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An ultracentrifuge study of silk fibroin
238
BIOCHIMICA ET BIOPHYSICA ACTA BBA 45 109
AN U L T R A C E N T R I F U G E STUDY OF S I L K F I B R O I N M. S. N A R A S I N G A RAO AND M. W. P A N D I T
Regzonal Research Laboratory, Hyderabad (India) (Received May 2ist, 1964)
SUMMARY
The molecular weight of silk fibroin in o.I M KNO 3 solution has been determined at various protein concentrations and p H values b y the Archibald method. The values calculated from the top meniscus decreased with time, reaching a limiting value of 16000 ± 2000. In sedimentation-velocity experiments, a single peak was observed at all protein concentrations and p H values studied. The sedimentation coefficient at infinite dilution had a value of 1.36 S and did not vary with pH. From 1.36 S, an approximate molecular weight of 21 ooo is calculated. Since this is close to the limiting value of 16000 it is concluded that the fibroin preparation consists mainly of this component. I t has been shown that the change of molecular weight with time was due to the presence of a high-molecular-weight impurity. This fraction was concentrated by preparative centrifugation at 35000 rev./min. I t had a sedimentation coefficient of 22.3 S approximating to a molecular weight of 1.2. lO6. Ultracentrifuge field-relaxation experiments also indicated the presence of heterogeneity and further suggested that the preparation contained essentially only two species, of molecular weight 16000 and about 1.2. IOe, without any intermediates.
INTRODUCTION
In a study 1 of the H+ equilibria of silk fibroin it was observed that when the titration data were analysed in terms of the Linderstr6m-Lang equation 2, treating the electrostatic interaction term as an empirical parameter, this term varied with pH. Above p H 4, the value increased to a m a x i m u m (around pH 7) and then decreased to a constant value beyond p H 9- A similar observation has been made in the titration study of insulin 3 and the interpretation has been given that this m a y be due to aggregation which the protein undergoes in the neutral p H range. If there is a p H dependent aggregation in the case of silk fibroin, molecular weight measurements should provide direct experimental evidence for the postulate. Thus we were led to determine the molecular weight of silk fibroin at various p H values and protein concentrations. If indeed there is an aggregation reaction, it was felt that these measurements might also explain the diversity in the reported molecular weight d a t # . Biochim. Biophys. Acta, 94 (1965) 238-247
AN
ULTRACENTRIFUGE
STUDY
OF
SILK
FIBROIN
239
EXPERIMENTAL
Silk fibroin The sample of silk used in this study was obtained through the courtesy of Dr. A. B. BISWAS, National Chemical Laboratory, Poona and was identical with that on which a study of H + equilibria has been made 1. The material had been purchased from the Mysore Sericulture Department from their silk breeding centre at Kunigal and was designated by them as "Mysore Pure Race". This silk belongs to the Bombyx mori group. The following procedure was used to prepare fibroin from the silk fibres: ioo g of silk obtained in reeled form was cut into small pieces, approx. I cm long, and washed in running water for several hours to remove foreign matter. The washed sample was hand-squeezed and boiled for 30 rain in 2 1 of Na~CO 3 (2 %) solution. This operation was carried out six times, each time using fresh, 2-1 aliquot of carbonate solution. The material was then successively washed with water and dilute acetic acid to remove the traces of carbonate. Finally the sample was washed thoroughly with distilled water and air dried.
Fibroin solution About 20 g of fibroin were dissolved in IOO ml of a saturated LiCNS solution by gently warnfing the solution. The resulting viscous solution was diluted with an equal volume of water and filtered through sintered glass. The filtrate was dialysed for 48 h in cold against distilled water to remove LiCNS and then passed through a column of Amberlite I.R. 12o and Amberlite I.R. 400 resins. The deionized fibroin solution had a pH of 4.20 ~ o.io and was stored in the cold.
Ultracentrifuge measurements These were made with a Spinco Model-E ultracentrifuge, fitted with a phaseplate and RTIC unit. Molecular weights were determined by the Archibald inethod 5. A standard I2-mm aluminium centre-piece was used. For obtaining the concentration of the protein solution in terms of the refractive index units, a synthetic-boundary cell of the capillary type (Spinco No. 5993) or KEGELES-type ~ was used. The ultracentrifuge measurements were made at room temperature (about 25°). The protein solutions were prepared in o.I M KNO 3 solution, to keep the composition identical with that used in H + equilibria studies 1. The pH of the solution was adjusted by the, addition of HNO 3 or KOH. The pH measurements were made with a Metrohm p H meter Model E396. For a false bottom, CC14 was used. Protein concentration was determined bv- measuring the absorption at 276 m/~, using a value of E 276 ~%m,a -11.5 -at pH 5.9 (see ref. I).
Partial specific volume For the calculation of the molecular weight of fibroin, a value of 0.75 has been :assumed for the partial specific volume. RESULTS
AND DISCUSSION
Molecular weight data The molecular weight of fibroin was determined by the Archibald method, both, as a function of concentration and of pH. At the isoionic pH (4.2o), measurements
Biochim. Biophys. Acta, 94 (1965) 238 247"
240
•.
S. NARASINGA RAO, M. W. PANDIT
were made with 0.5, ~, ~ and 4 % solutions and at pH values 3.o, 7.0 and 9.0 with a 1% solution. Normally, in each experiment three pictures were taken at 15 min, 30 min, and 45 min, respectively, after the attainment of the operating speed and the molecular weight calculated from these. It was observed (Fig. I) that the molecular weights calculated at various periods of centrifugation differed significantly. The values at t h e top meniscus decreased with time and those at the bottom meniscus increased. T h e gradients at the bottom meniscus were invariably very steep and extrapolation t o obtain the dc/dx value at the meniscus, needed for molecular weight calculations, was uncertain. In Fig. I, therefore, only molecular weight data at the top meniscus are given.
5.0
®
o
I~: 0
I
20
I
40
I
60
I
~
I
~D 0 120 Time(rnin)
I
140
I
160
'1 180
Fig. i. The molecular weight of fibroin at various concentrations and pFi ~ as a function of time. Q - - Q , pH 4.2, 1% solution (8766 rev./min) ; ®--®, pH 4.2, 2 % solx~Cdo~(7447 rev./min) ; Q - - I , pH 4.2, 4 ~o solution (3397 rev./min); @--@, pH 3.0, 1% soh~tiot~ t"7447 rev./min); ~, pH 9.0, 1% solution (9341 rev./min). -
-
The decrease in molecular weight with time was observed at a~ the concentrations and pH values used. While there is considerable scatter in the i5-min values obtained at different concentrations and pH, the agreement among the values improves with the period of centrifugation and these tend towards a limiting value. In one experiment where the solution was centrifuged for 18o min, the values obtained at 9° min and 18o min were the same within experimental error. The molecular weight value at 15 min is about 5oooo and the limiting value is 16oo0 4- 2ooo. It was also observed that the centrifugal field has an effect on the measured molecular weight. A majority of the experiments were made at speeds ranging from 7000 to 9000 rev./min when the molecular weight was observed to decrease with time. However, when solutions of the same concentration were centrifuged at higher speeds (12 59 ° or 15 220 rev./min), the molecular weight was found to be independent of time and corresponded to 16000, the limiting value obtained in low-speed experiments (Fig. 2). These results can be interpreted in two ways: (i) The fibroin contains a highmolecular-weight impurity which gets depleted at the top meniscus rapidly and t h u s causes the weight-average molecular weight to decrease with time. The effect of higher centrifugal field will be to enhance the rate of depletion. (2) The fibroin undergoes a concentration-dependent aggregation reaction. As the concentration at the t o p
Biochim. Biophys. Acta, 94 (I965) 238-247
AN ULTRACENTRIFUGE STUDY OF SILK FIBROIN
241
meniscus decreases with time, the molecular weight may be :expected to decrease if the rate of re-equilibration is slow compared to the rate of sedimentation. \ \\
4.0
\
?
\ \\
2.0
I
20
I
40 Time
I
60 (rain)
I
80
Fig. 2. The molecular Weight of fibroin d e t e r m i n e d a t higher speeds, as a function of time. • - - O , p H 4.2, o . 5 ~ o solution (12590 rev./min); O - - O , p H 4.2, I.O% solution (12590 rev./min); ® - - ® , p H 4.2, 2.O~o solution (15220 rev./min). The d a s h e d curve represents the d a t a at low speed.
Sedimentation-velocity
data
To decide between the two alternative explanations, sedimentation-velocity experiments were made. If the protein contains a heavier component in an appreciable proportion, the velocity pattern would reveal it as a separate peak. On the other hand, if there is an aggregation reaction, the values of s20, w would be expected to increase with concentration 7. Further, depending upon the degree of aggregation, the velocity pattern m a y consist of two peaks; the ratio of the areas under the two peaks will v a r y with protein concentration unlike in the case of heterogeneity s. At the is•ionic pH, sedimentation-velocity measurements were made with o.5, I, 2 and 4 % fibroin solutions. At p H 3.0 and 7.4 experiments were conducted with a I O//osolution. In all the cases a single svmmetrical~ peak was obtained (Fig. 3a, b). Further the s20,w values showed the normal concentration dependence of a nonaggregating protein system (Fig. 4). The data at various concentrations could be approximately fitted into the equation: 0
s20, w = s2o, w(I - - k C )
Fig. 3. The s e d i m e n t a t i o n - v e l o c i t y p a t t e r n of fibroin. (a) i ~o solution, p H 4.2 ; centrifugation at 59 780 rev./min for i 15 rain. (b) I ~o solution, p H 7.o ; centrifugation at 59 780 rev.]min for i i o rain. S e d i m e n t a t i o n proceeds from left to right.
Biochim. Biophys. Acta, 94 (1965) 238-247
242
M . S . NARASINGA RAO, M. W. PANDIT
The s~0,w value at infinite dilution, S°~o, w, was found to be 1.36 S and k, o.II (g/Ioo ml) -1. The values at different pH were also found to be the same within experimental error (Fig. 5). 1.4
1.2 1.4]
(~)
m
1.2
O.E
I 1.0
I 2,0 Concentration
I t 3.0 4.0 ( g/lOOml ) /
1.0
~
Q
I
I
4.0
6.0
pH
I 8.0
Fig. 4- The s e d i m e n t a t i o n coefficient of fibroin at various concentrations. Fig. 5. The s e d i m e n t a t i o n coefficient of i ~o fibroin solutions at various p H values.
An estimate of the molecular weight of the protein corresponding to 1.36 S can be made using the equation 9, M=
469 ° (s2o, w)a/2 [,/jl/2 (i -- ~p)m
where M is molecular weight, s20, w, sedimentation coefficient expressed in Svedberg units; [~], the intrinsic viscosity expressed in (g/Ioo ml) -z, ~, partial specific volume and p, density of solution. The intrinsic viscosity of fibroin at pH 4.2 has been measured and is o.127 (see ref. I). The molecular weight calculated from the above equation is 21000. This value approximates to the limiting value obtained by the Archibald method, namely, 16000. The sedimentation-velocity experiments clearly indicate that fibroin did not exhibit either a concentration-dependent or a pH-dependent aggregation reaction. The observed variations in the Archibald molecular weight data are therefore likely to have been due to a component which is heavier than 16000. Since this component had, as adjudged by Archibald experiments, considerable velocity even at low speeds, its molecular weight should be fairly high. To get direct experimental evidence for the existence of this heavier component and to determine its sedimentation behaviour, an attempt was made to concentrate this component from the mixture. Fibroin solution (13 ml of 5 % solution) was centrifuged at 35o00 rev./min for 3 h in a Type-4o Rotor in a Spinco Model-L centrifuge. At the end of the run the top 4 ml and the bottom 2 ml of the solution were collected separately. The bottom fraction was diluted with an equal volume of 0.2 M KNO 8 solution so that the protein concentration was approx. 1%, and a sedimentationvelocity experiment was made by centrifuging it at 25980 rev./min. The velocity pattern is given in Fig. 6. The sedimentation coefficient of this peak was found to be 22.3 S. On the assumption that molecular weight is proportional to (s) 3/~, an estimate of the molecular weight of the component can be made from the sedimentation coefficient. Since the 16000 mol. wt. component has a s~o,w value of 1.25 S at 1 % protein concentration, it follows that M
16000
or M
2o6 00o
LI.25J
Biochim. Biophys. Acta, 94 (1965) 238-247
AN ULTRACENTRIFUGE STUDY OF SILK FIBROIN
243
Thus the high-molecular-weight impurity in the fibroin preparation was nearly 80 times heavier than the major component. Since the molecular weights of both the fractions are known, their proportion in the mixture can be estimated if the true weight-average molecular weight is known. The data of Fig. I when extrapolated to zero time should yield this value. I t is seen that such an extrapolation is impossible with the data on 1 % and 2 % solutions. However, the values obtained with a 4 % solution, where the experiment had been made at a low speed, can be reasonably extrapolated to a value of IOOOOO.From this the proportion of the heavier component can be calculated to have been about 7 %.
Fig. 6. The sedimentation-velocity pattern of the heavier component. Protein concentration, approx. 1% ; centrifugation at 25 980 rev./min for 3o min. Sedimentation proceeds from left to right.
In sedimentation-velocity experiments with the fibroin preparation, it was impossible to detect this component. W~ did not notice any peak formation even with a 5 To solution when it was centrifuged for I h at 25 980 rev./min. Therefore the proportion of the heavier component cannot have been more than 2 or 3 %. The estimated value of 7 % can obviously be in gross error as it is based on approximate values for the true weight-average value and the molecular weight of the heavier component.
Field-relaxation experiments Recently, KEGELES AND SIA1° have shown that ultracentrifugal field-relaxation experiments provide a sensitive technique to detect high-molecular-weight impurities in a protein preparation and to distinguish heterogeneity from concentrationdependent, reversible, aggregation reactions. The experimental procedure consists in layering the solvent on the solution using a synthetic-boundary cell, and accelerating to a speed sufficient to cause sedimentation. This is followed by careful deceleration to a sufficiently low speed to permit horizontal extrapolation of the schlieren pattern to the image of the bottom of the fluid column. From an enlarged tracing of the pattern the weight-average molecular weight at the cell bottom is calculated with the equation : EFR TZb Mw ( I - - 7~p)(O 2 Xb(A1 +/t2) where E is the enlarger magnification factor; F, the magnification factor of the camera lens in the ultracentrifuge; Zb, the extrapolated deflection above the estimated base Biochim. Biophys. Acta, 94 (1965) 238-247
244
M.S. NARASINGA RAO, M. W. PANDIT
i
Fig. 7. Schlieren p a t t e r n s in ultracentrifugal field-relaxation experiments. (a) i % fibroin solution, p H 4.2. Centrifugation for 5 rain at 25980 rev./min, decelerated to 4267 rev./min; picture taken after I6 min at 4267 rev./min; b a r angle 80 °. (b) About i % solution of Fraction II. Centrifugation for 5 min at 25980 rev./min, decelerated to 3954 rev./min; picture taken after 16 min at 3964 rev./min; bar angle 80 °. (c) About i % solution of Fraction III. Centrifugation for 5 min at 25980 rev./min, decelerated to 9600 rev./min; picture taken after i i min at 9600 rev./min; bar angle 7 o°. Sedimentation proceeds from left to right.
line of the schlieren diagram at the bottom meniscus, as obtained from the enlarger projection; A 1 and A 2 are the areas under the free peak and near the cell bottom, as measured from the enlarger projection. The other terms in the equation have the usual meaning. The areas are measured with a planimeter. For a reversible aggregation reaction, the relaxation of the concentration build up at the cell bottom affects the molecular weight in a complex way. It is observed that in such a case the period of centrifugation at a higher speed has an effect on the molecular weight calculated. On the other hand, in the case of heterogeneity which does not involve gross impurity, the measured molecular weight is independent of the period of centrifugation at the higher speed. A few ultracentrifuge field-relaxation experiments were made with isoionic fibroin solutions with two objectives, v i z . , (I) to confirm the heterogeneity indicated by the Archibald experiments, (2) to determine if this heterogeneity is due to only one heavy impurity or a number of them. To test the reliability of the method and to arrive at satisfactory experimental conditions for fibroin solutions, an experiment was also made with crystalline lysozyme. The experimental details and the molecular weights obtained are given in Table I. A t(EGELES-type synthetic-boundary cell and CC14 for false bottom were used. The lysozyme was a Sigma product, lot No. L42B-247. In a sedimentationvelocity experiment, a 1% solution (o.I M phosphate buffer of pH 6.9) gave a single symmetrical peak with sso, w of 2.08 S, in satisfactory agreement with the literature v a l u e 11.
The molecular weight of lysozyme obtained by the field-relaxation method is 2019o compared to I5 585 from the Archibald method. This could be due to a heavy impurity in the lysozyme sample or to the limitations in our hands of the fieldrelaxation method for absolute molecular weight determinations. The fibroin preparation gives a molecular weight of 305000. This value is weighted more towards the high-molecular-weight component, which is in agreement with the observation of KEGELES AND SIA1°. Further, the period of centrigugation, 2 min or 5 min, at the higher speed has no effect on the measured molecular weight. This result suggests that the protein sample does not contain a number of heavy impurities. The sample (Fraction II) which was obtained as top fraction by centrifugation of the fibroin preparation for 3 h at 35000 rev./min in a preparative centrifuge still Biochim. Biophys. Acta, 94 (1965) 238-247
AN ULTRACENTRIFUGE STUDY OF SILK FIBROIN
245
TABLE I MOLECULAR W E I G H T
DATA FROM ULTRACENTRIFUGE
FIELD-RELAXATION EXPERIMENTS
T h e r a t e of deceleration in t h e e x p e r i m e n t s w a s a p p r o x . IOOO r e v . / m i n .
Sample
Period of centri- Lower speed and period fugation at of centrifugation 2598o rev./min (min)
Lysozyme, I ~/o s o l u t i o n i n o.I M p h o s p h a t e buffer (pH 6.9)
5
Fibroin, 1 % solution in o. i M KNO3
2 5
10560 r e v . / m i n ; 8 m i n io56o rev./min; ii min
4267 4267 3 555 3555
rev./min; rev./min; rev./min; rev./min;
Mol. wt.
2o52o 1986o
I6min 24 m i n I6min 17 m i n
322ooo 3o 3 ooo 294o00 3020o0
F r a c t i o n II, a b o u t 1 % s o l u t i o n in o.I M K N O 3
5
3954 r e v . / m i n ; I o m i n 3 954 r e v . / m i n ; 16 m i n
1082oo 99200
Fraction III, a b o u t 1 % s o l u t i o n in o.I M K N O 3
5
9600 r e v . / m i n ; 8 m i n 96oo r e v . / m i n ; 11 rain
23o6o 2148o
Mol. wt. at the bottom by the Archibald Method
15530 15564
13700 13980
exhibited heterogeneity although the molecular weight (lO3 7oo) is less than that obtained with the "unpurified" sample. This was also revealed in Archibald experiments. The molecular weights at the top meniscus, determined at low speeds, decreased with time; the rate of decrease was less than that observed with the "unpurified" sample. Another sample (Fraction III) obtained as the top fraction from centrifugation for 7 h at 35000 rev./min gave a molecular weight of 22270. This value m a y be considered to correspond to 16 ooo, the molecular weight of the light component obtained by the Archibald method. Further, this sample, in Archibald experiments at low speeds, did not show any time dependence of molecular weight. Thus, this fraction appeared to be completely free from the heavy component of molecular weight 1206 ooo. In the relaxation experiments a doubt has often been expressed that when the solution is centrifuged at the higher speed a gel or precipitate m a y be formed at the solution-immiscible liquid interface and that this m a y redissolve on deceleration. KEGELES AND SIA1° have pointed out that in the case of gel formation it will be impossible to make the gradient at the interface bend over even when decelerated to any practically attainable low speed. On the other hand such bending over is possible if there is no gel formation. I t is seen from Fig. 8 that the Fraction I I I , when decelerated to 4000 rev./min and held at that speed for IO min after centrifugation for 5 min at 25 980 rev./min, gives a schlieren pattern where the gradient at the interface is bent over. This experiment shows that there is no gel formation and the estimated molecular weight represents the weight-average value of the mixture present at the cell bottom at that moment. Biochim. Biophys. Acta, 94 (1965) 238-247
246
M.S. NARASINGA RAO, M. W. PANDIT
Both the Archibald and field-relaxation experiments suggest that the fibroin preparation consists essentially of only two components of molecular weight 16000 and approx. 1.2.io e, respectively, and that there are no intermediates present. If there were a number of heavy impurities, it is difficult to visualize that in the Archibald experiments a limiting value would be obtained and this would be identical with the value obtained in experiments involving the use of higher speeds and shorter periods of centrifugation. In the experiment using the field-relaxation technique, which appears to be a powerful tool for detecting heterogeneity, the sample which had been subjected to preparative centrifugation for 7 h gave a value close to 16000. All these results are compatible with the conclusion that the fibroin preparation consists of two components only.
Fig. 8. Schlieren p a t t e r n in ultracentrifugal field-relaxation experiment. About 1% solution of Fraction III. Centrifugation for 5 min a t 25980 rev./min decelerated to 4072 rev./min; picture t a k e n after IO min a t 4o72 rev./min; bar angle 7o°. Sedimentation proceeds from left to right,
Origin of the heterogeneity Fibroin as found in the silk fibre is insoluble in water and common inert solvents. The use of drastic methods to remove sericin from fibroin and of concentrated aqueous electrolyte solutions to dissolve the fibroin may cause degradation of the protein. The procedure we have employed to obtain aqueous fibroin solution is extraction of the silk fibres six times with boiling, 2 % Na2CO 8 solution, and dissolution of the fibroin in saturated LiCNS solution followed by removal of LiCNS by exhaustive dialysis and passage over ion-exchange resins. Two samples prepared at different times gave identical results. If these results can be considered representative, it may mean that fibroin consists of two fractions only. The higher-molecular-weight component may be an aggregate of the 16000 unit (or a lower unit), these units being held together by sericin. As the sericin is removed it may degrade to the 16000 unit by an "all or none" mechanism. The proportion of the heavier component in any preparation will then depend on the extent to which sericin has been removed. It is known that the last traces of sericin are intimately combined with the fibroin filament x2. Unless the sericin is completely removed, the fibroin preparation is likely to contain traces of the heavier impurity. The molecular weight of such a preparation determined by dynamic methods, such as velocity ultracentrifugation may not be sensitive to the presence of this impurity. On the other hand values determined by static methods such as osmotic pressure, light scattering and diffusion will be greatly influenced by the heavier component. Biochim. Biophys. Acta, 94 (1965) 238-247
AN ULTRACENTRIFUGE STUDY OF SILK FIBROIN
247
ACKNOWLEDGEMENTS
The authors are grateful to Dr. G. S. SIDHU for his encouragement. The Spinco Model-E ultracentrifuge used in this study was a gift from the Wellcome Trust London, to Dr. P. M. BHARGAVA. REFERENCES i 2 3 4 5 6 7 8 9
M. S. NARASINGA RAO, S. I4,. BATNI, D. G. TAKTE AND A. B. BISWAS, u n p u b l i s h e d results. C. TANFORD,Advan. Protein Chem., 17 (1962) 69. C. TANFORD AND J. EPSTEIN, J. Am. Chem. Soc., 76 (1954) 2163. F. LUCAS, J. T. B. SHAW AND S. G. SMITH, Advan. Protein Chem., 13 (1958) lO7. S. M. KLAINER AND G. KEGELES, J. Phys. Chem., 59 (1956) 952. G. KEGELES, J. Am. Chem. Soc., 74 (1952) 5532. G. W. SCHWERT, J. Biol. Chem., 179 (1949) 655. G. A. GILBERT, Discussions Faraday Soc., 20 (1955) 68. H. i{. SCHACHMAH, Ultracentrifugation in Biochemistry, Academic Press, New York, 1959, p. 242. Io G. KEGELES AND C. L. SIA, Biochemistry, 2 (1963) 19o6. i i L. R. WETTER AND H. F. DEUTSCH, J. Biol. Chem., 192 (1951) 237. i2 J. T. B. SHAW AND S. G. SMITH, Nature, 168 (1951) 745.
Biochim. Biophys. Acta, 94 (1965) 238-247
BIOCHIMICA ET BIOPHYSICA ACTA BBA 45 109
AN U L T R A C E N T R I F U G E STUDY OF S I L K F I B R O I N M. S. N A R A S I N G A RAO AND M. W. P A N D I T
Regzonal Research Laboratory, Hyderabad (India) (Received May 2ist, 1964)
SUMMARY
The molecular weight of silk fibroin in o.I M KNO 3 solution has been determined at various protein concentrations and p H values b y the Archibald method. The values calculated from the top meniscus decreased with time, reaching a limiting value of 16000 ± 2000. In sedimentation-velocity experiments, a single peak was observed at all protein concentrations and p H values studied. The sedimentation coefficient at infinite dilution had a value of 1.36 S and did not vary with pH. From 1.36 S, an approximate molecular weight of 21 ooo is calculated. Since this is close to the limiting value of 16000 it is concluded that the fibroin preparation consists mainly of this component. I t has been shown that the change of molecular weight with time was due to the presence of a high-molecular-weight impurity. This fraction was concentrated by preparative centrifugation at 35000 rev./min. I t had a sedimentation coefficient of 22.3 S approximating to a molecular weight of 1.2. lO6. Ultracentrifuge field-relaxation experiments also indicated the presence of heterogeneity and further suggested that the preparation contained essentially only two species, of molecular weight 16000 and about 1.2. IOe, without any intermediates.
INTRODUCTION
In a study 1 of the H+ equilibria of silk fibroin it was observed that when the titration data were analysed in terms of the Linderstr6m-Lang equation 2, treating the electrostatic interaction term as an empirical parameter, this term varied with pH. Above p H 4, the value increased to a m a x i m u m (around pH 7) and then decreased to a constant value beyond p H 9- A similar observation has been made in the titration study of insulin 3 and the interpretation has been given that this m a y be due to aggregation which the protein undergoes in the neutral p H range. If there is a p H dependent aggregation in the case of silk fibroin, molecular weight measurements should provide direct experimental evidence for the postulate. Thus we were led to determine the molecular weight of silk fibroin at various p H values and protein concentrations. If indeed there is an aggregation reaction, it was felt that these measurements might also explain the diversity in the reported molecular weight d a t # . Biochim. Biophys. Acta, 94 (1965) 238-247
AN
ULTRACENTRIFUGE
STUDY
OF
SILK
FIBROIN
239
EXPERIMENTAL
Silk fibroin The sample of silk used in this study was obtained through the courtesy of Dr. A. B. BISWAS, National Chemical Laboratory, Poona and was identical with that on which a study of H + equilibria has been made 1. The material had been purchased from the Mysore Sericulture Department from their silk breeding centre at Kunigal and was designated by them as "Mysore Pure Race". This silk belongs to the Bombyx mori group. The following procedure was used to prepare fibroin from the silk fibres: ioo g of silk obtained in reeled form was cut into small pieces, approx. I cm long, and washed in running water for several hours to remove foreign matter. The washed sample was hand-squeezed and boiled for 30 rain in 2 1 of Na~CO 3 (2 %) solution. This operation was carried out six times, each time using fresh, 2-1 aliquot of carbonate solution. The material was then successively washed with water and dilute acetic acid to remove the traces of carbonate. Finally the sample was washed thoroughly with distilled water and air dried.
Fibroin solution About 20 g of fibroin were dissolved in IOO ml of a saturated LiCNS solution by gently warnfing the solution. The resulting viscous solution was diluted with an equal volume of water and filtered through sintered glass. The filtrate was dialysed for 48 h in cold against distilled water to remove LiCNS and then passed through a column of Amberlite I.R. 12o and Amberlite I.R. 400 resins. The deionized fibroin solution had a pH of 4.20 ~ o.io and was stored in the cold.
Ultracentrifuge measurements These were made with a Spinco Model-E ultracentrifuge, fitted with a phaseplate and RTIC unit. Molecular weights were determined by the Archibald inethod 5. A standard I2-mm aluminium centre-piece was used. For obtaining the concentration of the protein solution in terms of the refractive index units, a synthetic-boundary cell of the capillary type (Spinco No. 5993) or KEGELES-type ~ was used. The ultracentrifuge measurements were made at room temperature (about 25°). The protein solutions were prepared in o.I M KNO 3 solution, to keep the composition identical with that used in H + equilibria studies 1. The pH of the solution was adjusted by the, addition of HNO 3 or KOH. The pH measurements were made with a Metrohm p H meter Model E396. For a false bottom, CC14 was used. Protein concentration was determined bv- measuring the absorption at 276 m/~, using a value of E 276 ~%m,a -11.5 -at pH 5.9 (see ref. I).
Partial specific volume For the calculation of the molecular weight of fibroin, a value of 0.75 has been :assumed for the partial specific volume. RESULTS
AND DISCUSSION
Molecular weight data The molecular weight of fibroin was determined by the Archibald method, both, as a function of concentration and of pH. At the isoionic pH (4.2o), measurements
Biochim. Biophys. Acta, 94 (1965) 238 247"
240
•.
S. NARASINGA RAO, M. W. PANDIT
were made with 0.5, ~, ~ and 4 % solutions and at pH values 3.o, 7.0 and 9.0 with a 1% solution. Normally, in each experiment three pictures were taken at 15 min, 30 min, and 45 min, respectively, after the attainment of the operating speed and the molecular weight calculated from these. It was observed (Fig. I) that the molecular weights calculated at various periods of centrifugation differed significantly. The values at t h e top meniscus decreased with time and those at the bottom meniscus increased. T h e gradients at the bottom meniscus were invariably very steep and extrapolation t o obtain the dc/dx value at the meniscus, needed for molecular weight calculations, was uncertain. In Fig. I, therefore, only molecular weight data at the top meniscus are given.
5.0
®
o
I~: 0
I
20
I
40
I
60
I
~
I
~D 0 120 Time(rnin)
I
140
I
160
'1 180
Fig. i. The molecular weight of fibroin at various concentrations and pFi ~ as a function of time. Q - - Q , pH 4.2, 1% solution (8766 rev./min) ; ®--®, pH 4.2, 2 % solx~Cdo~(7447 rev./min) ; Q - - I , pH 4.2, 4 ~o solution (3397 rev./min); @--@, pH 3.0, 1% soh~tiot~ t"7447 rev./min); ~, pH 9.0, 1% solution (9341 rev./min). -
-
The decrease in molecular weight with time was observed at a~ the concentrations and pH values used. While there is considerable scatter in the i5-min values obtained at different concentrations and pH, the agreement among the values improves with the period of centrifugation and these tend towards a limiting value. In one experiment where the solution was centrifuged for 18o min, the values obtained at 9° min and 18o min were the same within experimental error. The molecular weight value at 15 min is about 5oooo and the limiting value is 16oo0 4- 2ooo. It was also observed that the centrifugal field has an effect on the measured molecular weight. A majority of the experiments were made at speeds ranging from 7000 to 9000 rev./min when the molecular weight was observed to decrease with time. However, when solutions of the same concentration were centrifuged at higher speeds (12 59 ° or 15 220 rev./min), the molecular weight was found to be independent of time and corresponded to 16000, the limiting value obtained in low-speed experiments (Fig. 2). These results can be interpreted in two ways: (i) The fibroin contains a highmolecular-weight impurity which gets depleted at the top meniscus rapidly and t h u s causes the weight-average molecular weight to decrease with time. The effect of higher centrifugal field will be to enhance the rate of depletion. (2) The fibroin undergoes a concentration-dependent aggregation reaction. As the concentration at the t o p
Biochim. Biophys. Acta, 94 (I965) 238-247
AN ULTRACENTRIFUGE STUDY OF SILK FIBROIN
241
meniscus decreases with time, the molecular weight may be :expected to decrease if the rate of re-equilibration is slow compared to the rate of sedimentation. \ \\
4.0
\
?
\ \\
2.0
I
20
I
40 Time
I
60 (rain)
I
80
Fig. 2. The molecular Weight of fibroin d e t e r m i n e d a t higher speeds, as a function of time. • - - O , p H 4.2, o . 5 ~ o solution (12590 rev./min); O - - O , p H 4.2, I.O% solution (12590 rev./min); ® - - ® , p H 4.2, 2.O~o solution (15220 rev./min). The d a s h e d curve represents the d a t a at low speed.
Sedimentation-velocity
data
To decide between the two alternative explanations, sedimentation-velocity experiments were made. If the protein contains a heavier component in an appreciable proportion, the velocity pattern would reveal it as a separate peak. On the other hand, if there is an aggregation reaction, the values of s20, w would be expected to increase with concentration 7. Further, depending upon the degree of aggregation, the velocity pattern m a y consist of two peaks; the ratio of the areas under the two peaks will v a r y with protein concentration unlike in the case of heterogeneity s. At the is•ionic pH, sedimentation-velocity measurements were made with o.5, I, 2 and 4 % fibroin solutions. At p H 3.0 and 7.4 experiments were conducted with a I O//osolution. In all the cases a single svmmetrical~ peak was obtained (Fig. 3a, b). Further the s20,w values showed the normal concentration dependence of a nonaggregating protein system (Fig. 4). The data at various concentrations could be approximately fitted into the equation: 0
s20, w = s2o, w(I - - k C )
Fig. 3. The s e d i m e n t a t i o n - v e l o c i t y p a t t e r n of fibroin. (a) i ~o solution, p H 4.2 ; centrifugation at 59 780 rev./min for i 15 rain. (b) I ~o solution, p H 7.o ; centrifugation at 59 780 rev.]min for i i o rain. S e d i m e n t a t i o n proceeds from left to right.
Biochim. Biophys. Acta, 94 (1965) 238-247
242
M . S . NARASINGA RAO, M. W. PANDIT
The s~0,w value at infinite dilution, S°~o, w, was found to be 1.36 S and k, o.II (g/Ioo ml) -1. The values at different pH were also found to be the same within experimental error (Fig. 5). 1.4
1.2 1.4]
(~)
m
1.2
O.E
I 1.0
I 2,0 Concentration
I t 3.0 4.0 ( g/lOOml ) /
1.0
~
Q
I
I
4.0
6.0
pH
I 8.0
Fig. 4- The s e d i m e n t a t i o n coefficient of fibroin at various concentrations. Fig. 5. The s e d i m e n t a t i o n coefficient of i ~o fibroin solutions at various p H values.
An estimate of the molecular weight of the protein corresponding to 1.36 S can be made using the equation 9, M=
469 ° (s2o, w)a/2 [,/jl/2 (i -- ~p)m
where M is molecular weight, s20, w, sedimentation coefficient expressed in Svedberg units; [~], the intrinsic viscosity expressed in (g/Ioo ml) -z, ~, partial specific volume and p, density of solution. The intrinsic viscosity of fibroin at pH 4.2 has been measured and is o.127 (see ref. I). The molecular weight calculated from the above equation is 21000. This value approximates to the limiting value obtained by the Archibald method, namely, 16000. The sedimentation-velocity experiments clearly indicate that fibroin did not exhibit either a concentration-dependent or a pH-dependent aggregation reaction. The observed variations in the Archibald molecular weight data are therefore likely to have been due to a component which is heavier than 16000. Since this component had, as adjudged by Archibald experiments, considerable velocity even at low speeds, its molecular weight should be fairly high. To get direct experimental evidence for the existence of this heavier component and to determine its sedimentation behaviour, an attempt was made to concentrate this component from the mixture. Fibroin solution (13 ml of 5 % solution) was centrifuged at 35o00 rev./min for 3 h in a Type-4o Rotor in a Spinco Model-L centrifuge. At the end of the run the top 4 ml and the bottom 2 ml of the solution were collected separately. The bottom fraction was diluted with an equal volume of 0.2 M KNO 8 solution so that the protein concentration was approx. 1%, and a sedimentationvelocity experiment was made by centrifuging it at 25980 rev./min. The velocity pattern is given in Fig. 6. The sedimentation coefficient of this peak was found to be 22.3 S. On the assumption that molecular weight is proportional to (s) 3/~, an estimate of the molecular weight of the component can be made from the sedimentation coefficient. Since the 16000 mol. wt. component has a s~o,w value of 1.25 S at 1 % protein concentration, it follows that M
16000
or M
2o6 00o
LI.25J
Biochim. Biophys. Acta, 94 (1965) 238-247
AN ULTRACENTRIFUGE STUDY OF SILK FIBROIN
243
Thus the high-molecular-weight impurity in the fibroin preparation was nearly 80 times heavier than the major component. Since the molecular weights of both the fractions are known, their proportion in the mixture can be estimated if the true weight-average molecular weight is known. The data of Fig. I when extrapolated to zero time should yield this value. I t is seen that such an extrapolation is impossible with the data on 1 % and 2 % solutions. However, the values obtained with a 4 % solution, where the experiment had been made at a low speed, can be reasonably extrapolated to a value of IOOOOO.From this the proportion of the heavier component can be calculated to have been about 7 %.
Fig. 6. The sedimentation-velocity pattern of the heavier component. Protein concentration, approx. 1% ; centrifugation at 25 980 rev./min for 3o min. Sedimentation proceeds from left to right.
In sedimentation-velocity experiments with the fibroin preparation, it was impossible to detect this component. W~ did not notice any peak formation even with a 5 To solution when it was centrifuged for I h at 25 980 rev./min. Therefore the proportion of the heavier component cannot have been more than 2 or 3 %. The estimated value of 7 % can obviously be in gross error as it is based on approximate values for the true weight-average value and the molecular weight of the heavier component.
Field-relaxation experiments Recently, KEGELES AND SIA1° have shown that ultracentrifugal field-relaxation experiments provide a sensitive technique to detect high-molecular-weight impurities in a protein preparation and to distinguish heterogeneity from concentrationdependent, reversible, aggregation reactions. The experimental procedure consists in layering the solvent on the solution using a synthetic-boundary cell, and accelerating to a speed sufficient to cause sedimentation. This is followed by careful deceleration to a sufficiently low speed to permit horizontal extrapolation of the schlieren pattern to the image of the bottom of the fluid column. From an enlarged tracing of the pattern the weight-average molecular weight at the cell bottom is calculated with the equation : EFR TZb Mw ( I - - 7~p)(O 2 Xb(A1 +/t2) where E is the enlarger magnification factor; F, the magnification factor of the camera lens in the ultracentrifuge; Zb, the extrapolated deflection above the estimated base Biochim. Biophys. Acta, 94 (1965) 238-247
244
M.S. NARASINGA RAO, M. W. PANDIT
i
Fig. 7. Schlieren p a t t e r n s in ultracentrifugal field-relaxation experiments. (a) i % fibroin solution, p H 4.2. Centrifugation for 5 rain at 25980 rev./min, decelerated to 4267 rev./min; picture taken after I6 min at 4267 rev./min; b a r angle 80 °. (b) About i % solution of Fraction II. Centrifugation for 5 min at 25980 rev./min, decelerated to 3954 rev./min; picture taken after 16 min at 3964 rev./min; bar angle 80 °. (c) About i % solution of Fraction III. Centrifugation for 5 min at 25980 rev./min, decelerated to 9600 rev./min; picture taken after i i min at 9600 rev./min; bar angle 7 o°. Sedimentation proceeds from left to right.
line of the schlieren diagram at the bottom meniscus, as obtained from the enlarger projection; A 1 and A 2 are the areas under the free peak and near the cell bottom, as measured from the enlarger projection. The other terms in the equation have the usual meaning. The areas are measured with a planimeter. For a reversible aggregation reaction, the relaxation of the concentration build up at the cell bottom affects the molecular weight in a complex way. It is observed that in such a case the period of centrifugation at a higher speed has an effect on the molecular weight calculated. On the other hand, in the case of heterogeneity which does not involve gross impurity, the measured molecular weight is independent of the period of centrifugation at the higher speed. A few ultracentrifuge field-relaxation experiments were made with isoionic fibroin solutions with two objectives, v i z . , (I) to confirm the heterogeneity indicated by the Archibald experiments, (2) to determine if this heterogeneity is due to only one heavy impurity or a number of them. To test the reliability of the method and to arrive at satisfactory experimental conditions for fibroin solutions, an experiment was also made with crystalline lysozyme. The experimental details and the molecular weights obtained are given in Table I. A t(EGELES-type synthetic-boundary cell and CC14 for false bottom were used. The lysozyme was a Sigma product, lot No. L42B-247. In a sedimentationvelocity experiment, a 1% solution (o.I M phosphate buffer of pH 6.9) gave a single symmetrical peak with sso, w of 2.08 S, in satisfactory agreement with the literature v a l u e 11.
The molecular weight of lysozyme obtained by the field-relaxation method is 2019o compared to I5 585 from the Archibald method. This could be due to a heavy impurity in the lysozyme sample or to the limitations in our hands of the fieldrelaxation method for absolute molecular weight determinations. The fibroin preparation gives a molecular weight of 305000. This value is weighted more towards the high-molecular-weight component, which is in agreement with the observation of KEGELES AND SIA1°. Further, the period of centrigugation, 2 min or 5 min, at the higher speed has no effect on the measured molecular weight. This result suggests that the protein sample does not contain a number of heavy impurities. The sample (Fraction II) which was obtained as top fraction by centrifugation of the fibroin preparation for 3 h at 35000 rev./min in a preparative centrifuge still Biochim. Biophys. Acta, 94 (1965) 238-247
AN ULTRACENTRIFUGE STUDY OF SILK FIBROIN
245
TABLE I MOLECULAR W E I G H T
DATA FROM ULTRACENTRIFUGE
FIELD-RELAXATION EXPERIMENTS
T h e r a t e of deceleration in t h e e x p e r i m e n t s w a s a p p r o x . IOOO r e v . / m i n .
Sample
Period of centri- Lower speed and period fugation at of centrifugation 2598o rev./min (min)
Lysozyme, I ~/o s o l u t i o n i n o.I M p h o s p h a t e buffer (pH 6.9)
5
Fibroin, 1 % solution in o. i M KNO3
2 5
10560 r e v . / m i n ; 8 m i n io56o rev./min; ii min
4267 4267 3 555 3555
rev./min; rev./min; rev./min; rev./min;
Mol. wt.
2o52o 1986o
I6min 24 m i n I6min 17 m i n
322ooo 3o 3 ooo 294o00 3020o0
F r a c t i o n II, a b o u t 1 % s o l u t i o n in o.I M K N O 3
5
3954 r e v . / m i n ; I o m i n 3 954 r e v . / m i n ; 16 m i n
1082oo 99200
Fraction III, a b o u t 1 % s o l u t i o n in o.I M K N O 3
5
9600 r e v . / m i n ; 8 m i n 96oo r e v . / m i n ; 11 rain
23o6o 2148o
Mol. wt. at the bottom by the Archibald Method
15530 15564
13700 13980
exhibited heterogeneity although the molecular weight (lO3 7oo) is less than that obtained with the "unpurified" sample. This was also revealed in Archibald experiments. The molecular weights at the top meniscus, determined at low speeds, decreased with time; the rate of decrease was less than that observed with the "unpurified" sample. Another sample (Fraction III) obtained as the top fraction from centrifugation for 7 h at 35000 rev./min gave a molecular weight of 22270. This value m a y be considered to correspond to 16 ooo, the molecular weight of the light component obtained by the Archibald method. Further, this sample, in Archibald experiments at low speeds, did not show any time dependence of molecular weight. Thus, this fraction appeared to be completely free from the heavy component of molecular weight 1206 ooo. In the relaxation experiments a doubt has often been expressed that when the solution is centrifuged at the higher speed a gel or precipitate m a y be formed at the solution-immiscible liquid interface and that this m a y redissolve on deceleration. KEGELES AND SIA1° have pointed out that in the case of gel formation it will be impossible to make the gradient at the interface bend over even when decelerated to any practically attainable low speed. On the other hand such bending over is possible if there is no gel formation. I t is seen from Fig. 8 that the Fraction I I I , when decelerated to 4000 rev./min and held at that speed for IO min after centrifugation for 5 min at 25 980 rev./min, gives a schlieren pattern where the gradient at the interface is bent over. This experiment shows that there is no gel formation and the estimated molecular weight represents the weight-average value of the mixture present at the cell bottom at that moment. Biochim. Biophys. Acta, 94 (1965) 238-247
246
M.S. NARASINGA RAO, M. W. PANDIT
Both the Archibald and field-relaxation experiments suggest that the fibroin preparation consists essentially of only two components of molecular weight 16000 and approx. 1.2.io e, respectively, and that there are no intermediates present. If there were a number of heavy impurities, it is difficult to visualize that in the Archibald experiments a limiting value would be obtained and this would be identical with the value obtained in experiments involving the use of higher speeds and shorter periods of centrifugation. In the experiment using the field-relaxation technique, which appears to be a powerful tool for detecting heterogeneity, the sample which had been subjected to preparative centrifugation for 7 h gave a value close to 16000. All these results are compatible with the conclusion that the fibroin preparation consists of two components only.
Fig. 8. Schlieren p a t t e r n in ultracentrifugal field-relaxation experiment. About 1% solution of Fraction III. Centrifugation for 5 min a t 25980 rev./min decelerated to 4072 rev./min; picture t a k e n after IO min a t 4o72 rev./min; bar angle 7o°. Sedimentation proceeds from left to right,
Origin of the heterogeneity Fibroin as found in the silk fibre is insoluble in water and common inert solvents. The use of drastic methods to remove sericin from fibroin and of concentrated aqueous electrolyte solutions to dissolve the fibroin may cause degradation of the protein. The procedure we have employed to obtain aqueous fibroin solution is extraction of the silk fibres six times with boiling, 2 % Na2CO 8 solution, and dissolution of the fibroin in saturated LiCNS solution followed by removal of LiCNS by exhaustive dialysis and passage over ion-exchange resins. Two samples prepared at different times gave identical results. If these results can be considered representative, it may mean that fibroin consists of two fractions only. The higher-molecular-weight component may be an aggregate of the 16000 unit (or a lower unit), these units being held together by sericin. As the sericin is removed it may degrade to the 16000 unit by an "all or none" mechanism. The proportion of the heavier component in any preparation will then depend on the extent to which sericin has been removed. It is known that the last traces of sericin are intimately combined with the fibroin filament x2. Unless the sericin is completely removed, the fibroin preparation is likely to contain traces of the heavier impurity. The molecular weight of such a preparation determined by dynamic methods, such as velocity ultracentrifugation may not be sensitive to the presence of this impurity. On the other hand values determined by static methods such as osmotic pressure, light scattering and diffusion will be greatly influenced by the heavier component. Biochim. Biophys. Acta, 94 (1965) 238-247
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ACKNOWLEDGEMENTS
The authors are grateful to Dr. G. S. SIDHU for his encouragement. The Spinco Model-E ultracentrifuge used in this study was a gift from the Wellcome Trust London, to Dr. P. M. BHARGAVA. REFERENCES i 2 3 4 5 6 7 8 9
M. S. NARASINGA RAO, S. I4,. BATNI, D. G. TAKTE AND A. B. BISWAS, u n p u b l i s h e d results. C. TANFORD,Advan. Protein Chem., 17 (1962) 69. C. TANFORD AND J. EPSTEIN, J. Am. Chem. Soc., 76 (1954) 2163. F. LUCAS, J. T. B. SHAW AND S. G. SMITH, Advan. Protein Chem., 13 (1958) lO7. S. M. KLAINER AND G. KEGELES, J. Phys. Chem., 59 (1956) 952. G. KEGELES, J. Am. Chem. Soc., 74 (1952) 5532. G. W. SCHWERT, J. Biol. Chem., 179 (1949) 655. G. A. GILBERT, Discussions Faraday Soc., 20 (1955) 68. H. i{. SCHACHMAH, Ultracentrifugation in Biochemistry, Academic Press, New York, 1959, p. 242. Io G. KEGELES AND C. L. SIA, Biochemistry, 2 (1963) 19o6. i i L. R. WETTER AND H. F. DEUTSCH, J. Biol. Chem., 192 (1951) 237. i2 J. T. B. SHAW AND S. G. SMITH, Nature, 168 (1951) 745.
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