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Fractionation of the urinary mucoprotein of Tamm and Horsfall
ARCHIVES
OF
BIOCHEMISTRY
Fractionation
AND
89, 281-288
BIOPHYSICS
of the
Urinary
Mucoprotein
MYLES From
the Medical
Research
(1960)
of Tamm
and
MAXFIELD
Center, Brookhaven National Laboratory Long Island, New York
Received
Horsfall’
February
Upton,
15, 1960
The urinary mucoprotein of Tamm and Horsfall consisting of rod-shaped molecules of molecular weight 7 X lo6 has been broken into fragments of molecular weight one quarter as large as the original molecules. These fragments have been characterized by study with the ultracentrifuge, viscosity, light scattering, electrophoresis, and ultraviolet spectroscopy. The quarter molecules behave as a single component in the ultracentrifuge. They are rod-shaped, have a molecular weight of 1.7 X lo’, a width of 42 A., and a length of 1.5 X lo3 A. The quarter molecules are not monodisperse under electrophoresis but are divided equally into two species of slightly differing charge. The mucoprotein of Tamm is composed of an end-to-end aggregation of four quarter molecules, two of each species. The mucoprotein of Di Ferrante is composed of two of the quarter molecules, one of each species, joined end to end.
tein which was not, identical with that of Di Ferrante. It, is the puropse of this report to present the physicochemical characterization of this mucoprotein and to demonstrate its relationship to that, of Di Ferrant’e and of Tamm.
INTRODUCTION
The hemagglutinating activity of influenza, mumps, and Newcastle disease viruses is inhibited by the fibrous urinary mucoprotein isolated and characterized by Tamm and Horsfall (1, 2). A urinary mucoprotein with similar antiviral activity and ch’emical composit’ion has been isolat’ed by Di Ferrante and Popenoe (3). The Di Ferrante mucoprotein has been characterized by Maxfield (4) who showed that two molecules of the Di Ferrante mucoprotein linked end to end constitute one molecule of the Tamm mucoprotein. The mucoprotein is present in normal human urine in the form of the larger molecules and aggregates of these. It is desirable to further break down the Tamm mucoprotein into smaller wellcharacterized fragments more suitable for chemical analysis. In the course of our investigation of the Di Ferrante mucoprotein prepared directly from human urine, some samples of purified Tamm mucoprotein were used as starting material and passed through t’he Di Ferrante procedure. This yielded a mucopro1 Research supported Energy Commission.
by
the
U.
S.
MATERIALS
AND
METHODS
To 18 1. of pooled human urine collected from hypertensive patients was added NaCl to 0.58 M. As the precipitate settled in the course of 2-3 days, the supernatant was siphoned off. Occasional shaking caused the precipitate to form into a large ball which gradually contracted so that the supernatant could be cleanly siphoned off. When the volume reached about 1 l., the sediment was removed in a refrigerated International centrifuge, resuspended in distilled water, and dialyzed against repeated changes of water for 2 or 3 days. The solution was clarified by centrifugation, and the sediment was discarded. The supernatant was brought to 0.58 M with NaCl; the new precipitate was removed by centrifugation, redissolved, and dialyzed again against water. This final solution, after clarification by centrifuging at 30,000 r.p.m. in the No. 30 rotor of the Spinco preparative ultracentrifuge is a purified solution of the urinary mucoprotein of Tamm and Horsfall denoted by T&HE. To the solution of T&HE was added 0.5 g./l. cetpltrimethylammonium bromide. After stand-
Atomic 281
ing %3 days, the precipitate was removed by centrifugation, washed four times with 95y0 ethyl alcohol saturated with NaCl, dried for a few hours over paraffin chips, resuspended in water, and dialyzed against water. This dialysis must be continued until the alcohol is well removed or a precipitate will not form in the next step. The presence of small amounts of alcohol markedly increases the solubility of this preparation in salt solution. This solution was clarified in the centrifuge, and the supernatant was brought to 0.58 M with NaCl. The precipitate was redissolved and dialyzed against water. After final clarification in the Spinco preparative ultracentrifuge at 30,000 r.p.m. for 30 min. in the No. 30 rotor, this solution was designated T&HE --) DF. Solutions of the Di Ferrante mucoprotein were prepared as described in Ref. (4) and designated T&HDF. Viscosity measurements were made at 20°C. in one of two modified Ostwald capillary viscometers containing 7 ml. solution and having a flow time of 138.7 or 131.0 sec. with distilled water. Ultracentrifuge studies were made in a Spinco analytical ultracentrifuge at 20°C. Electrophoresis studies were performed in a Spinco electrophoresis apparatus of the Tiselius moving-boundary type. This apparatus turned out to be particularly well-adapted for the present task. Light scattering was performed in a BricePhoenix light-scattering photometer with light of wavelength 436 rnp. Some measurements were made at 546, 406, and 365 rnp. Ultraviolet spectra were obtained in a Beckman recording spectrophotometer.
FIG. 1. Ultracentrifuge patterns: Top: T&HE 8.min. intervals; bar angle 35”. Bottom: T&HE at 16.min. intervals; bar angle 45”. (Plate cracked
Refractive-index a Brice-Phoenix
measurements were differential refractometer.
made
in
RESULTS
The purity of the urinary mucoprotein of Tamm and Horsfall (T&HE) is attested to by the ultracentrifuge pattern of Fig. 1 top, which shows the mucoprotein sedimenting in water as a sharp knifelike peak. A similar high degree of puriby is demonstrated in Fig. 1 bottom, an ultracentrifuge pattern of T&HE -+ DF also in water. This peak is also single and broader than the peak of T&HE, and it sediments more slowly. Either of these patterns alone would be a fairly good criterion of purity. The fact that both patterns show single peaks, that only single peaks are visible over a range of concentrations in each solution, and that the sedimentation constants of the two preparations are significantly different render highly unlikely the possibility t’hat a contaminating component of different size or shape would remain undet’ected in any but very low concentration. Sedimentation data obt’ained from dilution series of T&HE + DF in water and in 0.03 M NaCl are plotted in Fig. 2. The mucoprotein is stable indefinitely in water solution. Such solutions are therefore easy to work with. More reliable physiochemical data can be obtained from solutions cgntaining an electrolyte. As was the case with
in water; + DF in at right.)
52,640 r.p.m.; 20°C.; photographs HgO; 52,640 r.p.m.; 20°C.; photographs
at
FRACTIONATION
OF
24 22 -
URINARY
Y E--DF 0 E-DF
MUCOPROTEIN
IN H,O IN 0.03M
NoCl
v, IO8-
6420
0
""1""1"""'1' a2 0.4
0.6
0.0
lo
12
CONCENTRATION
FIG.
2. Ultracentrifuge
I?4
I.6
IS
20
mg/ml
study of human T&HE --f DF.
90 &&I)
Y: -
60 50 40 30 20 IO n “0
x SAMPLE 0 SAMPLE
fIIfIIIIlIIIlI% 0.3
0.1
0.5
0.7
0.9
CONCENTRATION
FIG.
I.1
mg;:l
+27 *28
I.5
3. Viscosity of T&HE + DF in 0.03 M NaCl.
the Di Ferrante mucoprotein (4), salt solutions more concentrated than 0.03 M NaCl were found to causeaggregation of the mucoprotein in too short a period of time to complete the experiments. From this point of view, solutions of T&HE + DF were considerably more difficult to work with than solutions of T&HDF, though perhaps not as difficult as T&HE. The solubility, or better, the tendency to form tactoids, of these mucoproteins is slightly different. Extrapolation of the sedimentation data to zero mucoprotein concentration yields a sedimentation constant srOof 19.0 svedbergs (8). The sedimentation velocity both in water and in salt solution varies linearly with concentration, a behavior quite different from that of the Di Ferrante mucoprotein (4). This indicates
the presence of considerably less molecular interact’ion than in the casereported earlier. Viscosity data plotted in Fig. 3 as specific viscosity vs. concentration extrapolate to an intrinsic viscosity of slightly over 100. After studying the preparation procedure yielding T&HE -+ DF, one might expect to obtain the Di Ferrante mucoprotein as an end product. However, the sedimentation constant of T&HE -+ DF is somewhat lower than that of the Di Ferrante mucoprotein and the intrinsic viscosity is markedly lower. Therefore, the two mucoproteins cannot be identical. It is worthy of note that the viscosity of the Di FerranOe mucoprotein (4) drops at great dilution, suggesting dissociation of this mucoprotein on dilution. This
284
MAXFIELD
phenomenon has never been observed with T&HE -+ DF. The molecular weight of T&HE + DF calculated from the sediment’ation constant and the intrinsic viscosity is 1.7 X lo6 which is just one-half of t’he molecular weight of the mucoprot’ein of Di Ferrant’e and onequarter of that of the mucoprotein of Tamm and Horsfall. The axial rat,io of T&HE -+ DF calculated from the intrinsic viscosity is 36 which is also one-half of that of the Di Ferrante mucoprotein, and one-quart,er of that of the Tamm and Horsfall mucoprotein. It is concluded then that T&HE -+ DF is composed of molecules 42 A. in width and 1500 A. in length, all of similar size and shape. To make this characterization of the molecules of T&HE ---f DF more definitive, confirmation of these results was sought by an independent method. Light scattering was chosen as the method most suitable for the study of molecules of this size and shape and with these solubility properties. The light-scattering data are shown in Fig. 4 in the form of a Zimm plot. The molecular weight of T&HE + DF given by the reciprocal of the intercept on the ordinate is 1.7 X lo6 in excellent agreement with the results of ultracentrifuge and viscosity measurements. The light-scattering measurements were made in 0.03 M NaCl solution to compromise between the distortion of the Zimm plot obtained from distilled water
OO
0.10
solutions and the presence of tactoids which form in salt solution of T&HE --+ DF as they do in solutions of the Di Ferrante mucoprotein (4). Tactoids can be observed t#o grow in t’he light-scattering photometer at higher salt concent’rations and at higher mucoprotein concent’rations. In 0.03 M NaCl it is possible t’o study solutions of mucoprotein over a limited range of concentrations so low that several hours elapse before tactoids start to grow. The solutions must be checked before and after each light-scattering run for the growth of t,actoids, which is indicated by a gradually increasing intensity of scat’tered light, most marked at an angle of 30” from the transmitted beam. Data from all solutions giving evidence of the formation of tactoids were discarded and the solution was further diluted and cent’rifuged in the Spinco preparative ultracentrifuge until tactoids no longer formed during the experiment. The slope of the zero-angle line on the Zimm plot is negative, indicat,ing that the solution of mucoprotein in 0.03 M NaCl is not stable or that the solvent is a poor one. Thus the formation of a precipit’ate is to be expected. The slope of the zero concentration line of the Zimm plot combined with the intercept yields a radius of gyration of the molecule of T&HE + DF of somewhat less than 650 A. For a rod-shaped molecule, this implies a length in excess of 2000 A. far exceeding that obtained from viscosity meas-
/
I
I
I
I
I
0.20
0.30
040
a50
060
0.70
sin’
FIG.
4. Light-scattering
study
(Zimm
plot)
B + 1000
2
of T&HE
a80
C
--) DF
in 0.03 M NaCl
solution.
FRACTIONATION
OF
URINARY
urements. This discrepancy may be due to the distortion of the Zimm plot at low ionic strength. The Zimm plot obtained from T&HE + DF is similar in form but somewhat less distorted than that obtained from the Di Ferrante mucoprotein (4). It is much less distorted than that obtained from the mucoprotein of Tamm and Horsall (Tc?LHE).~ It is concluded on weighing the evidence that the molecule of T&HE --+ DF has a molecular weight of 1.7 X 106, a width of 42 A. and a length of 1500 A. In order to further examine the homogeneity of T&HE + DF, an electrophoretic study was made. To keep in solution the higher mucoprotein concentrations required for electrophoresis, the NaCl concentration was reduced to 0.005 M. This low ionic strength caused marked sharpening of the ascending and broadening of the descending boundary but should not cause splitting of the peaks. The low ionic strength and consequent low conduct’ivity of the solution resulted in a high potential gradient and therefore greater electrophoretic mobility of the mucoprotein than would be expected at higher more customarily used ionic st’rengths. The mucoprotein adhered strongly to the glass surfaces of the electrophoresis cell. This phenomenon was even more marked in the case of both the Tamm and the Di Ferrante mucoprotein. Because of this, the elecbrophoretic boundary must be compensated into view extremely slowly and a long waiting period provided after compensation and before the start of the electrophoresis current for the mucoprotein to drain from the sides of the cell. The progress of the drainage could be observed by removing the mask from before the ele&ophoresis cell and removing the cylindrical lens from the schlieren optical system to obt,ain an undistorted image of the entire width of the cell. After observing these precaut,ions, one obtained (Fig. 5) two incompletely resolved peaks of equal size in the electrophoresis pattern. This indicates that the molecules of T&HE -+ DF are equally divided int#o two species differing in charge but not detectably different in size or shape. 2 M.
Maxfield,
unpublished
results.
MUCOPROTEIN
285
FIG. 5. Electrophoresis pattern of T&HE + DF in 0.005 M NaCl solution; left, initial ascending and descending boundaries; right, ascending (bottom) and descending (top) boundaries after 68 min. electrophoresis time; current 3 ma. Protein solution pH 5.68. Protein migration is toward the positive electrode.
These results raised the question of whether only two or all four of the quarters of the mucoprotein of Tamm and Horsfall were present in the final preparations of T&HE + DF. In two preparations the amounts of T&HE in the start’ing material were 109 and 118 mg. The final yield of T&HE + DF in these preparations was 898 and 93 mg., respectively. Therefore, all four quart’ers must have been accounted for in the final solution. The existence of two species of T&HE -+ DF arising from t’he urinary mucoprotein of Tamm and Horsfall made it necessary to demonstrate the electrophoretic homogeneity of our preparations of T&HE. Figure 6 shows T&HE is indeed electrophoret,ically homogeneous. This implies that each molecule of T&HE is made up of two of each kind of T&HE -+ DF. It is possible to examine the structure of urinary mucoprotein still further. Since T&HE has been shown previously (4) to be composed of two molecules of t’he mucoprotein of Di Ferrant’e attached end t,o end, each molecule of the Di Ferrante mucoprotein must in turn be composed of two molecules of T&HE --$ DF. The latter two molecules might have the same or differing charge. Electrophoret,ic analysis of the Di Ferrante mucoprotein (Fig. 7) shows this component to be homogeneous. Therefore, each molecule of the Di Ferrante mucoprotein must consist of one molecule of the T&HE + DF of each charge.
286
MAXFIELD
FIG. 6. Electrophoresis tially 5.55.
(top) Protein
and after migration
pattern 106 min. is toward
of T&HE electrophoresis the positive
0.005 M NaCl time (bottom); electrode. in
The ultraviolet and visible spectrum of T&HE + DF is the same as that of T&HE and T&HDF. All three forms of mucoprotein stain positively with periodic acid Schiff stain. DISCUSSION
The physicochemical data obtained from the mucoprotein T&HE --+ DF in these experiments should be compared with the data from the mucoprotein of Di Ferrante and of Tamm and Horsfall (Table I) where the data for comparison are taken from Ref. (4). The molecules of T&HE --+ DF are of two speciesof differing charge (represented
solution; current
ascending boundary 2 ma. Protein solution
inipH
by solid lines and dashed lines in Table I), each of which are rods 42 A. in width, 1.5 X lo3 A. in length and of molecular weight 1.7 X 106. One molecule of each of these speciesis joined end to end to make a molecule of the Di Ferrante mucoprotein of molecular weight 3.5 X 106, width 42 A. and length 2.9 X lo3 A. Similarly one molecule of T&HE must consist of two molecules of each speciesof T&HE + DF. Between the two possible structures for T&HE designated (a) and (b) in Table I, no definite decision can be made. Perhaps the structural arrangement (a) is more likely to represent the true state of affairs because no evidence
FRACTIONATION
OF
URINARY
FIG. 7. Electrophoresis pattern of T&HDF in 0.005 and descending (top) initial boundary; right, ascending after 96 min. electrophoresis time; current 2 ma. Protein ward the positive electrode.
has been obtained for the existence of dimers of T&HE or trimers of T&HDF in normal human urine under physiological conditions. (Evidence for the presence of higher polymers does exist.) It is not apparent why a structure like (b) should not polymerize still further. The bond marked 0 in Table I is broken during the isolation of the Di Ferrante mucoprotein, while the bonds marked X are left intact. These bonds are therefore different in nature. The same procedure which breaks the bonds marked X in the preparation of T&HE -+ DF does not break these bonds in the preparation of T&HDF. The only difference is the starting material. This suggests the presence in urine of a substance which protects these bonds. It is of someinterest to determine at which step of the isolation procedure the bonds are broken. Cetyltrimethylammonium bromide (CTAB) precipitates the mucoprotein in such a way that even dialysis against water for a month will not remove enough CTAB
MUCOPROTEIN
M
NaCl (bottom) solution
287
solution; left, ascending (bottom) and descending (top) boundary pH 6.10. Protein migration is to-
to permit the mucoprotein to dissolve again. Therefore, it has not been possible to test the effect of this reagent alone on t’he bonds. On the other hand, ethyl alcohol alone does precipit’ate the mucoprotein and also breaks the bonds. This is of interest because ethyl alcohol is known to promote disulfide interchange reactions (6). It occasionally happens that mixtures of T&HDF and T&HE --+ DF are obtained from the isolation procedure. Such mixtures show single but very rapidly spreading boundaries in the ultracentrifuge. The presence of a mixture is, however, clearly indicated by a specific viscosity int’ermediate between that of T&HDF and that of T&HE + DF (7). When the st,arting material is T&HE, a sufficiently long exposure to alcohol insures the isolation of T&HE --+ DF. In the case of urine as a starting material, occasionally some T&HE + DF is produced. However, in this caselonger exposure to alcohol has not resulted in furtfher destruction of t’he T&HDF. This again suggest,sthe exist,ence of a subst,ance in urine
288
MAXFIELD TABLE
I
OF PHYSICOCHEMICAL
SUMMARY
Mucoproteins
DATA
in 0.03 M NaCl
T&HE - DF
T&HDF
DiFerrante
T&HE
Tamm and Horsfall
1. Structure
(a)
2. Intrinsic
3. 4. 5.
6. 7. 8. 9.
viscosity
lim I he1 - 1) c-0 Ce./ml. Sedimentation constant Si, Electrophoretic boundary Molecular ureight (a) Ultracentrifuge and viscosity (b) Light scattering Axial ratio (a) Viscosity Width of molecule Length of molecule Ultraviolet spectrum maximum
@I
100
19.0
x 10-13 Double
1.7 1.7
x x
106 106
36 42 A. 1.5 x 103 277 rnp
which protects the bonds marked X in Table I from the action of alcohol. REFERENCES 1.
TAMM, I., AND HORSFALL, F. L., JR., Proc. Exptl.
2.
TAMM, Med.
Biol. Med. 74, 108 (1950). I., AND HORSFALL, F. L.,JR., 96, 71 (1952).
Sot.
J. Exptl.
800 in water
325
22.3
3.5 3.1
X lo-l3 Single x x
29 x 10-13 Single
106 106
70 42 A. 2.9 x 103 277 rnp All obey Beer’s
7.0
[5.6
x
106
42 A. X 103; Ref. 277 mp
(5)]
law
3. DI FERRANTE, N., AND POPENOE, E. A., Stand J. Clin. Lab. Invest. 10, Suppl. 81,279 (1958). 4. MAXFIELD, M., Arch. B&hem. Biophys. 86, 382 (1959). 5. TAMM, I., BUGHER, J. C., AND HORSFALL, F.L., JR., J. Biol. Chem. 212, 125 (1955). 6. JENSEN, E. V., Science 130, 1319 (1959). 7. MAXFIELD, M., Science 128, 1087 (1958).
OF
BIOCHEMISTRY
Fractionation
AND
89, 281-288
BIOPHYSICS
of the
Urinary
Mucoprotein
MYLES From
the Medical
Research
(1960)
of Tamm
and
MAXFIELD
Center, Brookhaven National Laboratory Long Island, New York
Received
Horsfall’
February
Upton,
15, 1960
The urinary mucoprotein of Tamm and Horsfall consisting of rod-shaped molecules of molecular weight 7 X lo6 has been broken into fragments of molecular weight one quarter as large as the original molecules. These fragments have been characterized by study with the ultracentrifuge, viscosity, light scattering, electrophoresis, and ultraviolet spectroscopy. The quarter molecules behave as a single component in the ultracentrifuge. They are rod-shaped, have a molecular weight of 1.7 X lo’, a width of 42 A., and a length of 1.5 X lo3 A. The quarter molecules are not monodisperse under electrophoresis but are divided equally into two species of slightly differing charge. The mucoprotein of Tamm is composed of an end-to-end aggregation of four quarter molecules, two of each species. The mucoprotein of Di Ferrante is composed of two of the quarter molecules, one of each species, joined end to end.
tein which was not, identical with that of Di Ferrante. It, is the puropse of this report to present the physicochemical characterization of this mucoprotein and to demonstrate its relationship to that, of Di Ferrant’e and of Tamm.
INTRODUCTION
The hemagglutinating activity of influenza, mumps, and Newcastle disease viruses is inhibited by the fibrous urinary mucoprotein isolated and characterized by Tamm and Horsfall (1, 2). A urinary mucoprotein with similar antiviral activity and ch’emical composit’ion has been isolat’ed by Di Ferrante and Popenoe (3). The Di Ferrante mucoprotein has been characterized by Maxfield (4) who showed that two molecules of the Di Ferrante mucoprotein linked end to end constitute one molecule of the Tamm mucoprotein. The mucoprotein is present in normal human urine in the form of the larger molecules and aggregates of these. It is desirable to further break down the Tamm mucoprotein into smaller wellcharacterized fragments more suitable for chemical analysis. In the course of our investigation of the Di Ferrante mucoprotein prepared directly from human urine, some samples of purified Tamm mucoprotein were used as starting material and passed through t’he Di Ferrante procedure. This yielded a mucopro1 Research supported Energy Commission.
by
the
U.
S.
MATERIALS
AND
METHODS
To 18 1. of pooled human urine collected from hypertensive patients was added NaCl to 0.58 M. As the precipitate settled in the course of 2-3 days, the supernatant was siphoned off. Occasional shaking caused the precipitate to form into a large ball which gradually contracted so that the supernatant could be cleanly siphoned off. When the volume reached about 1 l., the sediment was removed in a refrigerated International centrifuge, resuspended in distilled water, and dialyzed against repeated changes of water for 2 or 3 days. The solution was clarified by centrifugation, and the sediment was discarded. The supernatant was brought to 0.58 M with NaCl; the new precipitate was removed by centrifugation, redissolved, and dialyzed again against water. This final solution, after clarification by centrifuging at 30,000 r.p.m. in the No. 30 rotor of the Spinco preparative ultracentrifuge is a purified solution of the urinary mucoprotein of Tamm and Horsfall denoted by T&HE. To the solution of T&HE was added 0.5 g./l. cetpltrimethylammonium bromide. After stand-
Atomic 281
ing %3 days, the precipitate was removed by centrifugation, washed four times with 95y0 ethyl alcohol saturated with NaCl, dried for a few hours over paraffin chips, resuspended in water, and dialyzed against water. This dialysis must be continued until the alcohol is well removed or a precipitate will not form in the next step. The presence of small amounts of alcohol markedly increases the solubility of this preparation in salt solution. This solution was clarified in the centrifuge, and the supernatant was brought to 0.58 M with NaCl. The precipitate was redissolved and dialyzed against water. After final clarification in the Spinco preparative ultracentrifuge at 30,000 r.p.m. for 30 min. in the No. 30 rotor, this solution was designated T&HE --) DF. Solutions of the Di Ferrante mucoprotein were prepared as described in Ref. (4) and designated T&HDF. Viscosity measurements were made at 20°C. in one of two modified Ostwald capillary viscometers containing 7 ml. solution and having a flow time of 138.7 or 131.0 sec. with distilled water. Ultracentrifuge studies were made in a Spinco analytical ultracentrifuge at 20°C. Electrophoresis studies were performed in a Spinco electrophoresis apparatus of the Tiselius moving-boundary type. This apparatus turned out to be particularly well-adapted for the present task. Light scattering was performed in a BricePhoenix light-scattering photometer with light of wavelength 436 rnp. Some measurements were made at 546, 406, and 365 rnp. Ultraviolet spectra were obtained in a Beckman recording spectrophotometer.
FIG. 1. Ultracentrifuge patterns: Top: T&HE 8.min. intervals; bar angle 35”. Bottom: T&HE at 16.min. intervals; bar angle 45”. (Plate cracked
Refractive-index a Brice-Phoenix
measurements were differential refractometer.
made
in
RESULTS
The purity of the urinary mucoprotein of Tamm and Horsfall (T&HE) is attested to by the ultracentrifuge pattern of Fig. 1 top, which shows the mucoprotein sedimenting in water as a sharp knifelike peak. A similar high degree of puriby is demonstrated in Fig. 1 bottom, an ultracentrifuge pattern of T&HE -+ DF also in water. This peak is also single and broader than the peak of T&HE, and it sediments more slowly. Either of these patterns alone would be a fairly good criterion of purity. The fact that both patterns show single peaks, that only single peaks are visible over a range of concentrations in each solution, and that the sedimentation constants of the two preparations are significantly different render highly unlikely the possibility t’hat a contaminating component of different size or shape would remain undet’ected in any but very low concentration. Sedimentation data obt’ained from dilution series of T&HE + DF in water and in 0.03 M NaCl are plotted in Fig. 2. The mucoprotein is stable indefinitely in water solution. Such solutions are therefore easy to work with. More reliable physiochemical data can be obtained from solutions cgntaining an electrolyte. As was the case with
in water; + DF in at right.)
52,640 r.p.m.; 20°C.; photographs HgO; 52,640 r.p.m.; 20°C.; photographs
at
FRACTIONATION
OF
24 22 -
URINARY
Y E--DF 0 E-DF
MUCOPROTEIN
IN H,O IN 0.03M
NoCl
v, IO8-
6420
0
""1""1"""'1' a2 0.4
0.6
0.0
lo
12
CONCENTRATION
FIG.
2. Ultracentrifuge
I?4
I.6
IS
20
mg/ml
study of human T&HE --f DF.
90 &&I)
Y: -
60 50 40 30 20 IO n “0
x SAMPLE 0 SAMPLE
fIIfIIIIlIIIlI% 0.3
0.1
0.5
0.7
0.9
CONCENTRATION
FIG.
I.1
mg;:l
+27 *28
I.5
3. Viscosity of T&HE + DF in 0.03 M NaCl.
the Di Ferrante mucoprotein (4), salt solutions more concentrated than 0.03 M NaCl were found to causeaggregation of the mucoprotein in too short a period of time to complete the experiments. From this point of view, solutions of T&HE + DF were considerably more difficult to work with than solutions of T&HDF, though perhaps not as difficult as T&HE. The solubility, or better, the tendency to form tactoids, of these mucoproteins is slightly different. Extrapolation of the sedimentation data to zero mucoprotein concentration yields a sedimentation constant srOof 19.0 svedbergs (8). The sedimentation velocity both in water and in salt solution varies linearly with concentration, a behavior quite different from that of the Di Ferrante mucoprotein (4). This indicates
the presence of considerably less molecular interact’ion than in the casereported earlier. Viscosity data plotted in Fig. 3 as specific viscosity vs. concentration extrapolate to an intrinsic viscosity of slightly over 100. After studying the preparation procedure yielding T&HE -+ DF, one might expect to obtain the Di Ferrante mucoprotein as an end product. However, the sedimentation constant of T&HE -+ DF is somewhat lower than that of the Di Ferrante mucoprotein and the intrinsic viscosity is markedly lower. Therefore, the two mucoproteins cannot be identical. It is worthy of note that the viscosity of the Di FerranOe mucoprotein (4) drops at great dilution, suggesting dissociation of this mucoprotein on dilution. This
284
MAXFIELD
phenomenon has never been observed with T&HE -+ DF. The molecular weight of T&HE + DF calculated from the sediment’ation constant and the intrinsic viscosity is 1.7 X lo6 which is just one-half of t’he molecular weight of the mucoprot’ein of Di Ferrant’e and onequarter of that of the mucoprotein of Tamm and Horsfall. The axial rat,io of T&HE -+ DF calculated from the intrinsic viscosity is 36 which is also one-half of that of the Di Ferrante mucoprotein, and one-quart,er of that of the Tamm and Horsfall mucoprotein. It is concluded then that T&HE -+ DF is composed of molecules 42 A. in width and 1500 A. in length, all of similar size and shape. To make this characterization of the molecules of T&HE ---f DF more definitive, confirmation of these results was sought by an independent method. Light scattering was chosen as the method most suitable for the study of molecules of this size and shape and with these solubility properties. The light-scattering data are shown in Fig. 4 in the form of a Zimm plot. The molecular weight of T&HE + DF given by the reciprocal of the intercept on the ordinate is 1.7 X lo6 in excellent agreement with the results of ultracentrifuge and viscosity measurements. The light-scattering measurements were made in 0.03 M NaCl solution to compromise between the distortion of the Zimm plot obtained from distilled water
OO
0.10
solutions and the presence of tactoids which form in salt solution of T&HE --+ DF as they do in solutions of the Di Ferrante mucoprotein (4). Tactoids can be observed t#o grow in t’he light-scattering photometer at higher salt concent’rations and at higher mucoprotein concent’rations. In 0.03 M NaCl it is possible t’o study solutions of mucoprotein over a limited range of concentrations so low that several hours elapse before tactoids start to grow. The solutions must be checked before and after each light-scattering run for the growth of t,actoids, which is indicated by a gradually increasing intensity of scat’tered light, most marked at an angle of 30” from the transmitted beam. Data from all solutions giving evidence of the formation of tactoids were discarded and the solution was further diluted and cent’rifuged in the Spinco preparative ultracentrifuge until tactoids no longer formed during the experiment. The slope of the zero-angle line on the Zimm plot is negative, indicat,ing that the solution of mucoprotein in 0.03 M NaCl is not stable or that the solvent is a poor one. Thus the formation of a precipit’ate is to be expected. The slope of the zero concentration line of the Zimm plot combined with the intercept yields a radius of gyration of the molecule of T&HE + DF of somewhat less than 650 A. For a rod-shaped molecule, this implies a length in excess of 2000 A. far exceeding that obtained from viscosity meas-
/
I
I
I
I
I
0.20
0.30
040
a50
060
0.70
sin’
FIG.
4. Light-scattering
study
(Zimm
plot)
B + 1000
2
of T&HE
a80
C
--) DF
in 0.03 M NaCl
solution.
FRACTIONATION
OF
URINARY
urements. This discrepancy may be due to the distortion of the Zimm plot at low ionic strength. The Zimm plot obtained from T&HE + DF is similar in form but somewhat less distorted than that obtained from the Di Ferrante mucoprotein (4). It is much less distorted than that obtained from the mucoprotein of Tamm and Horsall (Tc?LHE).~ It is concluded on weighing the evidence that the molecule of T&HE --+ DF has a molecular weight of 1.7 X 106, a width of 42 A. and a length of 1500 A. In order to further examine the homogeneity of T&HE + DF, an electrophoretic study was made. To keep in solution the higher mucoprotein concentrations required for electrophoresis, the NaCl concentration was reduced to 0.005 M. This low ionic strength caused marked sharpening of the ascending and broadening of the descending boundary but should not cause splitting of the peaks. The low ionic strength and consequent low conduct’ivity of the solution resulted in a high potential gradient and therefore greater electrophoretic mobility of the mucoprotein than would be expected at higher more customarily used ionic st’rengths. The mucoprotein adhered strongly to the glass surfaces of the electrophoresis cell. This phenomenon was even more marked in the case of both the Tamm and the Di Ferrante mucoprotein. Because of this, the elecbrophoretic boundary must be compensated into view extremely slowly and a long waiting period provided after compensation and before the start of the electrophoresis current for the mucoprotein to drain from the sides of the cell. The progress of the drainage could be observed by removing the mask from before the ele&ophoresis cell and removing the cylindrical lens from the schlieren optical system to obt,ain an undistorted image of the entire width of the cell. After observing these precaut,ions, one obtained (Fig. 5) two incompletely resolved peaks of equal size in the electrophoresis pattern. This indicates that the molecules of T&HE -+ DF are equally divided int#o two species differing in charge but not detectably different in size or shape. 2 M.
Maxfield,
unpublished
results.
MUCOPROTEIN
285
FIG. 5. Electrophoresis pattern of T&HE + DF in 0.005 M NaCl solution; left, initial ascending and descending boundaries; right, ascending (bottom) and descending (top) boundaries after 68 min. electrophoresis time; current 3 ma. Protein solution pH 5.68. Protein migration is toward the positive electrode.
These results raised the question of whether only two or all four of the quarters of the mucoprotein of Tamm and Horsfall were present in the final preparations of T&HE + DF. In two preparations the amounts of T&HE in the start’ing material were 109 and 118 mg. The final yield of T&HE + DF in these preparations was 898 and 93 mg., respectively. Therefore, all four quart’ers must have been accounted for in the final solution. The existence of two species of T&HE -+ DF arising from t’he urinary mucoprotein of Tamm and Horsfall made it necessary to demonstrate the electrophoretic homogeneity of our preparations of T&HE. Figure 6 shows T&HE is indeed electrophoret,ically homogeneous. This implies that each molecule of T&HE is made up of two of each kind of T&HE -+ DF. It is possible to examine the structure of urinary mucoprotein still further. Since T&HE has been shown previously (4) to be composed of two molecules of t’he mucoprotein of Di Ferrant’e attached end t,o end, each molecule of the Di Ferrante mucoprotein must in turn be composed of two molecules of T&HE --$ DF. The latter two molecules might have the same or differing charge. Electrophoret,ic analysis of the Di Ferrante mucoprotein (Fig. 7) shows this component to be homogeneous. Therefore, each molecule of the Di Ferrante mucoprotein must consist of one molecule of the T&HE + DF of each charge.
286
MAXFIELD
FIG. 6. Electrophoresis tially 5.55.
(top) Protein
and after migration
pattern 106 min. is toward
of T&HE electrophoresis the positive
0.005 M NaCl time (bottom); electrode. in
The ultraviolet and visible spectrum of T&HE + DF is the same as that of T&HE and T&HDF. All three forms of mucoprotein stain positively with periodic acid Schiff stain. DISCUSSION
The physicochemical data obtained from the mucoprotein T&HE --+ DF in these experiments should be compared with the data from the mucoprotein of Di Ferrante and of Tamm and Horsfall (Table I) where the data for comparison are taken from Ref. (4). The molecules of T&HE --+ DF are of two speciesof differing charge (represented
solution; current
ascending boundary 2 ma. Protein solution
inipH
by solid lines and dashed lines in Table I), each of which are rods 42 A. in width, 1.5 X lo3 A. in length and of molecular weight 1.7 X 106. One molecule of each of these speciesis joined end to end to make a molecule of the Di Ferrante mucoprotein of molecular weight 3.5 X 106, width 42 A. and length 2.9 X lo3 A. Similarly one molecule of T&HE must consist of two molecules of each speciesof T&HE + DF. Between the two possible structures for T&HE designated (a) and (b) in Table I, no definite decision can be made. Perhaps the structural arrangement (a) is more likely to represent the true state of affairs because no evidence
FRACTIONATION
OF
URINARY
FIG. 7. Electrophoresis pattern of T&HDF in 0.005 and descending (top) initial boundary; right, ascending after 96 min. electrophoresis time; current 2 ma. Protein ward the positive electrode.
has been obtained for the existence of dimers of T&HE or trimers of T&HDF in normal human urine under physiological conditions. (Evidence for the presence of higher polymers does exist.) It is not apparent why a structure like (b) should not polymerize still further. The bond marked 0 in Table I is broken during the isolation of the Di Ferrante mucoprotein, while the bonds marked X are left intact. These bonds are therefore different in nature. The same procedure which breaks the bonds marked X in the preparation of T&HE -+ DF does not break these bonds in the preparation of T&HDF. The only difference is the starting material. This suggests the presence in urine of a substance which protects these bonds. It is of someinterest to determine at which step of the isolation procedure the bonds are broken. Cetyltrimethylammonium bromide (CTAB) precipitates the mucoprotein in such a way that even dialysis against water for a month will not remove enough CTAB
MUCOPROTEIN
M
NaCl (bottom) solution
287
solution; left, ascending (bottom) and descending (top) boundary pH 6.10. Protein migration is to-
to permit the mucoprotein to dissolve again. Therefore, it has not been possible to test the effect of this reagent alone on t’he bonds. On the other hand, ethyl alcohol alone does precipit’ate the mucoprotein and also breaks the bonds. This is of interest because ethyl alcohol is known to promote disulfide interchange reactions (6). It occasionally happens that mixtures of T&HDF and T&HE --+ DF are obtained from the isolation procedure. Such mixtures show single but very rapidly spreading boundaries in the ultracentrifuge. The presence of a mixture is, however, clearly indicated by a specific viscosity int’ermediate between that of T&HDF and that of T&HE + DF (7). When the st,arting material is T&HE, a sufficiently long exposure to alcohol insures the isolation of T&HE --+ DF. In the case of urine as a starting material, occasionally some T&HE + DF is produced. However, in this caselonger exposure to alcohol has not resulted in furtfher destruction of t’he T&HDF. This again suggest,sthe exist,ence of a subst,ance in urine
288
MAXFIELD TABLE
I
OF PHYSICOCHEMICAL
SUMMARY
Mucoproteins
DATA
in 0.03 M NaCl
T&HE - DF
T&HDF
DiFerrante
T&HE
Tamm and Horsfall
1. Structure
(a)
2. Intrinsic
3. 4. 5.
6. 7. 8. 9.
viscosity
lim I he1 - 1) c-0 Ce./ml. Sedimentation constant Si, Electrophoretic boundary Molecular ureight (a) Ultracentrifuge and viscosity (b) Light scattering Axial ratio (a) Viscosity Width of molecule Length of molecule Ultraviolet spectrum maximum
@I
100
19.0
x 10-13 Double
1.7 1.7
x x
106 106
36 42 A. 1.5 x 103 277 rnp
which protects the bonds marked X in Table I from the action of alcohol. REFERENCES 1.
TAMM, I., AND HORSFALL, F. L., JR., Proc. Exptl.
2.
TAMM, Med.
Biol. Med. 74, 108 (1950). I., AND HORSFALL, F. L.,JR., 96, 71 (1952).
Sot.
J. Exptl.
800 in water
325
22.3
3.5 3.1
X lo-l3 Single x x
29 x 10-13 Single
106 106
70 42 A. 2.9 x 103 277 rnp All obey Beer’s
7.0
[5.6
x
106
42 A. X 103; Ref. 277 mp
(5)]
law
3. DI FERRANTE, N., AND POPENOE, E. A., Stand J. Clin. Lab. Invest. 10, Suppl. 81,279 (1958). 4. MAXFIELD, M., Arch. B&hem. Biophys. 86, 382 (1959). 5. TAMM, I., BUGHER, J. C., AND HORSFALL, F.L., JR., J. Biol. Chem. 212, 125 (1955). 6. JENSEN, E. V., Science 130, 1319 (1959). 7. MAXFIELD, M., Science 128, 1087 (1958).