of 1/'YJr on concentration in Table 3 are the weight intrinsic viscosities of the various ex-crystallins. In view of the standard errors of the slopes, there is highly significant differences between weight intrinsic viscosities of the bovine, canine and ovine ex-crystallins. Furthermore, the weight intrinsic viscosity of the bovine ex-crystallin is significantly increased upon alkaline modification. The difference (0·083-0·089) is not as highly significant as are the differences in the weight intrinsic viscosities of the intact ex-crystallin; however, the standard errors of the slope would indicate that the increase in weight intrinsic viscosity on alkaline modification is due less than two in ten to chance. The canine ex-crystallin has the largest weight intrinsic viscosity, whereas ovine ex-crystallin has the smallest. The weight intrinsic viscosity for bovine ex-crystallin is identical to that found by Perry & Koenig (1961) using 0·1 M Na 2HPO, as solvent (0·0824). The concentration dependence of the sedimentation coefficient is thought to be due in part to a viscosity effect, but not entirely (Schachman, 1959). One might expect the slopes of the regression lines of 8 20 on c to be similar in range to the weight intrinsic viscosities. This is not the case. Whereas the sedimentation coefficient of the ovine ex-crystallin has the greatest concentration dependence, the weight intrinsic viscosity on the other hand is the least. Intrinsic viscosity is a measure of effective volume of a molecule. The relationship between concentration
ISOLATION AND CHARACTERIZATION OF ex-CRYSTALLINS
159
dependence of the sedimentation coefficient and intrinsic viscosity is not clear. The dynamic conditions of the sedimentation process vs. the relatively static conditions of the viscosity determination may explain the difference. The relative rigidity of the various ex-crystallin molecules as well as the relative states of hydration may be reasons for differences in the sedimentation and viscosity behavior of the various ex-crystallins. Molecular weights determined by the sedimentation equilibrium method require no assumptions regarding shape of the molecule. There was evidence of heterogeneity of the ex-crystallins from the results of the sedimentation equilibrium studies. The major component was considered to be the major portion of the ex-crystallin material. Minor portions of either lighter or heavier material caused the heterogeneity. Molecular weights of the various ex-crystallins (Table 4) are of the same magnitude. The canine ex-crystallin is somewhat smaller. The molecular weight of the alkaline modified bovine ex-crystallin is half that of the intact bovine ex-crystallin. When the molecular weights are calculated from partial specific volume, sedimentation and viscosity data, assumptions must be made as to the shapes of the molecules. Molecular weights assuming prolate and oblate ellipsoidal and spherical models are given in Table 5. The molecular dimensions are given for these various models. In addition, the dimensions are given for a sphere having the average molecular weight of the prolate and oblate ellipsoids. In the case of the bovine the molecular weights for the oblate and sphere calculated from the ScheragaMandelkern equation agree most closely with the value from sedimentation equilibrium. The molecular weight of the alkaline-modified ex-crystallin assuming a prolate ellipsoid agrees best with the value by sedimentation equilibrium. The value of the molecular weight for the ovine ex-crystallin assuming the spherical model as calculated from the Scheraga-Mandelkern equation agrees best with the value obtained from sedimentation equilibrium. The canine ex-crystallin behaves similarly to the bovine in that either the oblate ellipsoid or the spherical model calculated by the Scheraga-Mandelkern equation gives optimal molecular weights. The assumption was made that the molecules were unhydrated. Actually, all models assumed give acceptable values for the molecular weights when compared to the values found by sedimentation equilibrium. Undoubtedly ex-crystallins are hydrated, and consideration of hydration might alter the effectiveness of the various models. The values of partial specific volume in Table 5 do show fundamental differences. The canine ex-crystallin would appear to be the most dense and the ovine and bovine ex-crystallins would appear to be the least dense. The value for the partial specific volume of the bovine is identical to that found by Perry & Koenig (1961) with 0·1 M Na 2HP0 4 as solvent. The molecular dimensions would suggest that when a prolate or oblate ellipsoid are assumed as models, the canine ex-crystallin is the most asymmetric, and ovine is the least asymmetric. These molecular dimensions are in a substantial measure dependent upon the volume intrinsic viscosities ([7]]). The molecular weights of
160
WILL E. FERGUSON AND VIRGIL L. KOENIG
the IX-crystallins are satisfied quite satisfactorily by the spherical model as calculated by the Scheraga-Mandelkern equation. It has become apparent that a bovine alkaline-modified IX-crystallin has been produced with characteristic electrophoretic, sedimentation, partial specific volume and viscosity properties. The value of molecular weight suggests that the original IX-crystallin has been cleaved into a fragment with half the original molecular weight. In addition, there may be some unfolding of the molecule. This transformation of the IX-crystallin appears to be time dependent as well as pH dependent as manifested in Fig. 2. The alkaline modification of the bovine IXcrystallin appears to be irreversible as far as reversal of pH is concerned. Acknowledgements-The authors are indebted to the Burton Brothers Abattoir in Houston and to the Houston Dog Pound for making available the bovine and canine eyes. The authors appreciate the technical assistance of Mrs. Suzanne McClain. The authors appreciate the advice of Professor E. T. Adams, Texas A. and M. University. REFERENCES AUGUSTEYN R. C. & SPECTOR A. (1971) ex-Crystallin. Fractionation of subunits and sequence studies on an isolated polypeptide. Biochem.J. 124, 345-355. BJORK I. (1960) Separation of calf-lens proteins by means of vertical column zone electrophoresis. Biochim. biophys. Acta 45, 372-374. BJORK I. (1968) Comparative studies of ex-crystallin from lenses of different mammalian species. Exp. Eye Res. 7, 129-133. BLOEMENDAL H., BONT W. S., JONGKIND J. F. & WISSE J. H. (1964) Isolation of ex-crystallin by gradient centrifugation. Biochim. biophys. Acta 82,191-194. BLOEMENDAL H. & TEN CATE G. (1959) Isolation and properties of ex-crystallin from the bovine lens. Archs Biochem. Biophys. 84, 512-527. COBB B. F. & KOENIG V. L. (1968) Free electrophoretic analysis in various buffers of the soluble proteins from the crystalline lens. Exp. Eye Res. 7, 91-102. CHERVENKA C. H. (1969) A Manual of Methods for the Analytical Ultracentrifuge. p. 56. Beckman Instruments Inc., Palo Alto, California. DE GROOT K, HOENDERS H. J., LEON A. & BLOEMENDAL H. (1970) Isolation of IX-crystallin by means of continuous flow electrophoresis in a liquid film. Exp. Eye Res. 10,71-74. DRUCKER C. (1941) Partial specific volumes in binary and ternary solutions. Arkiv. Kemi Mineral Geol. 14A, 1-4-8. FRANCOIS J., RABAEY M. & WIEME R. J. (1955) New method for fractionation of lens proteins. Archs Ophthal. 53,481-486. KOENIG V. L. (1950) Partial specific volumes for some porcine and bovine plasma protein fractions. Arch Biochem. 25, 241-245. MOERNER C. T. (1894) Untersuchungen der Protein-Substanzen in den lichtbrechenden Medien des Auges. Z. physiol. Chem. 18, 61-106. NIYOGI S. K. & KOENIG V. L. (1963) Physicochemical properties of ex-crystallin from ox lens. Biochim. biophys. Acta 69, 283-295. NIYOGI S. K. & KOENIG V. L. (1965) Physicochemical properties of ex-crystallin from calf lens compared to those from ox lens. Can.J. Biochem. 43, 331-340. PERRY ANN J. & KOENIG V. L. (1961) Some physicochemical properties of the watersoluble proteins of the bovine eye lens. Biochim. biophys. Acta 46, 413-422. RESNIK R. A. (1957) Lens proteins-I. ex-Crystallin of calf lens. Am.J. Ophthalm. 44, 357362. SCHACHMAN H. K (1959) Ultracentrifugation in Biochemistry. Academic Press, New York.
ISOLATION AND CHARACTERIZATION OF ex-CRYSTALLINS
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SPECTOR A. (1964) Methods of isolation of alpha, beta, and gamma crystallins and their subgroups. Invest. Ophthalm. 3,182-193. SPECTOR A., LI L. K., AUGUSTEYN R. C., SCHNEIDER A. & FREUND T. (1971) ex-Crystallin. The isolation and characterization of distinct macromolecular fractions. Biochem. J.
124, 337-343. VAN DAM A. F. & TEN CATE G. (1966) Isolation and some properties of bovine ex-crystallin.
Biochim. biophys. Acta 121, 183-186. Key Word Index-Bos taurus; Ovis aries; Canis familiaris; Eye proteins; ex-crystallins; lens protein.
6
ISOLATION AND CHARACTERIZATION OF BOVINE, CANINE AND OVINE a-CRYSTALLINS* W I L L E. F E R G U S O N and V I R G I L L. K O E N I G Department of Biochemistry, The University of Texas Medical Branch, Galveston, Texas 77550 (Received 18 ffanuary 1972)
Abstract--1. a-CrystaUins were isolated from bovine, canine and ovine ocular lenses by chemical methods. 2. An alkaline-modified bovine ¢-crystallin was prepared by subjecting normal bovine ¢-crystallin to pH > 9"5 with subsequent precipitation at pH 5.1. 3. Electrophoretic mobilities of the various ¢-crystallins were determined by moving boundary electrophoresis in Na-veronal and Tris-veronal buffers. Canine ~-crystallin had the highest mobility. 4. The a-crystallins were characterized by sedimentation, viscosity and partial specific volume determinations. 5. Molecular weights were determined by means of sedimentation equilibrium. The values were: bovine, 1,035,000; alkaline-modified bovine, 499,000; ovine, 1,047,000; and canine, 924,000. 6. Molecular weights and dimensions were calculated assuming prolate and oblate ellipsoidal and spherical models from sedimentation, viscosity and partial specific volume data. INTRODUCTION SINCE Moerner (1894) first studied systematically the proteins of the crystalline lens, the larger emphasis has been on the relatively easily isolatable c~-crystallin. Francois et al. (1955) prepared ~-crystallin from bovine lenses by means of isoelectric precipitation in the presence of low concentrations of ethanol. Later, workers have used starch block electrophoresis (Bloemendal & Ten Cate, 1959), vertical column electrophoresis (Bj6rk, 1960), density gradient centrifugation (Bloemendal et al., 1964), gel filtration (Van Dam & Ten Cate, 1966) and continuous flow electrophoresis (deGroot et al., 1970) to isolate c~-crystallin. Only small amounts of material may be isolated by the above specialized procedures, consequently analytical studies are performed on a variety of samples. Bj6rk (1968) prepared ~-crystallins from eight mammalian species including the cow, sheep, pig, horse, rabbit, fox, mink and rat by zone electrophoresis. While the molecular *The data in this paper are taken from a dissertation submitted by W. E. Ferguson to the Graduate Faculty of the University of Texas in partial fulfillment for the degree, Doctor of Philosophy, 1972. This investigation was supported in part by Grant EY-00283 from the National Eye Institute, U.S. Public Health Service. 151
152
WILL E. FERGUSONAND VIRGIL L. KOENIG
w e i g h t s a n d e l e c t r o p h o r e t i c m o b i l i t i e s differ f r o m species to species, the i m m u n o logical c h a r a c t e r i s t i c s are similar. T h e v a r i o u s ~ - c r y s t a l l i n s are d i s s o c i a t e d in 7 M u r e a , t h u s s u g g e s t i n g t h a t t h e y are m a d e u p o f s u b u n i t s . R e s n i k (1957) a n d P e r r y & K o e n i g (1961) w e r e a m o n g t h e first to r e p o r t p h y s i c o c h e m i c a l studies. N i y o g i & K o e n i g (1963, 1965) r e p o r t e d a d d i t i o n a l p h y s i c o c h e m i c a l p r o p e r t i e s of b o v i n e a - c r y s t a l l i n . Still f u r t h e r p h y s i c o c h e m i c a l s t u d i e s have b e e n r e c e n t l y m a d e b y S p e c t o r et al. (1971) a n d A u g u s t e y n a n d S p e c t o r (1971). T h e p r e s e n t i n v e s t i g a t i o n is c o n c e r n e d w i t h t h e isolation b y c h e m i c a l m e a n s of b o v i n e , ovine a n d c a n i n e c~-crystallins. I n t h e p r o c e s s o f p r e p a r i n g b o v i n e c~-crystallin, an alkaline m o d i f i e d a - c r y s t a l l i n r e s u l t e d . P h y s i c o - c h e m i c a l p r o p e r t i e s o f all these c~-crystallins were d e t e r m i n e d in o r d e r to c h a r a c t e r i z e a n d o b s e r v e differences t h a t m i g h t exist. MATERIALS AND METHODS A total of 700 bovine (Bos taurus) eyes were collected at the Burton Brothers Abattoir in Houston, Texas. Approximately 200 ovine (Ovis aries) eyes were purchased from Armour and Company, San Angelo, Texas. A total of 650 canine (Canis familiaris) eyes were collected at the Houston Dog Pound. T h e eyes were collected as soon after the death of the animal as possible and stored on dry ice. T h e lenses were later removed from the frozen eyes and decapsulated. Bovine and ovine lenses were extracted in distilled water at 4°C by mechanical stirring using 160 g of lenses to 1500 ml H , O . T h e insoluble albuminoid was removed by centrifugation. T h e supernatant was diluted to a protein concentration of 0"25%. T h e p H of the diluted solution was 7"2. The p H was adjusted to 5"1 by adding 1 N HC1 slowly through a capillary with rapid stirring. After adjustment of pH, ethanol was added slowly through a capillary to a concentration of 10% (v/v). T h e a-crystallin precipitate was allowed to settle for a short time, and then removed by centrifugation at 10,000 rev/min on the Sorvall RC2-B for 20 min. The precipitate was redissolved in distilled water by adding enough 0"04 M NaHCOa to maintain the p H at 7'4. The at-crystallin in each case was reprecipitated twice. T h e final a-crystallin solution was dialyzed free of NaHCO3 and lyophilized. The lyophilized samples were stored at 1 °C. Canine a-crystallin was prepared in a similar manner. The precipitating solution was 2 per cent with respect to protein concentration. In order to remove an impurity lighter than a-crystallin, the final a-crystallin solution was dialyzed against 0"2 M NaCl-0.067 M phosphate buffer, p H 6'8 and then placed on a Sephadex G-200 column equilibrated to the same buffer. T h e a-crystallin was recovered in the void volume, dialyzed free of buffer and lyophilized. T h e lyophilized samples were stored at 1 °C. In the process of the purification of bovine a-crystallin, the solution was inadvertently exposed to p H 10'0. T h e resulting c~-crystallin although precipitating at p H 5"1 was lighter upon sedimentation than a-crystallin not exposed to p H above 7"4. A quantity of the modified a-crystallin was prepared by exposing the solution to p H 10 for 24 hr. T h e modified ct-crystallin was precipitated at p H 5"1 as described above. The final solution was dialyzed free of electrolytes against distilled water and lyophilized. The dried samples were stored at 1 °C.
Eleetrophoretic analysis Moving boundary electrophoresis was performed on the Spinco Model H electrophoresis and diffusion apparatus. T h e procedures have been described by Cobb & Koenig (1968). Veronal buffer, p H 8"6 I'/2 0"1 ( K = 0"00293-0"00300 ~-1 cm-1) and Tris-veronal buffer, p H 8"2, 0"075 M ( K = 0"001030-0"001047/Y~ -1 cm -1) were used. The preparation of these buffers has been previously described by Cobb & Koenig (1968). Protein concentrations were 0"5 % as determined with a Goldberg refractometer.
ISOLATION AND CHARACTERIZATION OF Ot-CRYSTALLINS
153
Sedimentation analysis Velocity sedimentation analyses were performed on a Spinco Model E ultracentrifuge at a speed of 50,740 rev/min. The solvent was 0"2 M NaCI for all sedimentation studies. Sedimentation values were determined at no less than ten different concentrations and were corrected to water as solvent at 20°C. Regression lines for sedimentation coefficient (Svedberg units) vs. concentration (g/100 ml) were determined by the method of least squares. Sedimentation coefficients at infinite dilution were thereby obtained. Correlation coefficients and standard errors of the slopes and intercepts were calculated. Protein concentrations were determined by means of a Goldberg refractometer. The amounts of the 3S component were determined from the composition analyses of the sedimentation patterns and corrected for radial dilution. Meniscus depletion sedimentation equilibrium studies were performed as described by Chervenka (1969). The following modifications were made: FC-43 fluorocarbon oil was not used and the temperatures were regulated at 20°C. A titanium rotor (An-H) was used to minimize errors in speed control. Fringe displacement ( Y r - Yo) for each radial interval (r) was determined by means of a Nikon microcomparator with a 50 x lens. Log ( Y r - Yo) vs. r * was plotted over the entire length of the cell. Molecular weights were calculated for the equation, M = 2R____~Tx 2-303 d log( Y r - Yo) ( 1 - ~p)w ~ dr ~ where M is the molecular weight; R, the gas constant; T, the absolute temperature; ~, the partial specific volume of solute; p, the density of solution; w, the rotational velocity in rad/sec; Yr - Yo, the fringe displacement at any point, r; and r, the distance from the center of rotation in cm.
Viscosity determination Viscosity determinations were made on a series of protein concentrations in 0"2 M NaC1 for each a-crystallin preparation. The concentrations were obtained by serial dilution. The initial solutions dialyzed against 0"2 M NaC1 were centrifuged at 13,000 r.c.f, in a Lourdes centrifuge for 30 min in order to remove insoluble material. Flow times were measured in an Ostwald viscometer at 21°C. Densities of the solutions were measured in an Ostwald pycnometer also at 21°C. The concentrations of the solutions were determined from weights of dried residues from solutions and solvents evaporated at 105°C. The reciprocals of the relative viscosities were plotted against concentrations. Equations of the resulting straight lines were determined by the method of least squares: liar = 1 - K c , where ,/r is the relative viscosity; K, the weight intrinsic viscosity; and c, the protein coneentration (g/100 ml). Volume intrinsic viscosity (~) is K(100)/~.
Partial specific volume determination The method of Drucker (1941) was used to determine the apparent partial specific volumes of the ~-crystallins. At least four different concentrations of protein were used. Details of the procedure are the same as described by Koenig (1950). Concentrations of protein were determined from the weights of dried residues from solutions and solvents evaporated at 105°C. Calculation of molecular dimensions The calculation of the molecular dimensions from viscosity data were according to the methods described by Perry & Koenig (1961) and Niyogi & Koenig (1963). Both prolate and oblate ellipsoids of revolution were assumed as models.In addition dimensions were calculated for a spherical model, and the molecular weight and dimensions were calculated
154
WILL E. FERGUSONAND VIRGIL L. KOENIG
according to the Scheraga-Mandelkern equation as was done by Niyogi & Koenig (1963). Calculations of molecular weights from the frictional coefficients of the assumed models, sedimentation coeffÉcients and partial specific volumes were made by employing the Svedberg equation according to procedures described by Perry & Koenig (1961). RESULTS All ~-crystallins were homogeneous by moving boundary electrophoresis in sodium veronal and Tris-veronal buffers. The mobilities in both buffers are reported in Table 1 and are expressed in arbitrary units of 10 -5 cm2/V sec. The values reported are the average of two determinations. The average deviations from the mean values are also listed in the parentheses in Table 1. TABLE 1--ELECTBOPHORETICMOBILITIES
Species Bovine Bovine (alkaline-modified) Ovine Canine
Na-veronal, pH 8"6,
Tris-veronal, pH 8"2
(F/2 0"10)
(0-075 M)
5'26 5'33
( _+0"03) ( _+0"07)
5.56 5"79
( _+0'00) ( _+0'01)
5"05 6"74
( _+0'18) ( _+0"15)
5"69 6"31
( + 0'06) ( _+0"01)
Typical sedimentation patterns for the intact ~-crystallins are presented in Fig. 1. T h e bovine ~-crystallin had a small amount (4 per cent) of a lighter component (3S). Ovine o~-crystallin had a similar impurity amounting to 5 per cent in the 3S component. This lighter material could be removed completely by gel filtration on a Sephadex G-200 column. The canine ~-crystallin was completely free of the 3S component by sedimentation having been subjected to purification by gel filtration. The progressive modification of bovine a-crystallin upon exposure to alkali is illustrated in Fig. 2. Apparently the modification is time dependent and the pattern for bovine ~-crystallin exposed to pH > 9.5 for approximately 1 hr at 4°C shows an intermediate stage in the modification of the ~-crystallin. There is modified o~-crystallin (shoulder on main component) in the presence of unchanged a-crystallin (main component). The final pattern shows the modified ~-crystallin after exposure to pH > 9.5 for 24 hr at 4°C. The pattern for the modified ~crystallin shows the presence of the 3S component to the extent of 4 per cent. T h e sedimentation coefficient of the modified a-crystallin is substantially reduced (14-29 S). T h e lines of regression of Sz0, w on c are given in Table 2. The standard errors of the slopes (%) and the intercepts (%) are given. The correlation coefficient, r, the number of concentrations studied, and the range of concentrations are also listed. The equations for the lines of regression of the reciprocal of relative viscosity (1/~/,) on concentration are listed in Table 3. The standard errors of the slopes
1-41%, 1-62% 29 rain, 60 °
Canine
50,740 r e v / m i n
FIe;. 1. Sedimentation patterns for the normal ~-crystallins in 0-2 M NaCI.
Ovine 1.06%, 0"91% 14min, 50 =
2-22%, 1.92% 16min, 55 °
Bovine
50°740 rev/rain
59,780 rev/min
a - Crystollins
Bovine a -Crystallin 59,780 r e v / m i n
2.22, 192 % 16 rain, 55 °
Short exposure to pH>9"5 H 6 % , ITmin, 55 °
Alkaline modified I.B2, 1-41%; 19min, 60 ° Fro. 2. S e d i m e n t a t i o n p a t t e r n s s h o w i n g tile progressive modification of b o v i n e ,~crystallin exposed to p H > 9.5.
0·003 0·003 0·001 0·003
0·076-1·43 0,51-1,76 0·05-1·68 0·21-0·54
8 8 7 8
1/'1/, = l-o'083c 1/'1/, = l-o'089c 1/'1/, = l-o'068c 1/'1/, = l-o'094c
Bovine Bovine (alkaline-modified) Ovine Canine
a,
Equation
Determinations
Range of concentrations in g/100 ml
TABLE 3-VISCOSITY
Species
0·17 0'14
0·10-1·26 0·10-0·86
14 10
S.o.w = 21·90-2·12c Sso.w = 22'38-1'27c
0'13 0·27
0·10-2·53 0·10-1·82
26 10
Sso.w = 22'16-1'33c Sao.w = 14'29-1'57c
Bovine Bovine (alkaline-modified) Ovine Canine
a,
Determinations
Equation
Species
Range of concentrations in g/100 ml
TABLE 2-SEDIMENTATION
0'11 0·15
0·04 0·13
aI
0·999 0'995
0'996 1'000
T
0·971 0·949
0·990 0·947
T
VI VI
...
rn
...z
~ ~t"
n
I
R
"'I
0
~
...~
...~
~
lI:
n
~t:;j
z
0
~
t"
0
...rn
156
WILL
E.
FERGUSON AND VIRGIL
L.
KOENIG
(as) and the correlation coefficients, r, are also listed. The number of concentrations studied, and the range of concentrations are given. The slopes of the regression lines are the weight intrinsic viscosities. TABLE 4--MOLECULAR WEIGHTS BY SEDIMENTATION EQUILIBRIUM (MENISCUS DEPLETION METHOD)
Components
Range of concentrations Determination in g/100 ml
Species
Major
Bovine Bovine (alkaline-modified) Ovine Canine
1,035,000 499,000
518,000 705,000
4 3
0·05-0·15 0·10-0·20
1,047,000 924,000
1,497,000 1,309,000
3 3
0·025-0·06 0·033-0·06
Minor
The molecular weights by the method of meniscus depletion equilibrium sedimentation are listed in Table 4. The number of concentrations studied and the range of concentrations are listed. The heterogeneity of the
The
14·02
Canine
12·28
Bovine (alkalinemodified)
9·15
11·22
Bovine
Ovine
[1]]
Species Mol. wt.
24
33 28
0·743 859,000 ( ±0'009)
0-694 773,000 ( ±0-002)
32
281
240
237
272
a (A) b (A)
0·725 490,000 (± 0'004)
0·740 923,000 ( ±0'017)
v
Prolate ellipsoid
1-55
1·40
1-56
1·47
fifo
926,000
865,000
545,000
1,065,000
Mol. wt.
9
12
8
11
169
144
137
166
a(A) b(A)
Oblate ellipsoid
517,000
1·75
849,000
1·40 862,000
1-68
62
63
53
66
Mol. wt. r (A)
1·62 994,000
flfll
Sphere
935,000
64
66
54
536,000
989,000
69
r (A)
1,097,000
Mol. wt.
ScheragaMandelkem
TABLE 5-MoLECULAR WEIGHTS AND DIMENSIONS CALCULATED FROM SEDIMENTATION AND VISCOSITY DATA
~
VI
....
'"
t""
...Zt""
~
RI
'
0
Z
0
~ ...N~ ......;0>
:z:
tl 0
~
Z
0
~
'"0t""
...
158
WILL
E.
FERGUSON AND VIRGIL
L.
KOENIG
exposure of more charged groups during the alkaline modification of the bovine ex-crystallin. The canine ex-crystallin had the highest electrophoretic mobilities in both buffers. The ovine ex-crystallin had the lowest electrophoretic mobility in the Na-veronal buffer, whereas the bovine ex-crystallin had the lowest electrophoretic mobility in the Tris-veronal buffer. The sedimentation patterns in Fig. 1 suggest some heterogeneity in addition to the 38 component of the bovine and ovine ex-crystallins. The main ex-crystallin boundary is somewhat skewed on the heavy side of the boundary in all three ex-crystallins. This skewness on the heavy side of the boundary suggests the presence of heavy material at a concentration insufficient for resolution or having a spectrum of sedimentation coefficients incapable of resolution into a single component. The presence of the 38 impurity is in agreement with the finding by Spector (1964) of 10 per cent of the 38 component in a preparation of bovine ex-crystallin. The 38 component is readily removed by gel filtration on Sephadex G-200. There appears to be no significant differences in the sedimentation coefficients at infinite dilution (intercepts of the lines of regression for 8 20 on c) for the three intact ex-crystallins (Table 2). The sedimentation coefficient at infinite dilution was significantly reduced for the alkaline-modified ex-crystallin (from 22·16 to 14·298). There was somewhat more variation in the concentration dependence of the sedimentation coefficient as manifested in the slopes of the regression lines of 8 20 on c (Table 2). Ovine ex-crystallin had the greatest concentration dependence (largest slope). Alkaline modification of the bovine excrystallin caused an increase in the slope of the regression line over that of the intact bovine ex-crystallin. In view of the standard errors of the slopes, the difference would not seem significant. The slopes of the lines of regression of 1/'YJr on concentration in Table 3 are the weight intrinsic viscosities of the various ex-crystallins. In view of the standard errors of the slopes, there is highly significant differences between weight intrinsic viscosities of the bovine, canine and ovine ex-crystallins. Furthermore, the weight intrinsic viscosity of the bovine ex-crystallin is significantly increased upon alkaline modification. The difference (0·083-0·089) is not as highly significant as are the differences in the weight intrinsic viscosities of the intact ex-crystallin; however, the standard errors of the slope would indicate that the increase in weight intrinsic viscosity on alkaline modification is due less than two in ten to chance. The canine ex-crystallin has the largest weight intrinsic viscosity, whereas ovine ex-crystallin has the smallest. The weight intrinsic viscosity for bovine ex-crystallin is identical to that found by Perry & Koenig (1961) using 0·1 M Na 2HPO, as solvent (0·0824). The concentration dependence of the sedimentation coefficient is thought to be due in part to a viscosity effect, but not entirely (Schachman, 1959). One might expect the slopes of the regression lines of 8 20 on c to be similar in range to the weight intrinsic viscosities. This is not the case. Whereas the sedimentation coefficient of the ovine ex-crystallin has the greatest concentration dependence, the weight intrinsic viscosity on the other hand is the least. Intrinsic viscosity is a measure of effective volume of a molecule. The relationship between concentration
ISOLATION AND CHARACTERIZATION OF ex-CRYSTALLINS
159
dependence of the sedimentation coefficient and intrinsic viscosity is not clear. The dynamic conditions of the sedimentation process vs. the relatively static conditions of the viscosity determination may explain the difference. The relative rigidity of the various ex-crystallin molecules as well as the relative states of hydration may be reasons for differences in the sedimentation and viscosity behavior of the various ex-crystallins. Molecular weights determined by the sedimentation equilibrium method require no assumptions regarding shape of the molecule. There was evidence of heterogeneity of the ex-crystallins from the results of the sedimentation equilibrium studies. The major component was considered to be the major portion of the ex-crystallin material. Minor portions of either lighter or heavier material caused the heterogeneity. Molecular weights of the various ex-crystallins (Table 4) are of the same magnitude. The canine ex-crystallin is somewhat smaller. The molecular weight of the alkaline modified bovine ex-crystallin is half that of the intact bovine ex-crystallin. When the molecular weights are calculated from partial specific volume, sedimentation and viscosity data, assumptions must be made as to the shapes of the molecules. Molecular weights assuming prolate and oblate ellipsoidal and spherical models are given in Table 5. The molecular dimensions are given for these various models. In addition, the dimensions are given for a sphere having the average molecular weight of the prolate and oblate ellipsoids. In the case of the bovine the molecular weights for the oblate and sphere calculated from the ScheragaMandelkern equation agree most closely with the value from sedimentation equilibrium. The molecular weight of the alkaline-modified ex-crystallin assuming a prolate ellipsoid agrees best with the value by sedimentation equilibrium. The value of the molecular weight for the ovine ex-crystallin assuming the spherical model as calculated from the Scheraga-Mandelkern equation agrees best with the value obtained from sedimentation equilibrium. The canine ex-crystallin behaves similarly to the bovine in that either the oblate ellipsoid or the spherical model calculated by the Scheraga-Mandelkern equation gives optimal molecular weights. The assumption was made that the molecules were unhydrated. Actually, all models assumed give acceptable values for the molecular weights when compared to the values found by sedimentation equilibrium. Undoubtedly ex-crystallins are hydrated, and consideration of hydration might alter the effectiveness of the various models. The values of partial specific volume in Table 5 do show fundamental differences. The canine ex-crystallin would appear to be the most dense and the ovine and bovine ex-crystallins would appear to be the least dense. The value for the partial specific volume of the bovine is identical to that found by Perry & Koenig (1961) with 0·1 M Na 2HP0 4 as solvent. The molecular dimensions would suggest that when a prolate or oblate ellipsoid are assumed as models, the canine ex-crystallin is the most asymmetric, and ovine is the least asymmetric. These molecular dimensions are in a substantial measure dependent upon the volume intrinsic viscosities ([7]]). The molecular weights of
160
WILL E. FERGUSON AND VIRGIL L. KOENIG
the IX-crystallins are satisfied quite satisfactorily by the spherical model as calculated by the Scheraga-Mandelkern equation. It has become apparent that a bovine alkaline-modified IX-crystallin has been produced with characteristic electrophoretic, sedimentation, partial specific volume and viscosity properties. The value of molecular weight suggests that the original IX-crystallin has been cleaved into a fragment with half the original molecular weight. In addition, there may be some unfolding of the molecule. This transformation of the IX-crystallin appears to be time dependent as well as pH dependent as manifested in Fig. 2. The alkaline modification of the bovine IXcrystallin appears to be irreversible as far as reversal of pH is concerned. Acknowledgements-The authors are indebted to the Burton Brothers Abattoir in Houston and to the Houston Dog Pound for making available the bovine and canine eyes. The authors appreciate the technical assistance of Mrs. Suzanne McClain. The authors appreciate the advice of Professor E. T. Adams, Texas A. and M. University. REFERENCES AUGUSTEYN R. C. & SPECTOR A. (1971) ex-Crystallin. Fractionation of subunits and sequence studies on an isolated polypeptide. Biochem.J. 124, 345-355. BJORK I. (1960) Separation of calf-lens proteins by means of vertical column zone electrophoresis. Biochim. biophys. Acta 45, 372-374. BJORK I. (1968) Comparative studies of ex-crystallin from lenses of different mammalian species. Exp. Eye Res. 7, 129-133. BLOEMENDAL H., BONT W. S., JONGKIND J. F. & WISSE J. H. (1964) Isolation of ex-crystallin by gradient centrifugation. Biochim. biophys. Acta 82,191-194. BLOEMENDAL H. & TEN CATE G. (1959) Isolation and properties of ex-crystallin from the bovine lens. Archs Biochem. Biophys. 84, 512-527. COBB B. F. & KOENIG V. L. (1968) Free electrophoretic analysis in various buffers of the soluble proteins from the crystalline lens. Exp. Eye Res. 7, 91-102. CHERVENKA C. H. (1969) A Manual of Methods for the Analytical Ultracentrifuge. p. 56. Beckman Instruments Inc., Palo Alto, California. DE GROOT K, HOENDERS H. J., LEON A. & BLOEMENDAL H. (1970) Isolation of IX-crystallin by means of continuous flow electrophoresis in a liquid film. Exp. Eye Res. 10,71-74. DRUCKER C. (1941) Partial specific volumes in binary and ternary solutions. Arkiv. Kemi Mineral Geol. 14A, 1-4-8. FRANCOIS J., RABAEY M. & WIEME R. J. (1955) New method for fractionation of lens proteins. Archs Ophthal. 53,481-486. KOENIG V. L. (1950) Partial specific volumes for some porcine and bovine plasma protein fractions. Arch Biochem. 25, 241-245. MOERNER C. T. (1894) Untersuchungen der Protein-Substanzen in den lichtbrechenden Medien des Auges. Z. physiol. Chem. 18, 61-106. NIYOGI S. K. & KOENIG V. L. (1963) Physicochemical properties of ex-crystallin from ox lens. Biochim. biophys. Acta 69, 283-295. NIYOGI S. K. & KOENIG V. L. (1965) Physicochemical properties of ex-crystallin from calf lens compared to those from ox lens. Can.J. Biochem. 43, 331-340. PERRY ANN J. & KOENIG V. L. (1961) Some physicochemical properties of the watersoluble proteins of the bovine eye lens. Biochim. biophys. Acta 46, 413-422. RESNIK R. A. (1957) Lens proteins-I. ex-Crystallin of calf lens. Am.J. Ophthalm. 44, 357362. SCHACHMAN H. K (1959) Ultracentrifugation in Biochemistry. Academic Press, New York.
ISOLATION AND CHARACTERIZATION OF ex-CRYSTALLINS
161
SPECTOR A. (1964) Methods of isolation of alpha, beta, and gamma crystallins and their subgroups. Invest. Ophthalm. 3,182-193. SPECTOR A., LI L. K., AUGUSTEYN R. C., SCHNEIDER A. & FREUND T. (1971) ex-Crystallin. The isolation and characterization of distinct macromolecular fractions. Biochem. J.
124, 337-343. VAN DAM A. F. & TEN CATE G. (1966) Isolation and some properties of bovine ex-crystallin.
Biochim. biophys. Acta 121, 183-186. Key Word Index-Bos taurus; Ovis aries; Canis familiaris; Eye proteins; ex-crystallins; lens protein.
6