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Optical rotation and viscosity of native and denatured proteins. XI. Relationships between rotatory dispersion, ionization and configuration
ARCHIVES
OF
BIOCHEMISTRY
AND
BIOPHYSICS
74, 70-83 (1958)
Optical Rotation and Viscosity of Native and Denatured Proteins. XI. Relationships between Rotatory Dispersion, Ionization and Configuration1 B. Jirgensons From the University of I’exas M.D. Anderson Hospital Department of Biochemistry, Houston, Received
July
and Tumor Institute, Texas
29, 1957
It is the purpose of this publication to extend the previous work of this series (1) to include several more proteins, principally a group of wellcharacterized crystalline enzyme preparations. It is hoped that we may, by such extension, obtain a broad experimental basis for further studies on protein characterization, as well as the correlation of rotatory dispersion with configuration. The optical rotation of the proteins was studied as a function of incident wavelength, degree of ionization, and concentration by the technique of spectropolarimetry. The use in this study of shorter wavelength than can be obtained using the customary sodium lamp enabled us to reach a higher degree of precision than in earlier work (2). Those proteins showing anomalous rotatory behavior were also studied by viscometry to assess the magnitude of any possible changes in gross configuration. EXPERIMENTAL Matetials and Methods The following proteins were used in this study: 1. Pepsin. A three-times recrystallized pepsin preparation was obtained from Pentex Inc. (Kankakee, Illinois). The enzymic activity of the protein (lot 3709) was tested by the Anson hemoglobin digestion method (3,4), and it was found to be 0.29-0.32 unit/mg. pepsin nitrogen. This is about the same value as found by 1 Supported in part by a grant Institutes of Health, U. S. Public
from the National Cancer Institute, National Health Service, grant NCI-1785 (C4).
70
NATIVE
AND
DENATURED
PROTEINS.
XI
71
Northrop, Ku&z, and Herriott (4) in their best preparations of crystallized pepsin. 2. Trypsin. A crystallized trypsin preparation from Pentex Inc. (lot 3406) was used. Its enzymic activity was tested by the hemoglobin digestion method (3, 4) and found to be slightly lower than of the most active crystallized preparations described elsewhere (4). 3. The a-, &, and y-Chymotrypsins. Twice-recrystallized cu-chymotrypsin from Pentex Inc. (lot 2708), and similarly recrystallized B-chymotrypsin (lot 4401), and r-chymotrypsin (lot 5202) from the same source were employed. The enzymic activity of these proteins was not determined. 4. Trypsin Inhibitor. Two preparations of this protein were used. One, a twicerecrystallized trypsin inhibitor from soybeans was purchased from Pentex (lot A51012); the other, a five-times crystallized soybean inhibitor originated from Nutritional Biochemicals Corp. (lot 1461). The inhibitory activity of each was tested by the Anson method (3), and the activity was found satisfactory. The optical rotation of these preparations was found to be somewhat lower than that reported by Northrop and colleagues (4). The first preparation was found to be essentially homogeneous to electrophoresis and sedimentation. 5. Amylase and Taka-amylase. A crystallized preparation of pancreatic amylase was obtained from Pentex Inc. (lot 4401). Its enzymic activity was tested by the starch digestion-iodine method (5) and found satisfactory. The taka-amylase A was a three-times recrystallized preparation kindly presented by Professor Shiro Akabori of Osaka University. Both enzymes appeared essentially homogeneous on paper electrophoresis. The taka-amylase contained a small amount of a fast moving component in sedimentation studies. 6. Ribonuclease. A five-times recrystallized ribonuclease was obtained from Pentex Inc. (lot 3303). Since the specific rotation of this enzyme was the same as reported in the literature (6), no further descriptive studies were performed. 7. Phosvitin. This egg-yolk protein was obtained from Nutritional Biochemicals Corp. (lot 9423). It was found that it contains 9.4yo phosphorus. Sedimentation revealed one major component and a small amount of a slow-moving component. The specific rotation of this protein was found the same as reported by Mecham and Olcott (7). Several series of experiments were also done with papain (a crystallized preparation from Nutritional Biochemicals Corp.), Zysozyme (a three-times recrystallized enzyme from Pentex Inc.), chymotrypsinogen [see (l)], and bovine serum mercaplalbumin. The latter was used in the form of its mercury dimer, (Alb.S-) 2Hg; this was a ten-times recrystallized preparation from Pentex Inc., lot D8501. The optical rotation was measured by means of the Rudolph photoelectric ultraviolet-range spectropolarimeter. A Hanovia mercury lamp (type SH) was used as the light source, and the Hg spectral lines of 578, 546.1, 435.8,404.7, 366.3, 365.0, and 334.1 rnp were those mainly used for the rotation measurements. The rotation was determined by the method of symmetrical angles (8). All measurements were made in an air-conditioned room at 25°C. (11’). Semimicro tubes with glass windows were used, and the zero point was checked each day. The rotatory dispersion data were treated according to the method of Yang and Doty (6). The from the slope of the straight line, exdispersion constant, XO , was evaluated
72
JIRGENSONS
pressed by the modified
Drude
equation:
X2X [al = Xo2X [a] + K, where A is the wavelength, [a] the specific rotation, and K the intercept on the ordinate. The viscosity was measured by means of the Cannon-Ubbelohde semimicro dilution viscometer (9) at 28.6 f 0.03”C. The flow time for water at this temperature was 930.0 sec. The solutions were carefully freed of any large particles, and the viscometer was cleaned with sulfuric acid-bichromate mixture after each series of measurements. The regression lines for the dependence of the reduced specific viscosity, &c, on concentration, c, were calculated by the method of least squares. The ratio of the flow times was not multiplied by the density ratio, hence the viscosities reported in this paper are kinematic viscosities. The concentration of the protein solutions was determined by drying aliquots to constant weight at 108-llO”C., as described previously (1).
RESULTS
1. The Dependence of Specific Rotation on Concentration It is well known that the specific rotation is practically independent of protein concentration in relatively concentrated solutions of 2-8 % (2). At lower protein concentrations the readings are so small that the experimental scatter of observations makes it difficult to evaluate the function (2). Yang and Foster found (10) that the specific rotation of several proteins was either independent of concentration, or increased slightly with decreasing concentration. Their measurements were made with a sodium lamp, and the scatter of the points was considerable. Since the instrument we now have permits the use of short-wave light, it seemed desirable to recheck the concentration dependence in the low concentration range of 0.2-2 %. The results for three proteins are presented in Fig. 1. It is obvious that the specific rotation of lysozyme and pepsin is independent of protein concentration, while the specific rotation of chymotrypsinogen decreases slightly with increasing protein concentration. This is in agreement with the results of Yang and Foster (10). 2. Rotatory Dispersion and Viscosity of Pepsin at Various pH’s The results are presented in Table I and Fig. 2. The major part of the measurements was made with solutions with pH 4.8-9.0. The work with more acid solutions was avoided because of self-digestion of pepsin. The levorotation of pepsin at pH 6-9 is higher than that observed at pH 4.8-5.5 because of denaturation (11). At pH 4.8-5.5, the optical rotation results were quite reproducible, and the rotation did not change
NATIVE
AND
DENATURED
PROTEINS.
73
XI
with time. At pH > 8, however, the levorotation values did change somewhat in time. At pH values higher than 9.0, the observation was possible only in the beginning when very high rotation angles were measured; after 15-20 min., however, the rotation begins to decrease, and the observation is hampered by microgel formation. The rotation and dispersion data presented in Table I and Fig. 2 are of observations made 2 hr. after preparing the solutions. The fact is noteworthy that the dispersion constant, X0, of native pepsin is unusually low, and that the constant of the denatured pepsin is higher than that of the native pepsin. When the observations on the
300-0-0-0 -0
o- 3
250 1
200 1 -0-o-o
O- I
[a]:; C%
I 0.5
1 1.0
I 1.5
I 2.0
FIG. 1. The dependence of specific levorotation tration. Line 1, lysozyme in aqueous solutions; chymotrypsinogen in water.
TABLE Pep&n: PH
4.82 5.24 8.12 9.05 10.9
The Speci$c Rotation -r&s k. 226.9 231.0 286.1 301.4 314.0
of proteins on protein concenline 2, pepsin in water; line 3,
I
Values and Dispersion -bllp.7 aeg. 168.6 169.8 210.7 221.9 230.0
I 2.5
x0 ?I@ 212.8 216.3 221.0 223.4 222.0
(zkl) (f2) (fl) (f2) (f3)
Constants
at Various K 19.8 19.9 24.1 25.1 26.3
(10.05) (f0.07) (f0.05) (f0.10) (f0.15)
pH
74
JIRGENSONS
pepsin solutions of pH 8.1 were made 4 hr. after mixing, even a larger constant of 228 was found. It was also established that the pepsin which was kept in solutions having the pH values of 8.1 or 9.0 had no peptic activity. The changes in gross configuration of the macromolecules of the enzyme were studied by viscosity measurements. The results are presented in Fig. 3, and they are in complete accord with everything known about viscosity in relation to ionization and configuration. In aqueous medium (line 1) the viscosity is higher than in 0.125 sodium bromide solution (line 2). The intrinsic viscosity in both instances is low. At pH 9.0 (line 3), when pepsin is denatured, the viscosity is strongly enhanced, and the intrinsic viscosity of 0.098 (f0.003) was calculated. This indicates that the macromolecules are strongly expanded or uncoiled upon denaturation in such weakly alkaline solutions. 3. The Optical Rotatory Dispersion of Several Other Proteolytic Enzymes The rotatory dispersion data of trypsin, cy-, /?-, and y-chymotrypsins, and papain are presented in Table II. Some of the dispersion data are also
FIQ. 2. The rotatory dispersion of pepsin at four pH values. Line 1, 2.20y0 pepsin at pH 4.8; 2, 1.057c pepsin at pH 5.4; 3, 1.05% pepsin at pH 8.1; 4, 2.200/c pepsin at pH 9.0. The ionic strength in all instances was 0.05.
0.20
i 0.15o/‘o3
o ,o-/o’o’
oo5-~o.,-o
-~-o-o-o, o-o-~-o-o-~-
9 SP/c
C%
I I.0
2
I 2.0
1 3.0
a 40
I 5.0
Fro. 3. The dependence of the reduced specific viscosity $Jc of pepsin on its concentration. Line 1, pepsin in water, pH 5.10%; line 2, pepsin in 0.125 M sodium bromide, pH 5.24; line 3, pepsin in glycine buffer at pH 9.0, and ionic strength 0.05.
Dispersion Protein
T rypsin Trypsin a-Chymotrypsin a-Chymotrypsin @-Chymotrypsin r-Chymotrypsin 7.Chymotrypsin Papain Trypsin inhib. 2X tryst. Trypsin inhib. 5X tryst Amylase, pancr. Amylase, pancr. Taka-amylase
Constants
TABLE II of Several Proteins,
Enzymes
Concentra-
Solvent
Water 0.05 M CaCl, Water Glyc. buffer r/2 0.05 Water Water Glyc. buffer r/2 0.05 Glyc. buffer r/2 0.10 Glyc. buffer r/2 0.10 Glyc. buffer r/2 0.10 0.25 M NaBr Glyc. buffer r/2 0.12 0.125 M NaBr
Especially
tion
of the
protein
-
x0
PH
K
% 4.06 2.03 3.14 0.785
3.0 3.18 2.92 8.60
226.8 226.5 235.1 232.0
(&3) (f2) (fl) (f2)
14.7 14.9 18.5 17.7
3.06 3.32 1.06
3.12 3.30 3.96
234.5 237.0 233.3
(f2) (f2) (&3)
18.4 (jzO.1) 18.3 (fO.l) 22.1 (f0.15)
0.785
8.95
224.7
(~2)
14.8 (f0.1)
2.52
7.85
215.8
(f2)
23.8
1.06
8.82
208.3
(&2)
24.7 (f0.1)
3.86 1.93
5.75 7.0
218.6 218.6
(f4) (&4)
8.2 (f0.2) 7.8 (f0.2)
1.48
6.84
261.3
(f3)
4.8
75
mlr
(hO.15) (hO.1) (f0.05) (jzO.1)
(fO.l)
(f0.15)
76
JIRGENSONS
shown in Fig. 4. The dispersion constants, X0 , of all of these enzymes are of the same order of magnitude, although the specific rotation (and also the constant K) of the chymotrypsins is higher than that of trypsin or papain.
4. The Rotatory Dispersion and Viscosity of Trypsin Inhibitor Soybean trypsin inhibitor is another protein which possesses an abnormally low dispersion constant (208-220). The results are presented in Table II and Fig. 4. Solutions of this protein at pH 7.8 exhibited a dispersion constant of 215.8, a low viscosity (Fig. 5)) and showed inhibition of tryptic activity. It is also interesting to note that the slope of the viscosity regression line (line 2, Fig. 5) is near zero, yielding an intrinsic viscosity of only 0.051 at 0.05 ionic strength.
FIG. 4. The optical rotatory dispersion of several proteins. Line 1, 2.03% trypsin in 0.05M calcium chloride solution, pH 3.18; line 2,1.12% or-chymotrypsin in glycine buffer, pH 3.88, and ionic strength 0.05; line 3,0.785oj,papain in glycine buffer, pH 8.95,1’/2 0.10; line 4, trypsininhibitor (2X crystallized, fromsoybeans), c 2.52%, pH 7.85, in glycine buffer of r/2 0.10; line 5, 1.93% pancreatic amylase, pH 7.4, r/2 0.12; line 6, 1.48y0taka-amylase, pH 6.85,I?/2 0.12.
NATIVE
AND
DENATURED
PROTEINS.
77
XI
5. Pancreatic Amylase, and Taka-amylase A The rotatory
dispersion
behavior
of these enzymes
is shown in Fig. 4
and Table II. It is noteworthy that, although both enzymes possess low levorotation values, their dispersion constants differ widely. The takaamylase showed a normal or slightly high dispersion constant, whereas the pancreatic amylase is one more example of an enzyme with low dispersion constant. There is no reason to believe that the pancreatic amylase was denatured, since it was enzymically active in starch hydrolysis and was quite soluble, and, moreover, the viscosity of its solutions was very low (Fig. 5). The intrinsic kinematic viscosity of the amylase solution in glycine buffer of ionic strength 0.05 was found to be below 0.03. 6. Lysozyme and Ribonuclease Lysozyme and ribonuclease are both basic proteins with their isoelectric points at pH between 8 and 11. Since their rotatory dispersion
t I
0.20
-3
,o’O
: 00’
ON
O/O
t
o,05.~o.o.o-o-o-o-oq WC
o-0.0 I
I C%
1.0
2 -0-o
- I
I
I
I
2.0
3.0
4.0
FIG. 5. The dependence of the reduced specific viscosity q&c on protein conLine 1, pancreatic amylase in glycine buffer of ionic strength 0.05 and pH 7.70; line 2, trypsin inhibitor (2X crystallized from soybeans) at pH 7.8 and r/2 0.05; line 3, phosvitin at pH 4.7 and 1’/2 0.10; line4, phosvitin at pH 9.95 and r/2 0.10.
centration.
78
JIRGENSONS 2p~3
4
5
6
7
8
9
IO
II
12
-r4~4,7
FIG. 6. The dependence of specific rotation of proteins on pH (studied with light of short wavelength). Line 1, 1.97% lysozyme, I’/2 0.05; line 2, 1.72% ribonuclease, r/2 0.27; line3,1.150/, phosvitin, I’/2 0.12; line4,1.05% pepsin, 1’/2 0.05; line 5, 1.700/, bovine mercaptalbumin (Hg dimer) r/2 0.05.
properties have been investigated by other authors (6)) the present study is concerned chiefly with the pH dependence of the rotation, in order to have comparable data for more proteins of various composition. The dependence of the specific rotation on pH is presented in Fig. 6. The study was made using the short wavelength of 404.7 rnp which gives large rotation angles. Some other proteins also are included in this illustration for purpose of comparison. Although the specific rotation values of lysozyme and ribonuclease are different, they are not affected by varying the pH over rather wide limits. [This result with lysozyme is different from earlier reported data which were obtained on a relatively impure preparation with inferior optical technique (12) .] Serum albumin, which has been thoroughly studied in this laboratory (13, 14), shows a slight decrease of levorotation when the pH is raised from 7 to 9, and a pronounced increase at pH higher than 10.5 and lower than 4. At pH 4-7 the rotation has shown considerable variations from specimen to specimen. The data presented for the mercaptalbumin dimer possess a definite discontinuity in the rotation curve at pH 34.5. This is difficult to
NATIVE
AND
DENATURED
TABLE Specific
Rotation
Values and Dispersion
4.36 6.15 7.70 8.35 9.02 9.61
TABLE
PH
3.25 4.70 6.10 6.85 7.30 8.33 10.75
of Ribonuclease
ho n?P(*a 232.4 231.9 231.7 231.8 233.3 234.5
191.1 190.8 190.9 190.8 188.0 186.0
Rotation
and Dispersion
79
XI
III Constants
-blZ.,
PH
Specific
PROTEINS.
at Various
pH
K (fO.l) 21.0 21.1 21.1 21.2 20.6 20.3
IV
Constants
of Phosoitin
at Various
pH
-rGu
ho w (=w
K (fO.l)
130.6 129.6 149.0 168.0 184.5 195.6 203.9
225.0 225.1 216.2 213.6 210.0 209.8 207.6
14.5 14.4 17.3 19.9 21.8 23.4 24.6
establish using the conventional sodium light, but is quite explicit when the short waves are used. Figure 6 also demonstrates that the pH dependence of rotation for phosvitin (line 3) and pepsin (line4) is similar and quite different from that for lysozyme and ribonuclease. The rotatory dispersion data for ribonuclease are compiled in Table III. 7. The Rotatory Dispersion, Ionization
and Viscosity of Phosvitin
The specific rotation of phosvitin changes greatly as a function of pH, especially near neutrality. This is illustrated by line 3 in Fig. 6, as well as by the data of Table IV. The dispersion constant, X0 , of phosvitin changes with pH (see Fig. 7) in approximately the same way as does the specific rotation. The viscosity of phosvitin was studied at pH’s 4.7 and 9.9 (Fig. 5), and it was found that both the slope of the regression line and intrinsic viscosity in alkaline solution are greater than in acid solution. DISCUSSION
Summarizing the observations on 25 proteins studied, by the most precise method of spectropolarimetry, in t.his and in other laboratories
80
JIRGENSONS
pH
I
I
2
3
I
4.5
I
I I
I
I
6
7
8
9.5
I
12
FIG. 7. The dependence of the dispersion constant X0 on pH. Line 1, lysozyme, c 1.97%, r/2 0.05; line 2, 1.05% pepsin, r/2 0.05; line 3, 1.15’% phosvitin, I’/2 0.12.
(1,6), it seems useful to classify the proteins, according to the magnitude of their dispersion constants, X0 , into three groups: (CL)proteins which possess low dispersion constants (in the range 200-220 mp), (5) proteins which have dispersion constants in the medium range of 220-240, and (c) those with high dispersion constants. (a) Low X0: pancreatic amylase (this paper), Bence-Jones protein (l), y-globulin (l), /3-casein (l), pepsin (this paper), and trypsin inhibitor (this paper). Moreover, low dispersion constants are characteristic of many denatured proteins (1, 6, 15). (b) Medium XO: arachin (I), a-casein (l), chymotrypsinogen (l), the (Y-, fi-, and y-chymotrypsins (this paper), /3-lactoglobulin (1)) papain (this paper), ribonuclease [(6), and this paper], trypsin (this paper), and trypsinogen (1). (c) High X0: cyl-glycoprotein (l), metal-binding @-globulin (1)) serum albumin (1, 6)) fibrinogen (6)) insulin [(6), and this paper], ovalbumin (6)) and taka-amylase (this paper). In addition, phosvitin, as indicated in sec. 7, may belong either to group (a) or (5) depending on the pH of the solution. Earlier classifica-
NATIVE
AND
DENATURED
PROTEINS.
XI
81
tion of Linderstrem-Lang and Schellman (16)) in which the proteins with dispersion constants below 240 were classified as denatured, appears inadequate. Five of the six proteins noted in group (a) are native globular proteins, are quite soluble, and possess low intrinsic viscosity values; moreover, both amylase and pepsin which belong to this group are enzymically active. Only the /3-casein of this group might be regarded as a denatured, or at least moderately asymmetrical, protein. Most of the proteins of group (b) also have relatively low dispersion constants of 220-230 rnp. The interpretation, as put forward by Doty and associates (6, 17), requires that lowering of the dispersion constant must be accompanied by lowering of the specific rotation (in the absolute sense), i.e. increase in levorotation. This conforms with the behavior of several denatured proteins (15)) with the seven examples presented by Yang and Doty (6), as well as with our observations on phosvitin, taka-amylase, trypsin, the caseins (1) , and several other cases. However, the rotatory dispersion data of the y-globulins (l), Bence-Jones protein (1)) pepsin, and pancreatic amylase do not fit into this scheme: The dispersion constants of these globular proteins are low, and their specific rotation values are relatively close to zero. This indicates, if considered from the point of view of Yang and Doty (6), that either helices of opposite screw sense than normal are present in pepsin (and the other similarly behaving proteins) or that relatively compact configurations other than helical exist in the solutions. Interesting is also the case of trypsin inhibitor, where the optical rotatory properties (both dispersion constant and specific rotation) indicate denaturation, but the protein actually was in a native state. Chymotrypsinogen represents a similar instance (1) of relatively low dispersion constant and high levorotation. These exceptions seem to point out a considerable variety of configurations of protein molecules. A much more definitive situation exists correlating optical rotation and configuration with the ionization of proteins. Comparison of Figs. 5, 6, and 7, and Tables I, III, and IV shows that there is a considerable difference between the highly charged pepsin and phosvitin and the other proteins studied. The levorotation of both pepsin and phosvitin increases strongly with increasing ionization, i.e. rising pH. The levorotation of the other proteins, on the contrary, changes very little in the pH limits 4-9. Inasmuch as the intrinsic viscosity of pepsin and phosvitin also increases with increasing ionization, the rotation changes most
82
JIRGENSONS
probably are due to molecular expansion and/or change in molecular asymmetry. High intrinsic viscosities for pepsin and phosvitin at pH 8-9 were observed also in solutions which had ionic strength of 0.10-0.12. These data would seem to indicate that gross configurational changes, rather than electroviscous effects, are responsible for the increased viscosity of these solutions. It is also obvious that the changes in levorotation of pepsin and phosvitin at pH 5-9 are due to changes in gross configuration, the latter being caused most probably by electrostatic repulsion of the macromolecular segments. The macromolecules of pepsin and phosvitin apparently expand very readily, this probably being due partly to the high content of carboxyl (in pepsin) and phosphate groups (in phosvitin) , and partly to structural features, namely, absence of interchain bonding. The viscosity of the other proteins, however, changes much less in these pH limits. For example, the viscosity of ribonuclease, according to Buzzell and Tanford (18), does not change greatly as a function of pH, and this was attributed to the rather rigid configuration and cross-linking of the peptide chains in the macromolecules of this protein (18). Both the levorotation and the dispersion constants of ribonuclease change very little when the pH (i.e., ionization) is changed (Table III). Serum albumin represents an intermediate case. The macromolecules of this protein seem to be more flexible and more affected by ionization than those of ribonuclease and lysozyme, but they change very little in the pH limits 4-9 (19). A considerable expansion of this protein is thought to occur at pH below 4 (20) and above 10.5. The interesting changes of levorotation of albumin around pH’s 3.5-5 and 7-9 depend on the specimen (13, 14), and indicate the possibility of detectable individual variations in the amount of subfractions and/or compositional and structural differences. ACKNOWLEDGMENTS The author is indebted to Professor Shiro Akabori of Osaka University for a preparation of recrystallized taka-amylase. Also acknowledged is the cooperation of Mrs. E. C. Adams and Mr. John A. Cooper. SUMMARY
The optical rotations of pepsin, trypsin, the LY-,p-, and y-chymotrypsins, trypsin inhibitor, papain, pancreatic amylase, taka-amylase, lysozyme, ribonuclease, phosvitin, and several other proteins were studied. The rotatory dispersion constants of these proteins were determined by means of spectropolarimetry. It was found that pepsin,
NATIVE AND DENATURED PROTEINS. XI
83
pancreatic amylase, and soybean trypsin inhibitor have low dispersion constants. The concentration dependence of rotation of several proteins was studied. The pH dependence of the optical rotation was studied using mercaptalbumin, lysozyme, pepsin, ribonuclease, and phosvitin. The rotation of pepsin and phosvitin changed strongly when the pH of the solutions was changed in the range 4-9; the rotation of the other proteins changed very little in the same pH range. Viscosity measurements indicated that the change in levorotation of pepsin and phosvitin in the above-mentioned pH limits is due to changes in molecular gross configuration. It was also demonstrated that the intrinsic viscosity of native pepsin, pancreatic amylase, and trypsin inhibitor solutions was low and therefore incompatible with the assumption that these proteins might be denatured. This finding was substantiated by testing the enzymic activity of these substances. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9.
10. 11. 12. 13. 14.
15. 16. 17. 18. 19. 20.
JIRGENSONS, B., Arch. Biochem. Biophys. ‘74, 57 (1958). JIRGENSONS, B., Arch. Biochem. Biophys. 41, 333 (1952). ANSON, M. L., J. Gen. Physiol. 22, 79 (1938). NORTHROP, J. H., KUNITZ, M., AND HERRIOTT, R. M., “Crystalline
Enzymes,” 2nd ed. Columbia University Press, New York, 1948. SMITH, B. M., AND ROE, J. H., J. Biol. Chem. 179, 53 (1949). YANG, J. T., AND DOTY, P., J. Am. Chem. Sot. 79, 761 (1957). MECHAM, D. K., AND OLCOTT, H. S., J. Am. Chem. Sot. 71, 3670 (1949). RUI)OLPH, H., J. Opt. Sot. Am. 46, 50 (1955). CANNON, M. R., Ind. Eng. Chem., Anal. Ed. 16, 708 (1944). YANG, J. T., AND FOSTER, J. F., J. Am. Chem. Sot. 77,2374 (1955). GREEN, N. M., AND NEURATH, H., in “The Proteins” (Neurath, H. and Bailey, K., eds.), Vol. IIB, p. 1086. Academic Press Inc., New York, 1954. JIRGENSONS, B., Arch. Biochem. Biophys. 39, 261 (1952). JIRGENSONS, B., Arch. Biochem. Biophys. 69, 420 (1955). JIRGENSONS, B., Makromol. Chem. 21, 179 (1956). COIIEN, C., AND SZENT-GY~RGYI, A. G., J. Am. Chem. Sot. 79, 248 (1957). LINDERSTR$M-LANG, K., AND SCHELLMAN, J. A., Biochim. et Biophys. Acta 16, 156 (1954). DOTY, P., AND LUNDBERG, R. D., Proc. Natl. Acad. Sci. U. S. 43, 213 (1957). BUZZELL, J. G., AND TANFORD, C., J. Phys. Chem. 60,1204 (1956). TANFORD, C., AND BUZZELL, J. G., J. Phys. Chem. 60, 225 (1956). YANG, J. T., AND FOSTER, J. F., J. Am. Chem. Sot. 76, 1588 (1954).
OF
BIOCHEMISTRY
AND
BIOPHYSICS
74, 70-83 (1958)
Optical Rotation and Viscosity of Native and Denatured Proteins. XI. Relationships between Rotatory Dispersion, Ionization and Configuration1 B. Jirgensons From the University of I’exas M.D. Anderson Hospital Department of Biochemistry, Houston, Received
July
and Tumor Institute, Texas
29, 1957
It is the purpose of this publication to extend the previous work of this series (1) to include several more proteins, principally a group of wellcharacterized crystalline enzyme preparations. It is hoped that we may, by such extension, obtain a broad experimental basis for further studies on protein characterization, as well as the correlation of rotatory dispersion with configuration. The optical rotation of the proteins was studied as a function of incident wavelength, degree of ionization, and concentration by the technique of spectropolarimetry. The use in this study of shorter wavelength than can be obtained using the customary sodium lamp enabled us to reach a higher degree of precision than in earlier work (2). Those proteins showing anomalous rotatory behavior were also studied by viscometry to assess the magnitude of any possible changes in gross configuration. EXPERIMENTAL Matetials and Methods The following proteins were used in this study: 1. Pepsin. A three-times recrystallized pepsin preparation was obtained from Pentex Inc. (Kankakee, Illinois). The enzymic activity of the protein (lot 3709) was tested by the Anson hemoglobin digestion method (3,4), and it was found to be 0.29-0.32 unit/mg. pepsin nitrogen. This is about the same value as found by 1 Supported in part by a grant Institutes of Health, U. S. Public
from the National Cancer Institute, National Health Service, grant NCI-1785 (C4).
70
NATIVE
AND
DENATURED
PROTEINS.
XI
71
Northrop, Ku&z, and Herriott (4) in their best preparations of crystallized pepsin. 2. Trypsin. A crystallized trypsin preparation from Pentex Inc. (lot 3406) was used. Its enzymic activity was tested by the hemoglobin digestion method (3, 4) and found to be slightly lower than of the most active crystallized preparations described elsewhere (4). 3. The a-, &, and y-Chymotrypsins. Twice-recrystallized cu-chymotrypsin from Pentex Inc. (lot 2708), and similarly recrystallized B-chymotrypsin (lot 4401), and r-chymotrypsin (lot 5202) from the same source were employed. The enzymic activity of these proteins was not determined. 4. Trypsin Inhibitor. Two preparations of this protein were used. One, a twicerecrystallized trypsin inhibitor from soybeans was purchased from Pentex (lot A51012); the other, a five-times crystallized soybean inhibitor originated from Nutritional Biochemicals Corp. (lot 1461). The inhibitory activity of each was tested by the Anson method (3), and the activity was found satisfactory. The optical rotation of these preparations was found to be somewhat lower than that reported by Northrop and colleagues (4). The first preparation was found to be essentially homogeneous to electrophoresis and sedimentation. 5. Amylase and Taka-amylase. A crystallized preparation of pancreatic amylase was obtained from Pentex Inc. (lot 4401). Its enzymic activity was tested by the starch digestion-iodine method (5) and found satisfactory. The taka-amylase A was a three-times recrystallized preparation kindly presented by Professor Shiro Akabori of Osaka University. Both enzymes appeared essentially homogeneous on paper electrophoresis. The taka-amylase contained a small amount of a fast moving component in sedimentation studies. 6. Ribonuclease. A five-times recrystallized ribonuclease was obtained from Pentex Inc. (lot 3303). Since the specific rotation of this enzyme was the same as reported in the literature (6), no further descriptive studies were performed. 7. Phosvitin. This egg-yolk protein was obtained from Nutritional Biochemicals Corp. (lot 9423). It was found that it contains 9.4yo phosphorus. Sedimentation revealed one major component and a small amount of a slow-moving component. The specific rotation of this protein was found the same as reported by Mecham and Olcott (7). Several series of experiments were also done with papain (a crystallized preparation from Nutritional Biochemicals Corp.), Zysozyme (a three-times recrystallized enzyme from Pentex Inc.), chymotrypsinogen [see (l)], and bovine serum mercaplalbumin. The latter was used in the form of its mercury dimer, (Alb.S-) 2Hg; this was a ten-times recrystallized preparation from Pentex Inc., lot D8501. The optical rotation was measured by means of the Rudolph photoelectric ultraviolet-range spectropolarimeter. A Hanovia mercury lamp (type SH) was used as the light source, and the Hg spectral lines of 578, 546.1, 435.8,404.7, 366.3, 365.0, and 334.1 rnp were those mainly used for the rotation measurements. The rotation was determined by the method of symmetrical angles (8). All measurements were made in an air-conditioned room at 25°C. (11’). Semimicro tubes with glass windows were used, and the zero point was checked each day. The rotatory dispersion data were treated according to the method of Yang and Doty (6). The from the slope of the straight line, exdispersion constant, XO , was evaluated
72
JIRGENSONS
pressed by the modified
Drude
equation:
X2X [al = Xo2X [a] + K, where A is the wavelength, [a] the specific rotation, and K the intercept on the ordinate. The viscosity was measured by means of the Cannon-Ubbelohde semimicro dilution viscometer (9) at 28.6 f 0.03”C. The flow time for water at this temperature was 930.0 sec. The solutions were carefully freed of any large particles, and the viscometer was cleaned with sulfuric acid-bichromate mixture after each series of measurements. The regression lines for the dependence of the reduced specific viscosity, &c, on concentration, c, were calculated by the method of least squares. The ratio of the flow times was not multiplied by the density ratio, hence the viscosities reported in this paper are kinematic viscosities. The concentration of the protein solutions was determined by drying aliquots to constant weight at 108-llO”C., as described previously (1).
RESULTS
1. The Dependence of Specific Rotation on Concentration It is well known that the specific rotation is practically independent of protein concentration in relatively concentrated solutions of 2-8 % (2). At lower protein concentrations the readings are so small that the experimental scatter of observations makes it difficult to evaluate the function (2). Yang and Foster found (10) that the specific rotation of several proteins was either independent of concentration, or increased slightly with decreasing concentration. Their measurements were made with a sodium lamp, and the scatter of the points was considerable. Since the instrument we now have permits the use of short-wave light, it seemed desirable to recheck the concentration dependence in the low concentration range of 0.2-2 %. The results for three proteins are presented in Fig. 1. It is obvious that the specific rotation of lysozyme and pepsin is independent of protein concentration, while the specific rotation of chymotrypsinogen decreases slightly with increasing protein concentration. This is in agreement with the results of Yang and Foster (10). 2. Rotatory Dispersion and Viscosity of Pepsin at Various pH’s The results are presented in Table I and Fig. 2. The major part of the measurements was made with solutions with pH 4.8-9.0. The work with more acid solutions was avoided because of self-digestion of pepsin. The levorotation of pepsin at pH 6-9 is higher than that observed at pH 4.8-5.5 because of denaturation (11). At pH 4.8-5.5, the optical rotation results were quite reproducible, and the rotation did not change
NATIVE
AND
DENATURED
PROTEINS.
73
XI
with time. At pH > 8, however, the levorotation values did change somewhat in time. At pH values higher than 9.0, the observation was possible only in the beginning when very high rotation angles were measured; after 15-20 min., however, the rotation begins to decrease, and the observation is hampered by microgel formation. The rotation and dispersion data presented in Table I and Fig. 2 are of observations made 2 hr. after preparing the solutions. The fact is noteworthy that the dispersion constant, X0, of native pepsin is unusually low, and that the constant of the denatured pepsin is higher than that of the native pepsin. When the observations on the
300-0-0-0 -0
o- 3
250 1
200 1 -0-o-o
O- I
[a]:; C%
I 0.5
1 1.0
I 1.5
I 2.0
FIG. 1. The dependence of specific levorotation tration. Line 1, lysozyme in aqueous solutions; chymotrypsinogen in water.
TABLE Pep&n: PH
4.82 5.24 8.12 9.05 10.9
The Speci$c Rotation -r&s k. 226.9 231.0 286.1 301.4 314.0
of proteins on protein concenline 2, pepsin in water; line 3,
I
Values and Dispersion -bllp.7 aeg. 168.6 169.8 210.7 221.9 230.0
I 2.5
x0 ?I@ 212.8 216.3 221.0 223.4 222.0
(zkl) (f2) (fl) (f2) (f3)
Constants
at Various K 19.8 19.9 24.1 25.1 26.3
(10.05) (f0.07) (f0.05) (f0.10) (f0.15)
pH
74
JIRGENSONS
pepsin solutions of pH 8.1 were made 4 hr. after mixing, even a larger constant of 228 was found. It was also established that the pepsin which was kept in solutions having the pH values of 8.1 or 9.0 had no peptic activity. The changes in gross configuration of the macromolecules of the enzyme were studied by viscosity measurements. The results are presented in Fig. 3, and they are in complete accord with everything known about viscosity in relation to ionization and configuration. In aqueous medium (line 1) the viscosity is higher than in 0.125 sodium bromide solution (line 2). The intrinsic viscosity in both instances is low. At pH 9.0 (line 3), when pepsin is denatured, the viscosity is strongly enhanced, and the intrinsic viscosity of 0.098 (f0.003) was calculated. This indicates that the macromolecules are strongly expanded or uncoiled upon denaturation in such weakly alkaline solutions. 3. The Optical Rotatory Dispersion of Several Other Proteolytic Enzymes The rotatory dispersion data of trypsin, cy-, /?-, and y-chymotrypsins, and papain are presented in Table II. Some of the dispersion data are also
FIQ. 2. The rotatory dispersion of pepsin at four pH values. Line 1, 2.20y0 pepsin at pH 4.8; 2, 1.057c pepsin at pH 5.4; 3, 1.05% pepsin at pH 8.1; 4, 2.200/c pepsin at pH 9.0. The ionic strength in all instances was 0.05.
0.20
i 0.15o/‘o3
o ,o-/o’o’
oo5-~o.,-o
-~-o-o-o, o-o-~-o-o-~-
9 SP/c
C%
I I.0
2
I 2.0
1 3.0
a 40
I 5.0
Fro. 3. The dependence of the reduced specific viscosity $Jc of pepsin on its concentration. Line 1, pepsin in water, pH 5.10%; line 2, pepsin in 0.125 M sodium bromide, pH 5.24; line 3, pepsin in glycine buffer at pH 9.0, and ionic strength 0.05.
Dispersion Protein
T rypsin Trypsin a-Chymotrypsin a-Chymotrypsin @-Chymotrypsin r-Chymotrypsin 7.Chymotrypsin Papain Trypsin inhib. 2X tryst. Trypsin inhib. 5X tryst Amylase, pancr. Amylase, pancr. Taka-amylase
Constants
TABLE II of Several Proteins,
Enzymes
Concentra-
Solvent
Water 0.05 M CaCl, Water Glyc. buffer r/2 0.05 Water Water Glyc. buffer r/2 0.05 Glyc. buffer r/2 0.10 Glyc. buffer r/2 0.10 Glyc. buffer r/2 0.10 0.25 M NaBr Glyc. buffer r/2 0.12 0.125 M NaBr
Especially
tion
of the
protein
-
x0
PH
K
% 4.06 2.03 3.14 0.785
3.0 3.18 2.92 8.60
226.8 226.5 235.1 232.0
(&3) (f2) (fl) (f2)
14.7 14.9 18.5 17.7
3.06 3.32 1.06
3.12 3.30 3.96
234.5 237.0 233.3
(f2) (f2) (&3)
18.4 (jzO.1) 18.3 (fO.l) 22.1 (f0.15)
0.785
8.95
224.7
(~2)
14.8 (f0.1)
2.52
7.85
215.8
(f2)
23.8
1.06
8.82
208.3
(&2)
24.7 (f0.1)
3.86 1.93
5.75 7.0
218.6 218.6
(f4) (&4)
8.2 (f0.2) 7.8 (f0.2)
1.48
6.84
261.3
(f3)
4.8
75
mlr
(hO.15) (hO.1) (f0.05) (jzO.1)
(fO.l)
(f0.15)
76
JIRGENSONS
shown in Fig. 4. The dispersion constants, X0 , of all of these enzymes are of the same order of magnitude, although the specific rotation (and also the constant K) of the chymotrypsins is higher than that of trypsin or papain.
4. The Rotatory Dispersion and Viscosity of Trypsin Inhibitor Soybean trypsin inhibitor is another protein which possesses an abnormally low dispersion constant (208-220). The results are presented in Table II and Fig. 4. Solutions of this protein at pH 7.8 exhibited a dispersion constant of 215.8, a low viscosity (Fig. 5)) and showed inhibition of tryptic activity. It is also interesting to note that the slope of the viscosity regression line (line 2, Fig. 5) is near zero, yielding an intrinsic viscosity of only 0.051 at 0.05 ionic strength.
FIG. 4. The optical rotatory dispersion of several proteins. Line 1, 2.03% trypsin in 0.05M calcium chloride solution, pH 3.18; line 2,1.12% or-chymotrypsin in glycine buffer, pH 3.88, and ionic strength 0.05; line 3,0.785oj,papain in glycine buffer, pH 8.95,1’/2 0.10; line 4, trypsininhibitor (2X crystallized, fromsoybeans), c 2.52%, pH 7.85, in glycine buffer of r/2 0.10; line 5, 1.93% pancreatic amylase, pH 7.4, r/2 0.12; line 6, 1.48y0taka-amylase, pH 6.85,I?/2 0.12.
NATIVE
AND
DENATURED
PROTEINS.
77
XI
5. Pancreatic Amylase, and Taka-amylase A The rotatory
dispersion
behavior
of these enzymes
is shown in Fig. 4
and Table II. It is noteworthy that, although both enzymes possess low levorotation values, their dispersion constants differ widely. The takaamylase showed a normal or slightly high dispersion constant, whereas the pancreatic amylase is one more example of an enzyme with low dispersion constant. There is no reason to believe that the pancreatic amylase was denatured, since it was enzymically active in starch hydrolysis and was quite soluble, and, moreover, the viscosity of its solutions was very low (Fig. 5). The intrinsic kinematic viscosity of the amylase solution in glycine buffer of ionic strength 0.05 was found to be below 0.03. 6. Lysozyme and Ribonuclease Lysozyme and ribonuclease are both basic proteins with their isoelectric points at pH between 8 and 11. Since their rotatory dispersion
t I
0.20
-3
,o’O
: 00’
ON
O/O
t
o,05.~o.o.o-o-o-o-oq WC
o-0.0 I
I C%
1.0
2 -0-o
- I
I
I
I
2.0
3.0
4.0
FIG. 5. The dependence of the reduced specific viscosity q&c on protein conLine 1, pancreatic amylase in glycine buffer of ionic strength 0.05 and pH 7.70; line 2, trypsin inhibitor (2X crystallized from soybeans) at pH 7.8 and r/2 0.05; line 3, phosvitin at pH 4.7 and 1’/2 0.10; line4, phosvitin at pH 9.95 and r/2 0.10.
centration.
78
JIRGENSONS 2p~3
4
5
6
7
8
9
IO
II
12
-r4~4,7
FIG. 6. The dependence of specific rotation of proteins on pH (studied with light of short wavelength). Line 1, 1.97% lysozyme, I’/2 0.05; line 2, 1.72% ribonuclease, r/2 0.27; line3,1.150/, phosvitin, I’/2 0.12; line4,1.05% pepsin, 1’/2 0.05; line 5, 1.700/, bovine mercaptalbumin (Hg dimer) r/2 0.05.
properties have been investigated by other authors (6)) the present study is concerned chiefly with the pH dependence of the rotation, in order to have comparable data for more proteins of various composition. The dependence of the specific rotation on pH is presented in Fig. 6. The study was made using the short wavelength of 404.7 rnp which gives large rotation angles. Some other proteins also are included in this illustration for purpose of comparison. Although the specific rotation values of lysozyme and ribonuclease are different, they are not affected by varying the pH over rather wide limits. [This result with lysozyme is different from earlier reported data which were obtained on a relatively impure preparation with inferior optical technique (12) .] Serum albumin, which has been thoroughly studied in this laboratory (13, 14), shows a slight decrease of levorotation when the pH is raised from 7 to 9, and a pronounced increase at pH higher than 10.5 and lower than 4. At pH 4-7 the rotation has shown considerable variations from specimen to specimen. The data presented for the mercaptalbumin dimer possess a definite discontinuity in the rotation curve at pH 34.5. This is difficult to
NATIVE
AND
DENATURED
TABLE Specific
Rotation
Values and Dispersion
4.36 6.15 7.70 8.35 9.02 9.61
TABLE
PH
3.25 4.70 6.10 6.85 7.30 8.33 10.75
of Ribonuclease
ho n?P(*a 232.4 231.9 231.7 231.8 233.3 234.5
191.1 190.8 190.9 190.8 188.0 186.0
Rotation
and Dispersion
79
XI
III Constants
-blZ.,
PH
Specific
PROTEINS.
at Various
pH
K (fO.l) 21.0 21.1 21.1 21.2 20.6 20.3
IV
Constants
of Phosoitin
at Various
pH
-rGu
ho w (=w
K (fO.l)
130.6 129.6 149.0 168.0 184.5 195.6 203.9
225.0 225.1 216.2 213.6 210.0 209.8 207.6
14.5 14.4 17.3 19.9 21.8 23.4 24.6
establish using the conventional sodium light, but is quite explicit when the short waves are used. Figure 6 also demonstrates that the pH dependence of rotation for phosvitin (line 3) and pepsin (line4) is similar and quite different from that for lysozyme and ribonuclease. The rotatory dispersion data for ribonuclease are compiled in Table III. 7. The Rotatory Dispersion, Ionization
and Viscosity of Phosvitin
The specific rotation of phosvitin changes greatly as a function of pH, especially near neutrality. This is illustrated by line 3 in Fig. 6, as well as by the data of Table IV. The dispersion constant, X0 , of phosvitin changes with pH (see Fig. 7) in approximately the same way as does the specific rotation. The viscosity of phosvitin was studied at pH’s 4.7 and 9.9 (Fig. 5), and it was found that both the slope of the regression line and intrinsic viscosity in alkaline solution are greater than in acid solution. DISCUSSION
Summarizing the observations on 25 proteins studied, by the most precise method of spectropolarimetry, in t.his and in other laboratories
80
JIRGENSONS
pH
I
I
2
3
I
4.5
I
I I
I
I
6
7
8
9.5
I
12
FIG. 7. The dependence of the dispersion constant X0 on pH. Line 1, lysozyme, c 1.97%, r/2 0.05; line 2, 1.05% pepsin, r/2 0.05; line 3, 1.15’% phosvitin, I’/2 0.12.
(1,6), it seems useful to classify the proteins, according to the magnitude of their dispersion constants, X0 , into three groups: (CL)proteins which possess low dispersion constants (in the range 200-220 mp), (5) proteins which have dispersion constants in the medium range of 220-240, and (c) those with high dispersion constants. (a) Low X0: pancreatic amylase (this paper), Bence-Jones protein (l), y-globulin (l), /3-casein (l), pepsin (this paper), and trypsin inhibitor (this paper). Moreover, low dispersion constants are characteristic of many denatured proteins (1, 6, 15). (b) Medium XO: arachin (I), a-casein (l), chymotrypsinogen (l), the (Y-, fi-, and y-chymotrypsins (this paper), /3-lactoglobulin (1)) papain (this paper), ribonuclease [(6), and this paper], trypsin (this paper), and trypsinogen (1). (c) High X0: cyl-glycoprotein (l), metal-binding @-globulin (1)) serum albumin (1, 6)) fibrinogen (6)) insulin [(6), and this paper], ovalbumin (6)) and taka-amylase (this paper). In addition, phosvitin, as indicated in sec. 7, may belong either to group (a) or (5) depending on the pH of the solution. Earlier classifica-
NATIVE
AND
DENATURED
PROTEINS.
XI
81
tion of Linderstrem-Lang and Schellman (16)) in which the proteins with dispersion constants below 240 were classified as denatured, appears inadequate. Five of the six proteins noted in group (a) are native globular proteins, are quite soluble, and possess low intrinsic viscosity values; moreover, both amylase and pepsin which belong to this group are enzymically active. Only the /3-casein of this group might be regarded as a denatured, or at least moderately asymmetrical, protein. Most of the proteins of group (b) also have relatively low dispersion constants of 220-230 rnp. The interpretation, as put forward by Doty and associates (6, 17), requires that lowering of the dispersion constant must be accompanied by lowering of the specific rotation (in the absolute sense), i.e. increase in levorotation. This conforms with the behavior of several denatured proteins (15)) with the seven examples presented by Yang and Doty (6), as well as with our observations on phosvitin, taka-amylase, trypsin, the caseins (1) , and several other cases. However, the rotatory dispersion data of the y-globulins (l), Bence-Jones protein (1)) pepsin, and pancreatic amylase do not fit into this scheme: The dispersion constants of these globular proteins are low, and their specific rotation values are relatively close to zero. This indicates, if considered from the point of view of Yang and Doty (6), that either helices of opposite screw sense than normal are present in pepsin (and the other similarly behaving proteins) or that relatively compact configurations other than helical exist in the solutions. Interesting is also the case of trypsin inhibitor, where the optical rotatory properties (both dispersion constant and specific rotation) indicate denaturation, but the protein actually was in a native state. Chymotrypsinogen represents a similar instance (1) of relatively low dispersion constant and high levorotation. These exceptions seem to point out a considerable variety of configurations of protein molecules. A much more definitive situation exists correlating optical rotation and configuration with the ionization of proteins. Comparison of Figs. 5, 6, and 7, and Tables I, III, and IV shows that there is a considerable difference between the highly charged pepsin and phosvitin and the other proteins studied. The levorotation of both pepsin and phosvitin increases strongly with increasing ionization, i.e. rising pH. The levorotation of the other proteins, on the contrary, changes very little in the pH limits 4-9. Inasmuch as the intrinsic viscosity of pepsin and phosvitin also increases with increasing ionization, the rotation changes most
82
JIRGENSONS
probably are due to molecular expansion and/or change in molecular asymmetry. High intrinsic viscosities for pepsin and phosvitin at pH 8-9 were observed also in solutions which had ionic strength of 0.10-0.12. These data would seem to indicate that gross configurational changes, rather than electroviscous effects, are responsible for the increased viscosity of these solutions. It is also obvious that the changes in levorotation of pepsin and phosvitin at pH 5-9 are due to changes in gross configuration, the latter being caused most probably by electrostatic repulsion of the macromolecular segments. The macromolecules of pepsin and phosvitin apparently expand very readily, this probably being due partly to the high content of carboxyl (in pepsin) and phosphate groups (in phosvitin) , and partly to structural features, namely, absence of interchain bonding. The viscosity of the other proteins, however, changes much less in these pH limits. For example, the viscosity of ribonuclease, according to Buzzell and Tanford (18), does not change greatly as a function of pH, and this was attributed to the rather rigid configuration and cross-linking of the peptide chains in the macromolecules of this protein (18). Both the levorotation and the dispersion constants of ribonuclease change very little when the pH (i.e., ionization) is changed (Table III). Serum albumin represents an intermediate case. The macromolecules of this protein seem to be more flexible and more affected by ionization than those of ribonuclease and lysozyme, but they change very little in the pH limits 4-9 (19). A considerable expansion of this protein is thought to occur at pH below 4 (20) and above 10.5. The interesting changes of levorotation of albumin around pH’s 3.5-5 and 7-9 depend on the specimen (13, 14), and indicate the possibility of detectable individual variations in the amount of subfractions and/or compositional and structural differences. ACKNOWLEDGMENTS The author is indebted to Professor Shiro Akabori of Osaka University for a preparation of recrystallized taka-amylase. Also acknowledged is the cooperation of Mrs. E. C. Adams and Mr. John A. Cooper. SUMMARY
The optical rotations of pepsin, trypsin, the LY-,p-, and y-chymotrypsins, trypsin inhibitor, papain, pancreatic amylase, taka-amylase, lysozyme, ribonuclease, phosvitin, and several other proteins were studied. The rotatory dispersion constants of these proteins were determined by means of spectropolarimetry. It was found that pepsin,
NATIVE AND DENATURED PROTEINS. XI
83
pancreatic amylase, and soybean trypsin inhibitor have low dispersion constants. The concentration dependence of rotation of several proteins was studied. The pH dependence of the optical rotation was studied using mercaptalbumin, lysozyme, pepsin, ribonuclease, and phosvitin. The rotation of pepsin and phosvitin changed strongly when the pH of the solutions was changed in the range 4-9; the rotation of the other proteins changed very little in the same pH range. Viscosity measurements indicated that the change in levorotation of pepsin and phosvitin in the above-mentioned pH limits is due to changes in molecular gross configuration. It was also demonstrated that the intrinsic viscosity of native pepsin, pancreatic amylase, and trypsin inhibitor solutions was low and therefore incompatible with the assumption that these proteins might be denatured. This finding was substantiated by testing the enzymic activity of these substances. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9.
10. 11. 12. 13. 14.
15. 16. 17. 18. 19. 20.
JIRGENSONS, B., Arch. Biochem. Biophys. ‘74, 57 (1958). JIRGENSONS, B., Arch. Biochem. Biophys. 41, 333 (1952). ANSON, M. L., J. Gen. Physiol. 22, 79 (1938). NORTHROP, J. H., KUNITZ, M., AND HERRIOTT, R. M., “Crystalline
Enzymes,” 2nd ed. Columbia University Press, New York, 1948. SMITH, B. M., AND ROE, J. H., J. Biol. Chem. 179, 53 (1949). YANG, J. T., AND DOTY, P., J. Am. Chem. Sot. 79, 761 (1957). MECHAM, D. K., AND OLCOTT, H. S., J. Am. Chem. Sot. 71, 3670 (1949). RUI)OLPH, H., J. Opt. Sot. Am. 46, 50 (1955). CANNON, M. R., Ind. Eng. Chem., Anal. Ed. 16, 708 (1944). YANG, J. T., AND FOSTER, J. F., J. Am. Chem. Sot. 77,2374 (1955). GREEN, N. M., AND NEURATH, H., in “The Proteins” (Neurath, H. and Bailey, K., eds.), Vol. IIB, p. 1086. Academic Press Inc., New York, 1954. JIRGENSONS, B., Arch. Biochem. Biophys. 39, 261 (1952). JIRGENSONS, B., Arch. Biochem. Biophys. 69, 420 (1955). JIRGENSONS, B., Makromol. Chem. 21, 179 (1956). COIIEN, C., AND SZENT-GY~RGYI, A. G., J. Am. Chem. Sot. 79, 248 (1957). LINDERSTR$M-LANG, K., AND SCHELLMAN, J. A., Biochim. et Biophys. Acta 16, 156 (1954). DOTY, P., AND LUNDBERG, R. D., Proc. Natl. Acad. Sci. U. S. 43, 213 (1957). BUZZELL, J. G., AND TANFORD, C., J. Phys. Chem. 60,1204 (1956). TANFORD, C., AND BUZZELL, J. G., J. Phys. Chem. 60, 225 (1956). YANG, J. T., AND FOSTER, J. F., J. Am. Chem. Sot. 76, 1588 (1954).