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Optical rotation and viscosity of native and denatured proteins. III. The Bence-Jones protein and human γ-globulin
Optical Rotation and Viscosity of Native and Denatured Proteins. III. The Bence- Jones Protein and Human y-Globulin1 B. Jirgensons From The University
of Texas, M. D. Anderson Houston,
Hospital
for Cancer Research,
Texas
Received April 9, 1953 INTRODUCTION
The optical rotation and viscosity of proteins increases upon denaturation. This observation has been made by several authors, and the respective publications are quoted, in the two previous papers of this series. Our studies, as reported in the two papers (1, 2), were directed toward the application of the optical rotation and viscosity methods for the characterization of blood and urinary proteins in disease states. The resistance of various proteins, such as bovine serum albumin, y-globulin, pepsin, lysozyme, and Bence-Jones protein toward the denaturing influence of guanidine hydrochloride, heat, potassium thiocyanate, and other agents was studied. It was found that the various proteins differ considerably in their resistance toward the denaturing agents. BenceJones protein was found to be very sensitive toward denaturing agents. The amino acid composition of a number of samples of Bence-Jones protein which were isolated from urine of one patient by different methods and in various periods of time has been reported elsewhere (3). In the present communication, results are presented which were obtained from the study of several samples of Bence-Jones protein isolated from urine of two more patients with multiple myeloma. In all instances it was possible to obtain fractions of this protein. The fractions differed in their sedimentation constants? but were similar in other properties, 1 This investigation was supported by grants from the National Cancer Institute, National Institutes of Health, U. S. Public Health Service, and from the American Cancer Society. *We are greatly indebted to Dr. John D. Ferry and Mr. Ignacio Tinoro, Jr. of the Department of Chemist.ry of the University of Wisconsin for carrying out 154
especially in t,heir amino acid composition. In comparing the Bcnw.lones protein with other proteins we found that the amino acid composition of our samples of Bence-Jones protein resemtjled that, of y-globulin. (‘l’hcso results will be reported in another paper.) The optical rotation and visc&ty of these nc~- samples Ivere also found to be nearly the same as the optical rotat’ion and viscosity of r-globulin under t,he same csondit,iolls. l-Io\rever, some differences lvere found in the resistance to\\ard tlemtturing agents between the Bence-Jones samples ant1 the hum:ul r-globulin, >md also among the Berlce-Joncs snmplcs thcmsclves from t,hc t,wo patients. EXPERIMENTAL
lklaterials and Methods The Bence-Jones protein samples were isolated from the urine of two patients with multiple myeloma3 by precipitation with ammonium sulfate. Patient I, produced the samples denoted by A, B, DI, EI, and EII. The other patient S produced samples SI, 611, HI, and KII. The concentration of the anomalous protein in the urine of both patients was 2-4 g./l. The samples A and B were precipitated from two fresh urine specimens with saturated ammonium sulfate at 50y0 saturntion. The precipitate was washed with 50y0 saturated ammonium sulfate, dissolved in water, and dialyzed until free from sulfate. The Bence-Jones protein, which remained in solution, was precipitated with acet~oneat -5°C. The precipitates were washed with cold acetone and cold ether, and dried under vacuum. Sample DI was precipitated by very slow addition of ammonium sulfate at 34% saturation, and two other fractions could be obtained from the filtrate. Sample DT wa.s purified like the former samples. Samples EI and EII were isolated by careful fractionation from one urine specimen, EI appearing at 41% and EII at 49% saturation. From the urine of the other patient (S), the fraction SI was isolated at -J1.5% saturation, and from the filt,rate SII at 5101,saturation; KI appeared at. 367,, and KII at 497, saturation. All were purified like A, B, etc. The Berice-Jones protein samples were almost colorless substances, soluble in water, and the solution flocculated at 5+55”C., if the pH of t.hc solutions was in the limits of pH 4.5-6.5. The precipitates dissolved part,ially upon boiling. The isoelectric point of the samples was determined by precipit,ation with sodium lauryl sulfate (4), and found to be at pH 4.9 for sample B, 4.7 forDI, 4.6 for SI, and 4.7 for SII. The nitrogen content varied slightly from sample to sample; the lowest value for N was 14.6% (in the sample KI); KII contained 15.7’% N; SI, 15.8%; and SII, 15.5%. The nitrogen content of the Bence-Jones protein from the these determinations and the evaluation of the sedimentation con&ants. The Svedberg-type oil-turbine ultracentrifugr (under thr supervision of Professor J. W. Williams) was employed. 3 The cooperation of Drs. C. D. Howe and C. C. Shullenberger of &I. D. Anderson Hospital in providing the clinical dat,a and fhe urine samples is ncknon-lcdgetl.
156
IS. JIRGENRONS
other patient was somewhat higher: 16.570 in iz, 17.00/oin B, 16.5y0 in DI, 16.1% in EI, and 15.8% in EII (all values calculated for ash- and moisture-free material). The sedimentation revealed that samples DI, EI, and EII were homogeneous. The samples were dissolved in glycine buffer of ionic strength 0.1 and pH 8.6. The sedimentation constants si at 25°C. were: DI, 2.99; El, 2.58; and EII, 2.51. The amino acid composit)ion was determined hy microbiological methods, and it, will be reported in another paper. All t,he Bence-Jones samples had low values for histidine (0.41.5%) and methionine (0.2-1.1%); the latter was determined also by chemical methods. The methionine content from the Hence-Jones protein from patient 1, was higher (0.51.1%) than that of the patient S (0.2-0.4%). High values for serine (9.1-11.3%) and valine (8.4-11.670) were obtained in all cases. The human r-globulin used in the experiments was a sample from Dr. R. Cohn’s laboratory (Harvard University), denoted Fraction II, “Run Goli.” The optical rotation was determined with a Schmidt & Haensch precision polarimeter which permitted readings with an accuracy of 0.002”. The mean value of rotation was calculated from ten readings. All measurements were made in a constant-temperature room at 24-25°C. A sodium lamp was the light source. Only freshly prepared solutions of the protein samples which had been stored in a refrigerator were used, and the possible changes of the rotation with time were checked. The viscosity was measured with two Ostwald-type capillary viscometers in a constant-temperature bath at 28.4”C. The upper bulb of the viscometers had a volume of 0.9 ml. The flow time of 2.0 ml. water in the viscometer used in most determinations was 151.8 sec. The detergent (Aerosol)-containing solutions were measured with another viscometer in which 3.0 ml. of solution was introduced; the flow time of water for this tube was 174.0 sec. The viscosity number or reduced viscosity was calculated as follows: The flow time of the solution was divided by the flow time of the solvent; from the resulting relative viscosity value was subtracted 1.09, and the resulting specific viscosity was divided by the concentration of the protein (grams of protein/100 ml. solution). The pH was determined by means of a glass-electrode Beckman pH meter. Glytine buffers of ionic strength 0.1 were employed. Double-distilled water and precision-calibrated volumetric equipment of Pyrex glass were used. All reagents used were reagent grade.
Dependence of Optical Rotation on pH of the Protein Solutions The dependence of the optical rotation
on pH for two samples of is shown in Fig. 1. The rotaof about 4-10, and in more strongly acid or alkaline solutions the levorotation of the proteins increases [see Ref. (1) and (2) and the literature cited therein]. Two facts are noteworthy in this case. First, the rotation curve of the sample DI is nearly identical with the rotation curve of human y-globulin. Second, the rotation values for the sample SI are lower than those of DI. The samples obtained formerly from a patient who produced large amounts Bence-Jones protein and human r-globulin tion values are constant in the pH limits
of Bence-.Jonr:s protein were \-cry stable in alkaline solutiolls, and did not show an increase in rotation even at pH 1 I.2 (1, 3) t,hough the rotation values in nearly neutral solutions were the same as for t,he sample ST. The changes of the rotatory power with time were checked, especially for t,hc ac*id and alkaline solutions. The changes, if they were found at, XII, were \:ery small. The ilwrease of rotation at very low and very high pH values is due
FIG. 1. The dependence of levorotation teins, 1.0%. Curve 1, Bence-Jones protein curve 3, Bence-Jones protein sample SI.
on pH. The concentrations of the prosample DI; curve 2, human y-globulin;
to denat.uration. This happens at about, pH’s 2-3 alrd 11.513. At still higher concentrations of acid or alkali, hydrolytic, cleavage occurs also. Optical Rotation of Solutions Containing
Guanidine Salt
This dependenre is shown in Fig. 2. The abscissa represents the concentration of the added guanidine hydrochloride moles per liter of the mixture, the ordinate the value of the specific rotat~ion. The readings wrc made instantly after preparing the mixtrwcs. .\ 5 .U stoc*k solution of gllanidine hydrochloride was employed, and this solution was adtlrtl to 2 yO solut,iolw of the protzeins dissolved ilk :L weakly alknlilrf~ buf’er.
158
B.
JIRGENSONS
The rotation values of these solutions containing guanidine salt changed in time very slowly. The readings plotted are mean values obtained about 30 min. after mixing the solutions. In several hours the rotation values increased to 3-5 $%only. Figure 2 shows that the rotation values of Bence-Jones protein are higher than those of human r-globulin, in the presence of equal amounts Cd
0.5
1.0
1.5
2.0
2.5
on the concentration of guanidine hydroFIG. 2. Dependence of levorotation chloride (moles per liter). Curve 1, human r-globulin; curve 2, Bence-Jones protein SII; curve 3, Bence-Jones protein sample DI. Concentration of all proteins, 1.0%.
of guanidine hydrochloride.
This indicates that the Bence-Jones protein influence of guanidine salt than the r-globulin. The minimum concentration of guanidine salt which causes a definite increase in rotation is smaller for Bence-Jones protein than for human r-globulin. The data obtained for the optical rotation of bovine y-globulin (1) show no substantial differences between the human and bovine globulin. The various samples of the Bence-Jones protein from the same or different patients are also very samples are more sensitive toward the denaturing
similar
in respect
to their
guanidine hydrochloride.
resistance
toward
the denaturing
influenre
of
l”ig\ircl :5 sho\vs t)ho dependence of t,hc viscosit,y number (rr:tl~~wd \.iswsity) 011pfl for samples 111, MI, and human r-globulin. The wrves for sample DI and for globulin are very similar, but the viscosit,ies for sample 1~11 are higher. It is also noteworthy that in t.hc p1-I limits of
0.110 0.100 -
00900 08000700 0600 050-
vc II
1
2
3 4 P"
5
6
7
8
91011
FIO. 3. The dependence of reduced viscosity on pH. Curve 1, 1% solutions of DI; curve 2,0.475% solutions of KII; curve 3, 1% solutions of human r-globulin. All proteins dissolved in glycine buffers of ionic strengt,h 0.1.
J-9 the reduced viscosities are very low, especially for the sample DI and globulin, and the same low viscosities were found for other samples obtained from the urine of the patient L. This means that the Bence.Joncs proteins used were in the native state. A pecwliar change of the viscosity in time was observed in alkaline solutions, a phenomenon noticed already earlier with some other proteins (5, 6). At a pH of about IO-11 the viscosity of a solution of BenceJones protein increases in time, reaches a maximum, and then decreases again. Thisis illustrated in Fig. 4 for the sample KII. The first reading was made just aft’er t,he solution attained the bath temperature, and
160
R.
JIRGESSONS
the measurements were cont~inued in time intervals as indicated on the graph. -r-Globulin did not show any definite maximum in time at this pH. However, a slow increase in viscosity was observed at pH 11.3, and aft,er several hours the viscosity derreascd again slowly (Fig. 4). The viscosity of various samples of Benre-Jones protein dissolved in guanidine salt solutions is higher than in the absence of this denaturing reagent. T-Globulin behaved similarly. There is a definite increase in viscosity only in the presence of about 1.0 iii guanidine hydrochloride,
0.240 x
0220
0
:
0.200 ,--o----o-----
0.180
\
-____ ---0
,
y
2
--:“--,
0.160 p”“.
0.140 20 4060
120
240 TIME
FIG.
360
~YINUISS)
The change of viscosity in time in alkaline solutions. Curve 1,0.475% solution of Ii11 at pH 10.9; curve 2, 1% human r-globulin at pH 11.3. 4.
and the reduced viscosity is higher at higher concentrations of this denaturing agent. In the presence of 1.0 M guanidine salt the reduced viscosity of the various samples of the proteins was 0.082-0.094; it was 0.0964.106 with the same reagent taken 1.5 M and about 0.130-0.160 in the presence of 2.5 M guanidine hydrochloride. Y!o significant differences were found in this respect between the various samples of the Bence-Jones protein and the r-globulin. The pH of these solutions was 5.0-6.3. The Increase in Rotation and Viscosity in the Presence of Aerosol OT Aerosol OT (a nearly 100 y0 pure dioctyl sodium sulfosuccinate from the American Cyanamid Company) is a very powerful denaturing re-
agcllt. I’;vcn in 0.5 y0 solutions it acts strongly on the J3enw-Jones protein and y-globulin causing an increase in viscosity and optical rotat,ion. The proteins were dissolved directly in a 0.5 % solution of the ;2erosol solut,iotl. The results are presented in Table 1. The pH of all solutions was nearly the same, namely, G.54.8. In the al)selwe of t,he denaturing agent, at t,his pH the specifics rot,atiotl \xlues are about 15-50” only (see Fig. I), and the reduced viwositiex arc near 0.0474.OGO (Fig. 3). Hence there is a caonsiderable iwrease itr viwosity a11d optical rotation in the presence of 0.5 y0 .\erosol. In I .Ov0 .2erosol t hc specific rotation values are the same as in O..i % ,4crosol, bitt the \-iswsity rises up to 0.155-0.165. The results shown in Table I indicate that samples ;‘I, 13, ET, and Err TABLE The Oplicrrl
SZLmpk!:
Rotation
A
and T’iscositu
=T
I
of Bence-Jones
Protein
arltl y-Globt~li~c
in the Presence of 0.60/, .-lero.sol m;--B
i
EI
El1
~~~_. __-
SI
KlI
) r-Globulin
~~~ ~
Hpccific robatioll Reduwd viscosit)
’ -62.5” 0.082
-65.1” 0.083
/ -65.5” 0.080 ~__
~ -63.8”
~ -51.8”
/ 0.08’2 / ~
I
/ --j&
- fj() ‘)‘J a /
0.100 i ~~
0.110
! ~~~
0. I60 /
(patiellt L) have a higher specific rotation and a lower viscosity than samples ST and KII (patient S). The specific rotation of y-globulin is bet,n-ecn that, of both groups of the Berwe-Jones protein samples, but thcl viwosity number of the globulin is higher than that) of all the Henw ,Jows spwimcns in such solutions. The Dermturation of Hence-Jones Protein bg Plopanol Flwculntion of proteins with propanol has been used as a means of characterization (3, 7). If c*onst,ant amounts of a protein (at a constant pH and constant temperat’ure) are flocculated in series with increasing amounts of propanol, the flocculation may or may not be associated with dcnatwttion. .\ very derisive fact,or that determines the denaturation is t,he Icmperatuw. At temperatures of near OY‘., no clenal~~~rationo(~(‘urs, and prokiw can be precipitated ~4th alcohols wit’hout twing denatured. I)enat,uratio~ owurs at higher temperatures, depending largely on the sensitivity of a particular protein toward denaturation in general. Fig-
VOL-%
PROPANOL
FIG. 5. The flocculation of Bence-Jones protein sample B with n-propanol. Curve 1, turbidity readings after 24 hr. standing at 37°C. Curve 2, turbidity readings after 16 hr. standing at 8°C. The concentration of the protein in all mixtures was 0.042%, the pH was 5.4. Curve 3, turbidity readings for 0.05% r-globulin after 16 hr. standing at 37°C.; pH = 5.6.
0.6 >
03 02 0.1
PROPANOL
VOL-%
FIG. 6. The flocculation of SI (concentration 0.085%, pH 4.9) with n-propanol. Curve 1, readings after 22 hr. standing at 37”; curve 2, after 16 hr. standing at 6”. 162
N.kTIVE
AND
DENATURED
PROTXTNS.
III
163
urcs .5and 6 show the behavior of samples B and SI. On t(he abscissas is plotted the concentration of propanol in volume per cent in the miztures, ou the ordinates the turijidity (read as reciprocal transmittance by means of a Coleman 6.1 spectrophotometer). The magnitude of t#he turbidity readings for a particular protein depends on t,he pH and concent ration, hut, tOhegeneral shape of the curve rcwaills t,he same and is ~‘cpro-
VOL-%
PROPANOL
Fro. 7. Solubility of Bence-Jones protein I!, in water-propanol mixtures. Curve 1, at 8”; curve 2, at 37”. Curve 3, soluhility of r-globulin at 37”; curve 4, solubility of r-globulin nt 8°C.
ducible. Comparison of Figs. 5 and 6 shows that there is a considerable difference between B and SI; the same was obtained in the flocculation experiments with other samples from patients L and S. Flocculation of human -y-globulin revealed that this protein flocculates like B at low temperatures (Fig. 5). T-Globulin showed no turbidity maximum at 15-20 vol. yOpropanol neither at low temperature nor at 37°C. The high turbidity of the Bence-Jones samples at low propanol concentrations indicates that these proteins are much easier denatured by propanol than y-globulin. The sample SI (as well as SII, KI, and KTI) thereby is more labile than B and others from patient L, as the SI solutions br-
164
B.
JIRGEKSONS
came turbid with 15-20 y0 propanol already at f$-PC., but B only at 37°C. (also at room temperature of 23-25°C. though very slowly). The relatively low turbidity values at high concentrations of propanol do not necessarily suggest a weak denaturation, as turbidity depends mostly on the optical properties of the dispersed phase. This was ascertained also by solubility experiments. Aliquots of Bence-Jones protein B (0.100 g.) were kept for 24 hr. with 10.0 ml. of propanol-water mixtures of various composition either at 8 or 37°C. All samples were stirred in the same fashion. They were then filtered, and the concent,ration of protein dissolved was determined by evaporation and drying at 110°C. The results are presented in Fig. 7, in which the amount of the protein dissolved (in per cent from the original amount) is plotted against the volume per cent of the propanol in the mixture. Curve 1 shows that at 8°C. the solubility decreases rapidly with increasing concentration of the propanol. However, at 37°C. (curve 2) there is a solubility maximum at about 40 vol. Y0 of the propanol. This indicates that such water-propanol mixtures at a certain temperature possess a certain solubilizing ability for the Bence-Jones protein. The determination of optical rotation of the solution of Bence-Jones protein in 40 vol. % propanol showed that the protein certainly is denatured in this solvent at that temperature. The specific rotation for B was found to be -70.2”. The solubility curves indicate that the solubility is very low in the mixtures containing a large proportion of propanol; hence, the rather low turbidity values at about 750/, propanol are due merely to the high optical translucency of the dispersed phase in the flocculating systems (Figs. 5 and 6). DISCUSSION Denaturation is the distortion of the original, unique configuration of the molecules of native proteins. The increase of viscosity on denaturation was believed to suggest unfolding of the spheroprotein molecules (8), though this may not always be the case. Doty and Katz (9) could not find any conclusive evidence of a change in shape of bovine serum albumin molecules denatured by concentrated urea. Their work on light scattering leads to the conclusion that serum albumin molecules do not unfold but undergo an isotropic swelling in concentrated urea. Riley and Arndt (10) recently arrived at a similar conclusion regarding the heat denaturation of serum albumin as investigated by means of x-ray
wattcriIlg. They found that t,he a-helix of the native molec&? remailts intact after dena,turation. The unfolding thus seems to be very incomplete. Hence, the reason for the viscosity increase may be partial unfolding and swelling. The increase in optical rotation upon such “short, Mrgc~" cahanges in the configuration of the protein molecaules is consistent with the picture of welling and very incomplete unfolding. The similarity in the amino acid composition foulrd between the various samples of Bence-Jones protein and r-globulin suggested this comparslirc study of optical robation, viscosity, and flowulation with propanol as well. The experimental part shows that regarding the dependencies of specific rotation on pH, as well as of reduced viscosity on pH, t,he Beuce-,Jones protein has nearly the same properties as -y-globulin. However, bhe Bence-Jones protein samples are more sensitive toward guanidine hydrochloride than y-globulin, if the changes are estimated hy means of optical rotation (Fig. 2). All the Bence-Jones protein samples are also more sensitive than r-globulin toward the denaturing influence of propanol. The differentiation between the Bewe-Jones spec+mells themxelves was best shown by means of denaturation with Aerosol OT (Table I). ‘I’hrl molecular size of Bence-Jones proteins investigated is smaller than t)hc molecular size of y-globulin, the sedimentat’ion c~onstants of the former being about 2.5-3.0 (uncorr.) but of t,he latter about 7 and higher (11, 12). This may explain their differences in sensitivity toward the denaturing influences of guanidine salt as determined by optic,aI rotat,iotl. For t#hesame weight concentrations in solut’ions there are more Belice-.Jones protein molecules t’han r-globulin molecules. The formel t,hus ha\-e a larger surface than the latter. Because of this larger surface t’he small Rence-*Jones molecules interact with the denaturing agent more readily than the large globulin molecules. It is thereby assumed that no cwmplrt.ck unfolding occurs, whic*h is reasonable if one cbonsiders t,he modorat,r ilwwase in the viscosity of these solutions. The rclat,ions in solutiorls wntSainillg .\erosol seem to be more complicated, but it is possible that also in this csasethe molecular size is a very important fact,or; the high viscosity of r-globulin in this case may be due to the formation of aggregatw between the large molecules of the globulin and t’he detergent. ‘l’hc outlined approach seems to be of a certain value for t,he chara(bterizatioll arid differentiation of serum and tissue proteitls. The methods are now itI use in our st)udies of blood prot’eins from cwwct’ pati(~ttts.
166
B. JIRGENSONS ACKNOWLEDGMENT
The author is cordially in this investigation.
grateful
to Dr. Jorge Awapara
for his interest
and care
SUMMARY
Several samples of Bence-Jones protein were isolated in native state from the urine of two patients with multiple myeloma, and their denaturation was studied. Acid, alkali, guanidine hydrochloride, Aerosol OT, and n-propanol were used as denaturing agents. Denaturation was followed by means of optical rotation and viscosity measurements, and flocculation with propanol was observed. Human y-globulin, which resembles in amino acid composition Bence-Jones proteins, was taken for comparison, and studied by the same methods. The dependence of the optical rotation and viscosity on pH for y-globulin and some samples of Bence-Jonesprotein can be expressedby equal curves, though some other samples of Bence-Jonesprotein (from other patient) showed lower values for levorotation and higher viscosities than the former. All Bence-Jones protein samples were more sensitive toward denaturation with guanidine hydrochloride and propanol than y-globulin. This difference can be explained as caused by a difference in the molecular size. REFERENCES 1. JIRGENSONS, B., Arch. Biochem. and Biophys. 39, 261 (1952). 2. JIRGENSONS, B., Arch. Biochem. and Biophys. 41, 333 (1952). 3. JIRGENSONS, B., LANDUA, A. J., AND AWAPARA, J., Biochim. et Biophys. Acta 9, 625 (1952). 4. NEURATH, H., AND PUTNAM, F. W., J. Am. Chem. Sot. 66, 692 (1944). 5. JIRQENSONS, B., Makromol. Chem. 2, 201 (1948). 6. GR~H, J., Kolloid-2.107, 67 (1944). 7. JIRQENSONS, B., Makromol. Chem. 10, 78 (1953). 8. NEURATH, H., GREENSTEIN, J., PUTNAM, F. W., AND ERICKSON, J. O., Chem. Revs. 34, 157 (1944). 9. DOTY, P., AND KATZ, S., Abstracts, Chicago Meeting Amer. Chem. Sot., Sept., 1950; DOTY, P., AND EDSALL, J. T., Advances in Protein Chem. 6, 72 (1951). 10. RILEY, D. P., AND ARNDT, U. W., Proc. Roy. Sot. (London) B141, 93 (1953). 11. DEUTSCH, H. F., ALBERTY, R. A., AND GOSTING, L. J., J. Biol. Chem. 166, 21 (1946) . 12. ONCLEY, J. L., SCATCHARD, G.. AND BROWN, A., J. Phys. & Colloid Chem. 61, 184 (1947).
of Texas, M. D. Anderson Houston,
Hospital
for Cancer Research,
Texas
Received April 9, 1953 INTRODUCTION
The optical rotation and viscosity of proteins increases upon denaturation. This observation has been made by several authors, and the respective publications are quoted, in the two previous papers of this series. Our studies, as reported in the two papers (1, 2), were directed toward the application of the optical rotation and viscosity methods for the characterization of blood and urinary proteins in disease states. The resistance of various proteins, such as bovine serum albumin, y-globulin, pepsin, lysozyme, and Bence-Jones protein toward the denaturing influence of guanidine hydrochloride, heat, potassium thiocyanate, and other agents was studied. It was found that the various proteins differ considerably in their resistance toward the denaturing agents. BenceJones protein was found to be very sensitive toward denaturing agents. The amino acid composition of a number of samples of Bence-Jones protein which were isolated from urine of one patient by different methods and in various periods of time has been reported elsewhere (3). In the present communication, results are presented which were obtained from the study of several samples of Bence-Jones protein isolated from urine of two more patients with multiple myeloma. In all instances it was possible to obtain fractions of this protein. The fractions differed in their sedimentation constants? but were similar in other properties, 1 This investigation was supported by grants from the National Cancer Institute, National Institutes of Health, U. S. Public Health Service, and from the American Cancer Society. *We are greatly indebted to Dr. John D. Ferry and Mr. Ignacio Tinoro, Jr. of the Department of Chemist.ry of the University of Wisconsin for carrying out 154
especially in t,heir amino acid composition. In comparing the Bcnw.lones protein with other proteins we found that the amino acid composition of our samples of Bence-Jones protein resemtjled that, of y-globulin. (‘l’hcso results will be reported in another paper.) The optical rotation and visc&ty of these nc~- samples Ivere also found to be nearly the same as the optical rotat’ion and viscosity of r-globulin under t,he same csondit,iolls. l-Io\rever, some differences lvere found in the resistance to\\ard tlemtturing agents between the Bence-Jones samples ant1 the hum:ul r-globulin, >md also among the Berlce-Joncs snmplcs thcmsclves from t,hc t,wo patients. EXPERIMENTAL
lklaterials and Methods The Bence-Jones protein samples were isolated from the urine of two patients with multiple myeloma3 by precipitation with ammonium sulfate. Patient I, produced the samples denoted by A, B, DI, EI, and EII. The other patient S produced samples SI, 611, HI, and KII. The concentration of the anomalous protein in the urine of both patients was 2-4 g./l. The samples A and B were precipitated from two fresh urine specimens with saturated ammonium sulfate at 50y0 saturntion. The precipitate was washed with 50y0 saturated ammonium sulfate, dissolved in water, and dialyzed until free from sulfate. The Bence-Jones protein, which remained in solution, was precipitated with acet~oneat -5°C. The precipitates were washed with cold acetone and cold ether, and dried under vacuum. Sample DI was precipitated by very slow addition of ammonium sulfate at 34% saturation, and two other fractions could be obtained from the filtrate. Sample DT wa.s purified like the former samples. Samples EI and EII were isolated by careful fractionation from one urine specimen, EI appearing at 41% and EII at 49% saturation. From the urine of the other patient (S), the fraction SI was isolated at -J1.5% saturation, and from the filt,rate SII at 5101,saturation; KI appeared at. 367,, and KII at 497, saturation. All were purified like A, B, etc. The Berice-Jones protein samples were almost colorless substances, soluble in water, and the solution flocculated at 5+55”C., if the pH of t.hc solutions was in the limits of pH 4.5-6.5. The precipitates dissolved part,ially upon boiling. The isoelectric point of the samples was determined by precipit,ation with sodium lauryl sulfate (4), and found to be at pH 4.9 for sample B, 4.7 forDI, 4.6 for SI, and 4.7 for SII. The nitrogen content varied slightly from sample to sample; the lowest value for N was 14.6% (in the sample KI); KII contained 15.7’% N; SI, 15.8%; and SII, 15.5%. The nitrogen content of the Bence-Jones protein from the these determinations and the evaluation of the sedimentation con&ants. The Svedberg-type oil-turbine ultracentrifugr (under thr supervision of Professor J. W. Williams) was employed. 3 The cooperation of Drs. C. D. Howe and C. C. Shullenberger of &I. D. Anderson Hospital in providing the clinical dat,a and fhe urine samples is ncknon-lcdgetl.
156
IS. JIRGENRONS
other patient was somewhat higher: 16.570 in iz, 17.00/oin B, 16.5y0 in DI, 16.1% in EI, and 15.8% in EII (all values calculated for ash- and moisture-free material). The sedimentation revealed that samples DI, EI, and EII were homogeneous. The samples were dissolved in glycine buffer of ionic strength 0.1 and pH 8.6. The sedimentation constants si at 25°C. were: DI, 2.99; El, 2.58; and EII, 2.51. The amino acid composit)ion was determined hy microbiological methods, and it, will be reported in another paper. All t,he Bence-Jones samples had low values for histidine (0.41.5%) and methionine (0.2-1.1%); the latter was determined also by chemical methods. The methionine content from the Hence-Jones protein from patient 1, was higher (0.51.1%) than that of the patient S (0.2-0.4%). High values for serine (9.1-11.3%) and valine (8.4-11.670) were obtained in all cases. The human r-globulin used in the experiments was a sample from Dr. R. Cohn’s laboratory (Harvard University), denoted Fraction II, “Run Goli.” The optical rotation was determined with a Schmidt & Haensch precision polarimeter which permitted readings with an accuracy of 0.002”. The mean value of rotation was calculated from ten readings. All measurements were made in a constant-temperature room at 24-25°C. A sodium lamp was the light source. Only freshly prepared solutions of the protein samples which had been stored in a refrigerator were used, and the possible changes of the rotation with time were checked. The viscosity was measured with two Ostwald-type capillary viscometers in a constant-temperature bath at 28.4”C. The upper bulb of the viscometers had a volume of 0.9 ml. The flow time of 2.0 ml. water in the viscometer used in most determinations was 151.8 sec. The detergent (Aerosol)-containing solutions were measured with another viscometer in which 3.0 ml. of solution was introduced; the flow time of water for this tube was 174.0 sec. The viscosity number or reduced viscosity was calculated as follows: The flow time of the solution was divided by the flow time of the solvent; from the resulting relative viscosity value was subtracted 1.09, and the resulting specific viscosity was divided by the concentration of the protein (grams of protein/100 ml. solution). The pH was determined by means of a glass-electrode Beckman pH meter. Glytine buffers of ionic strength 0.1 were employed. Double-distilled water and precision-calibrated volumetric equipment of Pyrex glass were used. All reagents used were reagent grade.
Dependence of Optical Rotation on pH of the Protein Solutions The dependence of the optical rotation
on pH for two samples of is shown in Fig. 1. The rotaof about 4-10, and in more strongly acid or alkaline solutions the levorotation of the proteins increases [see Ref. (1) and (2) and the literature cited therein]. Two facts are noteworthy in this case. First, the rotation curve of the sample DI is nearly identical with the rotation curve of human y-globulin. Second, the rotation values for the sample SI are lower than those of DI. The samples obtained formerly from a patient who produced large amounts Bence-Jones protein and human r-globulin tion values are constant in the pH limits
of Bence-.Jonr:s protein were \-cry stable in alkaline solutiolls, and did not show an increase in rotation even at pH 1 I.2 (1, 3) t,hough the rotation values in nearly neutral solutions were the same as for t,he sample ST. The changes of the rotatory power with time were checked, especially for t,hc ac*id and alkaline solutions. The changes, if they were found at, XII, were \:ery small. The ilwrease of rotation at very low and very high pH values is due
FIG. 1. The dependence of levorotation teins, 1.0%. Curve 1, Bence-Jones protein curve 3, Bence-Jones protein sample SI.
on pH. The concentrations of the prosample DI; curve 2, human y-globulin;
to denat.uration. This happens at about, pH’s 2-3 alrd 11.513. At still higher concentrations of acid or alkali, hydrolytic, cleavage occurs also. Optical Rotation of Solutions Containing
Guanidine Salt
This dependenre is shown in Fig. 2. The abscissa represents the concentration of the added guanidine hydrochloride moles per liter of the mixture, the ordinate the value of the specific rotat~ion. The readings wrc made instantly after preparing the mixtrwcs. .\ 5 .U stoc*k solution of gllanidine hydrochloride was employed, and this solution was adtlrtl to 2 yO solut,iolw of the protzeins dissolved ilk :L weakly alknlilrf~ buf’er.
158
B.
JIRGENSONS
The rotation values of these solutions containing guanidine salt changed in time very slowly. The readings plotted are mean values obtained about 30 min. after mixing the solutions. In several hours the rotation values increased to 3-5 $%only. Figure 2 shows that the rotation values of Bence-Jones protein are higher than those of human r-globulin, in the presence of equal amounts Cd
0.5
1.0
1.5
2.0
2.5
on the concentration of guanidine hydroFIG. 2. Dependence of levorotation chloride (moles per liter). Curve 1, human r-globulin; curve 2, Bence-Jones protein SII; curve 3, Bence-Jones protein sample DI. Concentration of all proteins, 1.0%.
of guanidine hydrochloride.
This indicates that the Bence-Jones protein influence of guanidine salt than the r-globulin. The minimum concentration of guanidine salt which causes a definite increase in rotation is smaller for Bence-Jones protein than for human r-globulin. The data obtained for the optical rotation of bovine y-globulin (1) show no substantial differences between the human and bovine globulin. The various samples of the Bence-Jones protein from the same or different patients are also very samples are more sensitive toward the denaturing
similar
in respect
to their
guanidine hydrochloride.
resistance
toward
the denaturing
influenre
of
l”ig\ircl :5 sho\vs t)ho dependence of t,hc viscosit,y number (rr:tl~~wd \.iswsity) 011pfl for samples 111, MI, and human r-globulin. The wrves for sample DI and for globulin are very similar, but the viscosit,ies for sample 1~11 are higher. It is also noteworthy that in t.hc p1-I limits of
0.110 0.100 -
00900 08000700 0600 050-
vc II
1
2
3 4 P"
5
6
7
8
91011
FIO. 3. The dependence of reduced viscosity on pH. Curve 1, 1% solutions of DI; curve 2,0.475% solutions of KII; curve 3, 1% solutions of human r-globulin. All proteins dissolved in glycine buffers of ionic strengt,h 0.1.
J-9 the reduced viscosities are very low, especially for the sample DI and globulin, and the same low viscosities were found for other samples obtained from the urine of the patient L. This means that the Bence.Joncs proteins used were in the native state. A pecwliar change of the viscosity in time was observed in alkaline solutions, a phenomenon noticed already earlier with some other proteins (5, 6). At a pH of about IO-11 the viscosity of a solution of BenceJones protein increases in time, reaches a maximum, and then decreases again. Thisis illustrated in Fig. 4 for the sample KII. The first reading was made just aft’er t,he solution attained the bath temperature, and
160
R.
JIRGESSONS
the measurements were cont~inued in time intervals as indicated on the graph. -r-Globulin did not show any definite maximum in time at this pH. However, a slow increase in viscosity was observed at pH 11.3, and aft,er several hours the viscosity derreascd again slowly (Fig. 4). The viscosity of various samples of Benre-Jones protein dissolved in guanidine salt solutions is higher than in the absence of this denaturing reagent. T-Globulin behaved similarly. There is a definite increase in viscosity only in the presence of about 1.0 iii guanidine hydrochloride,
0.240 x
0220
0
:
0.200 ,--o----o-----
0.180
\
-____ ---0
,
y
2
--:“--,
0.160 p”“.
0.140 20 4060
120
240 TIME
FIG.
360
~YINUISS)
The change of viscosity in time in alkaline solutions. Curve 1,0.475% solution of Ii11 at pH 10.9; curve 2, 1% human r-globulin at pH 11.3. 4.
and the reduced viscosity is higher at higher concentrations of this denaturing agent. In the presence of 1.0 M guanidine salt the reduced viscosity of the various samples of the proteins was 0.082-0.094; it was 0.0964.106 with the same reagent taken 1.5 M and about 0.130-0.160 in the presence of 2.5 M guanidine hydrochloride. Y!o significant differences were found in this respect between the various samples of the Bence-Jones protein and the r-globulin. The pH of these solutions was 5.0-6.3. The Increase in Rotation and Viscosity in the Presence of Aerosol OT Aerosol OT (a nearly 100 y0 pure dioctyl sodium sulfosuccinate from the American Cyanamid Company) is a very powerful denaturing re-
agcllt. I’;vcn in 0.5 y0 solutions it acts strongly on the J3enw-Jones protein and y-globulin causing an increase in viscosity and optical rotat,ion. The proteins were dissolved directly in a 0.5 % solution of the ;2erosol solut,iotl. The results are presented in Table 1. The pH of all solutions was nearly the same, namely, G.54.8. In the al)selwe of t,he denaturing agent, at t,his pH the specifics rot,atiotl \xlues are about 15-50” only (see Fig. I), and the reduced viwositiex arc near 0.0474.OGO (Fig. 3). Hence there is a caonsiderable iwrease itr viwosity a11d optical rotation in the presence of 0.5 y0 .\erosol. In I .Ov0 .2erosol t hc specific rotation values are the same as in O..i % ,4crosol, bitt the \-iswsity rises up to 0.155-0.165. The results shown in Table I indicate that samples ;‘I, 13, ET, and Err TABLE The Oplicrrl
SZLmpk!:
Rotation
A
and T’iscositu
=T
I
of Bence-Jones
Protein
arltl y-Globt~li~c
in the Presence of 0.60/, .-lero.sol m;--B
i
EI
El1
~~~_. __-
SI
KlI
) r-Globulin
~~~ ~
Hpccific robatioll Reduwd viscosit)
’ -62.5” 0.082
-65.1” 0.083
/ -65.5” 0.080 ~__
~ -63.8”
~ -51.8”
/ 0.08’2 / ~
I
/ --j&
- fj() ‘)‘J a /
0.100 i ~~
0.110
! ~~~
0. I60 /
(patiellt L) have a higher specific rotation and a lower viscosity than samples ST and KII (patient S). The specific rotation of y-globulin is bet,n-ecn that, of both groups of the Berwe-Jones protein samples, but thcl viwosity number of the globulin is higher than that) of all the Henw ,Jows spwimcns in such solutions. The Dermturation of Hence-Jones Protein bg Plopanol Flwculntion of proteins with propanol has been used as a means of characterization (3, 7). If c*onst,ant amounts of a protein (at a constant pH and constant temperat’ure) are flocculated in series with increasing amounts of propanol, the flocculation may or may not be associated with dcnatwttion. .\ very derisive fact,or that determines the denaturation is t,he Icmperatuw. At temperatures of near OY‘., no clenal~~~rationo(~(‘urs, and prokiw can be precipitated ~4th alcohols wit’hout twing denatured. I)enat,uratio~ owurs at higher temperatures, depending largely on the sensitivity of a particular protein toward denaturation in general. Fig-
VOL-%
PROPANOL
FIG. 5. The flocculation of Bence-Jones protein sample B with n-propanol. Curve 1, turbidity readings after 24 hr. standing at 37°C. Curve 2, turbidity readings after 16 hr. standing at 8°C. The concentration of the protein in all mixtures was 0.042%, the pH was 5.4. Curve 3, turbidity readings for 0.05% r-globulin after 16 hr. standing at 37°C.; pH = 5.6.
0.6 >
03 02 0.1
PROPANOL
VOL-%
FIG. 6. The flocculation of SI (concentration 0.085%, pH 4.9) with n-propanol. Curve 1, readings after 22 hr. standing at 37”; curve 2, after 16 hr. standing at 6”. 162
N.kTIVE
AND
DENATURED
PROTXTNS.
III
163
urcs .5and 6 show the behavior of samples B and SI. On t(he abscissas is plotted the concentration of propanol in volume per cent in the miztures, ou the ordinates the turijidity (read as reciprocal transmittance by means of a Coleman 6.1 spectrophotometer). The magnitude of t#he turbidity readings for a particular protein depends on t,he pH and concent ration, hut, tOhegeneral shape of the curve rcwaills t,he same and is ~‘cpro-
VOL-%
PROPANOL
Fro. 7. Solubility of Bence-Jones protein I!, in water-propanol mixtures. Curve 1, at 8”; curve 2, at 37”. Curve 3, soluhility of r-globulin at 37”; curve 4, solubility of r-globulin nt 8°C.
ducible. Comparison of Figs. 5 and 6 shows that there is a considerable difference between B and SI; the same was obtained in the flocculation experiments with other samples from patients L and S. Flocculation of human -y-globulin revealed that this protein flocculates like B at low temperatures (Fig. 5). T-Globulin showed no turbidity maximum at 15-20 vol. yOpropanol neither at low temperature nor at 37°C. The high turbidity of the Bence-Jones samples at low propanol concentrations indicates that these proteins are much easier denatured by propanol than y-globulin. The sample SI (as well as SII, KI, and KTI) thereby is more labile than B and others from patient L, as the SI solutions br-
164
B.
JIRGEKSONS
came turbid with 15-20 y0 propanol already at f$-PC., but B only at 37°C. (also at room temperature of 23-25°C. though very slowly). The relatively low turbidity values at high concentrations of propanol do not necessarily suggest a weak denaturation, as turbidity depends mostly on the optical properties of the dispersed phase. This was ascertained also by solubility experiments. Aliquots of Bence-Jones protein B (0.100 g.) were kept for 24 hr. with 10.0 ml. of propanol-water mixtures of various composition either at 8 or 37°C. All samples were stirred in the same fashion. They were then filtered, and the concent,ration of protein dissolved was determined by evaporation and drying at 110°C. The results are presented in Fig. 7, in which the amount of the protein dissolved (in per cent from the original amount) is plotted against the volume per cent of the propanol in the mixture. Curve 1 shows that at 8°C. the solubility decreases rapidly with increasing concentration of the propanol. However, at 37°C. (curve 2) there is a solubility maximum at about 40 vol. Y0 of the propanol. This indicates that such water-propanol mixtures at a certain temperature possess a certain solubilizing ability for the Bence-Jones protein. The determination of optical rotation of the solution of Bence-Jones protein in 40 vol. % propanol showed that the protein certainly is denatured in this solvent at that temperature. The specific rotation for B was found to be -70.2”. The solubility curves indicate that the solubility is very low in the mixtures containing a large proportion of propanol; hence, the rather low turbidity values at about 750/, propanol are due merely to the high optical translucency of the dispersed phase in the flocculating systems (Figs. 5 and 6). DISCUSSION Denaturation is the distortion of the original, unique configuration of the molecules of native proteins. The increase of viscosity on denaturation was believed to suggest unfolding of the spheroprotein molecules (8), though this may not always be the case. Doty and Katz (9) could not find any conclusive evidence of a change in shape of bovine serum albumin molecules denatured by concentrated urea. Their work on light scattering leads to the conclusion that serum albumin molecules do not unfold but undergo an isotropic swelling in concentrated urea. Riley and Arndt (10) recently arrived at a similar conclusion regarding the heat denaturation of serum albumin as investigated by means of x-ray
wattcriIlg. They found that t,he a-helix of the native molec&? remailts intact after dena,turation. The unfolding thus seems to be very incomplete. Hence, the reason for the viscosity increase may be partial unfolding and swelling. The increase in optical rotation upon such “short, Mrgc~" cahanges in the configuration of the protein molecaules is consistent with the picture of welling and very incomplete unfolding. The similarity in the amino acid composition foulrd between the various samples of Bence-Jones protein and r-globulin suggested this comparslirc study of optical robation, viscosity, and flowulation with propanol as well. The experimental part shows that regarding the dependencies of specific rotation on pH, as well as of reduced viscosity on pH, t,he Beuce-,Jones protein has nearly the same properties as -y-globulin. However, bhe Bence-Jones protein samples are more sensitive toward guanidine hydrochloride than y-globulin, if the changes are estimated hy means of optical rotation (Fig. 2). All the Bence-Jones protein samples are also more sensitive than r-globulin toward the denaturing influence of propanol. The differentiation between the Bewe-Jones spec+mells themxelves was best shown by means of denaturation with Aerosol OT (Table I). ‘I’hrl molecular size of Bence-Jones proteins investigated is smaller than t)hc molecular size of y-globulin, the sedimentat’ion c~onstants of the former being about 2.5-3.0 (uncorr.) but of t,he latter about 7 and higher (11, 12). This may explain their differences in sensitivity toward the denaturing influences of guanidine salt as determined by optic,aI rotat,iotl. For t#hesame weight concentrations in solut’ions there are more Belice-.Jones protein molecules t’han r-globulin molecules. The formel t,hus ha\-e a larger surface than the latter. Because of this larger surface t’he small Rence-*Jones molecules interact with the denaturing agent more readily than the large globulin molecules. It is thereby assumed that no cwmplrt.ck unfolding occurs, whic*h is reasonable if one cbonsiders t,he modorat,r ilwwase in the viscosity of these solutions. The rclat,ions in solutiorls wntSainillg .\erosol seem to be more complicated, but it is possible that also in this csasethe molecular size is a very important fact,or; the high viscosity of r-globulin in this case may be due to the formation of aggregatw between the large molecules of the globulin and t’he detergent. ‘l’hc outlined approach seems to be of a certain value for t,he chara(bterizatioll arid differentiation of serum and tissue proteitls. The methods are now itI use in our st)udies of blood prot’eins from cwwct’ pati(~ttts.
166
B. JIRGENSONS ACKNOWLEDGMENT
The author is cordially in this investigation.
grateful
to Dr. Jorge Awapara
for his interest
and care
SUMMARY
Several samples of Bence-Jones protein were isolated in native state from the urine of two patients with multiple myeloma, and their denaturation was studied. Acid, alkali, guanidine hydrochloride, Aerosol OT, and n-propanol were used as denaturing agents. Denaturation was followed by means of optical rotation and viscosity measurements, and flocculation with propanol was observed. Human y-globulin, which resembles in amino acid composition Bence-Jones proteins, was taken for comparison, and studied by the same methods. The dependence of the optical rotation and viscosity on pH for y-globulin and some samples of Bence-Jonesprotein can be expressedby equal curves, though some other samples of Bence-Jonesprotein (from other patient) showed lower values for levorotation and higher viscosities than the former. All Bence-Jones protein samples were more sensitive toward denaturation with guanidine hydrochloride and propanol than y-globulin. This difference can be explained as caused by a difference in the molecular size. REFERENCES 1. JIRGENSONS, B., Arch. Biochem. and Biophys. 39, 261 (1952). 2. JIRGENSONS, B., Arch. Biochem. and Biophys. 41, 333 (1952). 3. JIRGENSONS, B., LANDUA, A. J., AND AWAPARA, J., Biochim. et Biophys. Acta 9, 625 (1952). 4. NEURATH, H., AND PUTNAM, F. W., J. Am. Chem. Sot. 66, 692 (1944). 5. JIRQENSONS, B., Makromol. Chem. 2, 201 (1948). 6. GR~H, J., Kolloid-2.107, 67 (1944). 7. JIRQENSONS, B., Makromol. Chem. 10, 78 (1953). 8. NEURATH, H., GREENSTEIN, J., PUTNAM, F. W., AND ERICKSON, J. O., Chem. Revs. 34, 157 (1944). 9. DOTY, P., AND KATZ, S., Abstracts, Chicago Meeting Amer. Chem. Sot., Sept., 1950; DOTY, P., AND EDSALL, J. T., Advances in Protein Chem. 6, 72 (1951). 10. RILEY, D. P., AND ARNDT, U. W., Proc. Roy. Sot. (London) B141, 93 (1953). 11. DEUTSCH, H. F., ALBERTY, R. A., AND GOSTING, L. J., J. Biol. Chem. 166, 21 (1946) . 12. ONCLEY, J. L., SCATCHARD, G.. AND BROWN, A., J. Phys. & Colloid Chem. 61, 184 (1947).