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Optical rotation and viscosity of native and denatured proteins. IV. Fractions of serum albumin and γ-globulin from various sources
Optical Rotation and Viscosity of Native and Denatured Proteins. IV. Fractions of Serum Albumin and T-Globulin from Various Sources1 B. Jirgensons From The University
and S. Sirotzky2
of Texas, M. D. Anderson Hospital for Cancer Houston, Texas
Received February 16,
Research,
1954
INTRODUCTION
Optical rotation gives valuable information about possible small structural differences and changes in the protein molecule. The viscosity, on the other hand, may give hints on possible changes in molecular shape, as well as about the interactions of the protein with the solvent. We used lately both the optical rotation and viscosity method to test the stability of various proteins toward denaturing agents (1)) to check the reversibility of denaturation (a), as well as to characterize specimensof the BenceJones protein (3). Important papers on kinetics of protein denaturation, using both the optical rotation and viscosity method, were recently published by Kauzmann and his associates (4-7). The main purpose of this study was to find possible differences in the same kind of proteins which were isolated from various sources, e.g., serum albumin fractions from normal serum and from sera of sick individuals. Some preliminary data on the optical rotation of albumin and y-globulin isolated from blood of cancer patients were reported recently (8). The specific rotation of the serum albumin fractions from cancer patients was found to be a little lower than that of normal subjects, especially at pH 5.0-5.2. The work has now been extended to other casesof cancer, and to some diseasesrelated to cancer. Several specimens of the proteins were isolated from blood of pregnant women and tested for comparison. As single data of rotation are of little value, the meas1 Supported in part by grants from the National U. S. Public Health Service, and from the American 2 With the technical assistance of Elouise Oliver. 400
Institutes Cancer
of Health Society.
(C-1785))
h-:\TIVE
.\SD
DENATURED
PROTEINS.
IV
401
urementa were made on series of solutions lvhich had difierent pH. The A-incosity of the solutions of both native proteins and proteins denatured with guanidine hydrochloride was also determined. EXPERIMENTAL
Materials
and ill ethods
The proteins were isolated by Cohn’s method No. 10 (9. 10) from human blood serum. Since individual specimens of blood which can be drawn are too small to obtain sufficient amounts of proteins, the sera of two or three individuals usually were pooled. (The specimens of the proteins from patients with lymphosarcoma and Hodgkin’s disease were not pooled.) Only sera from patients with t,he same condition were pooled, such as those obtained from three individuals with cancer of the lung. All the procedures of precipitation, washing, and centrifugation were performed at -5”. The treatment of the albumin fraction with Xalcitc and the dialysis were carried out in the refrigerator at j-8“. The completeness of dialysis was tested 4-\: means of conductivity measurements. The pure solutions were finally lyophlhzed and stored in the refrigerator. All samples of sera were handled in the same fashion. The purity of the protein preparations ~v:ts tested by means of electrophoresis on paper, and the proteins were found to 1)~ wlatively pure by this criterion. Small amounts of a- and &globulins could 1~ detected (Fig. 1). The ~-globulins were found to be completely free of :tn~- fast-moving components (Fig. 1). No differences were found bctxvcen thrl rlcctr~~phorrtic patterns of our
FIG. 1. The paper-electrophoresis fractions. AH. the albumin from and SP1 are the albumin fractions other A-fractions are those from globulin fraction.
patterns of some albumin and r-globulin Harvard University (WY0 albumin) ; APl. A1’2, isolated from the blood of pregnant women; the patients with tumors; ??: is an example of a y-
402
B.
JIRGENSONS
AND
S.
SIROTZKY
albumin specimens and a 97% serum albumin obtained from Harvard University. The moisture and ash content of the specimens was determined, and the specific rotation values were calculated for moisture-free material. The ash content in all cases was found to be very low ((rO.5%). The specimen of the crystalline bovine serum albumin was obtained from Armour & Co. (lot N.66706). All the chemicals used were reagent grade. The Molisch tests revealed that all of the seven albumin fractions tested contained some carbohydrate. The specimens showing high optical rotation, however, gave a color of the same intensity as those which had low rotation values of 50-55”. A 97T0 pure albumin from Harvard (the fraction IV-8) showed color of about the same intensity as our samples. Furthermore, we tested some of our albumin fractions for the presence of lipides by extracting them with ether. Only traces of lipides were found. The concentration of the protein was determined by evaporating the aqueous solutions and drying at lOb108”, or by determining the moisture of the lyophilized specimens (by drying at the above mentioned temperature), and dissolving a weighed amount of the not-dried material either in water or in a buffer solution. All specimens were dried in the same fashion. The pH was varied with glycine buffers, the ionic strength in the mixtures being 0.05. The final pH was measured with a Beckman glass-electrode pH meter. PH.
4
5
6
FIG. 2. The dependence of specific albumin isolated from sera of pregnant AP3, 1.10%; AP4, 1.00%.
a
rotation women.
9
10
on pH for various API, 0.7201 albumin;
specimens of AP2,0.9201,;
N.lTIVE
AND
DEN.kTCRED
PROTEINS.
403
IV
The optical rotation was measured with a Schmidt and Haensch precision polarimeter (maximum accuracy of readings 0.002”) in a thermostatted room at, 25°C:. Sodium light was used for all determinations. The values reported are mean values from 10 to 26 readings; in each case the total range of variation is indicated by the vertical line through the appropriate point. In most cases the accuracy of the hnally calculated specific rotation was 0.5”. The viscosity was measured with an Ostwald capillary viscometer at BX.S”C.. as described in a previous paper (3).
1. Difiwvaccs
in the Dependence of Levorotation on pH for Various Specimensof Serum Albumin Fractions
Figure 2 shows this dependence for four specimensof albumin isolated from blood of pregnant women. Each albumin specimen was obtained from t,he pooled sera of two individuals. In all instances the pregnancy PH 3
4
5
6
1
9
9
10
t:
FIG. 3. The dependence of specific rotation on pH for serum albumin from various sources. Curve 1: Serum albumin from patients with multiple myeloma, 0.470/c; Curve 2: Albumin of Hodgkin’s disease, 0.7OoJo; Curve 3: Normal human albumin, 0.61%; Curve 4: Normal bovine albumin (Armour), 0.80%.
404
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AND
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was in the latest stages, and the persons were in good health. The curves reveal definite differences in the optical activity of these albumin specimens. The specific rotation is not constant in the pH limits of 4-10, as assumed formerly (ll), but the curves in most cases are saddleshaped [see also Ref. (S)]. We did not investigate the optical activity of solutions possessing very high or very low pH because of lack of material. However, it is well known that the levorotation of such solutions is always greater than near the neutrality point (dotted continuation of the curves). In Fig. 3 are presented data about albumin fractions isolated from blood of patients with multiple myeloma (two patients), and Hodgkin’s disease (two specimens at different times from the same individual). Curves for normal human albumin (pooled from three persons) and
FIG. 4. The dependence of specific rotation on pH for serum albumin from cancer patients. Curve 1: Albumin, 0.73%, cancei of lung; Curve 2: Albumin, l.OS%, gastrointestinal cancer; Curve 3: Albumin, 0.8401,, cancer of cervix; Curve 4: Albumin, 0.869& cancer of face; Curve 5: Albumin, 0.46?&, cancer of mouth; Curve 6: Normal albumin, 1.06%.
SITIVE
.4ND
DENATURED
PROTEIKS.
405
IV
bovine albumin (Armour) also are presented for comparison. The specific rotation values of the “pathological” albumins are considerably smaller than those of the “normals.” Figure 4 gives information on the behavior of albumins isolated from blood of rawer patients. Another pooled normal human albumin is included again for comparison. Most of the readings in t,he cases of malignancy are lower (the points in the negative plot higher) t,han for the normal albumin or t,he “pregnancy albumin” in Fig. 2. However, in some instIantw the albumin fraction from the serum of canc’er patient’s has about the same specific rot,ation values as the normal albumin. .Igain, the dift’erences in t,he various cwrves in this figure are remarkable. C~omparison of Figs. 3 and 4 shows that, the albumin from normal subjects (from various sources) also differs considerably, as the various “pregnancy albumins” do. Several experiments with various buffer concentrat)ions were made (at the same pH) to clarify the importanre of ionir strength. The result) was t’hat the buffw cunrentration has no significant, influence on the spwitiv
Spa hen
Concentration of the protrin
Solvent
pH
Buffer Water Buffer Buffer Buffer Buffer Buffer Buffer Water Buffer Buffer Wat,er Buffer Water Water Buffer Water Buffer Buffer Buffer Buffer
7.Y
1 .o
5.5 4.0 s.5 8.6 4.9 x.3 8.8 5.5 6.0 x.4 5.6 8.X 5.7 5.9 8.6 5.9 4.0 4.1 6.4 8.7
0.66 0.49 0.49 0.49 0.43 0.43 0.43 0.66 0.43 0 .43 0 (93 0.i-l 0.71 2.2 1.1 2.5 1.25 1.25 1.25 1.25
g.,
1. iX ormal , Harvard 2. Pregnancy, 2 Do. Do.
(Run
DO.
3. Pregnancy, 3 Do. Ih. 1. Cnnrer, breast 5. Canwr, Ih.
6. 7. 8. 9.
wrvis
Cancer, gastrointest~inal Cancer, face Cancer, mouth Lymphosarcoma Do. 10. Hodgkins disease DO. DO. DO.
I)o.
Goli)
JO0
ml.
iprcilic rotation deg.
-51.2 -53.3 -52.X -32.3 -52.8 -51.6 -53.6 -51.6 ---I!).$ -- 50, :i -49.1 -50.4 -50.2 -49.1 -51.4 -51.3 -51.6 -51.9 -52.1 -.51 .fi -51 .-I
406
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SIROTZKY
rotation of albumin. Additional measurements in the presence of 0.1-0.4 M barium chloride or 0.5-1.0 M sodium bromide showed that even such high salt concentrations did not influence significantly the optical activity of serum albumin. The influence of the protein concentration on the rotation (at constant pH and ionic strength) also is very slight and has no bearing on the differences between the samples. It seemed necessary also to check the reproducibility of the measurements. For example, specimens were taken from a protein which was lyophilized in various degrees, the rotation was measured, and the protein concentration was determined by evaporation and weighing. The data were plotted, and fitted well on the curves. Several solutions were preserved in the refrigerator, and, after several days, the optical activity was measured. No changes were observed with time. This pertains also to albumin solutions which were kept in a frozen state at - 5°C. Moreover, we checked the reproducibility of the fractionation procedure. Three albumin fractions, which were isolated from three batches of the same normal serum, gave almost identical rotation curves. All this supports the conclusion that the differences in the optical activity between the various samples are independent of the experimental procedure but that they are inherent to the protein itself.
FIG. 5. The dependence of reduced viscosity on concentration mens of human serum albumin. AP3, albumin in pregnancy; ACU, of uterus; ACL, albumin, cancer of lung.
for three albumin,
specicancer
NATIVE
2. The Optical Activity
AND
DENATCRED
of Various
PROTEINS.
407
IV
Specimens of y-Globulin
Fractions
The small amounts of the globulin fract’ions available made it impossible to invest,igate the dependence of the opt,ical rot’ation on pH as completely as was done with the albumin specimens. Some data are presented in Table I. The data indicate that the opt’ical activity of the various samples is not quite the same. 3. The Viscosity
of 1’arious Specimens of Native Albumin
In Fig. 5 is shown the change of the reduced viscosity (specific viscosity divided by concentration of the albumin in grams/l00 ml.) as caused by change of concentration. Extrapolation to zero concentration gives the so-called intrinsic viscosity value, which for the presented cases of “cancer albumin” is only about 0.0364I.037. The “pregnancy albumin” gave the intrinsic viscosity of 0.0404.041, which is the same as for pooled samples of normal albumin (12). However, considerable variations were found in the viscosity readings for different specimens, as indicated in Table II. TABLE l’iscosities
of Various
Specimen
Specimens
II
of
Human
Serum
Solvent
PII
Water Water Buffer
5.1 4.9 6.4 6.4 6.4
Albumin
Concentration of albumin gJ100
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. l-4. 15. 16.
Normal, Xormal, Normal, Pregnancy, Do. Pregnancy, Do. Pregnancy, Do. Cancer, Cancer, Cancer, Do. Cancer, Cancer, Cancer, Cancer, Cancer, Cancer, Multiple
Harvard 1 2 1
Buffer Buffer
2 3 prostate breast cervix,
1
cervix, 2 uterus lung, 1 lung, 2 gastrointestinal face myeloma
Buffer Buffer Buffer Buffer Buffer Buffer Buffer Water Water Buffer Buffer Buffer Buffer
6.i
6.i 6.9 6.9 7.i 6.4 4.2 4.2 6.0 5.0 4.9 7.5 6.7 7.6 4.6
0.47 0.275 0.63 0.54 0.41 0.92 0.69 0.83 0.62 0.50 1.0 1.25 0.62 0.67 0.48 0.66 0.n 0.50 0.86 0.47
Reduced viscosity
ml. 0.044 0.044
0.039 0.053 0.051 0.052 0.048 0.0-u 0.045 0.030 0.030 0.040 0.032 0.043 0.039 0.039 0.052 0.041 O.Oi8 0.032
108
R.
JIRGENSOSS
ASD
TABLE Optical
Rotation
and Viscosity Specimen
1. 2. 3. 1. 5. 6. 7. 8. 9. 10. 11. 12. 13.
Normal Pregnancy, Pregnancy, Cancer, Cancer, Cancer, Cancer, Cancer, Cancer, Cancer, Cancer, Lymphatic Multiple
1 2 breast., 1 breast, 2 breast, 3 cervix, 1 lung, 1 lung, 2 gastrointestinal face leukemia myeloma
S.
SIROTZliY
III
of Albumin Denatured 2.0 M; PH 5.2-5.6 Concentration of the albumin .$.:I00 ml.
IrithGun,bidineH!Jtl~(~chl~~i(~(,. Specific rotat1on dq.
0. 63
-74.6
0.36 0. 92
-TJ.-l - ii.8
O.%Tl
-64.2 -61.5 - 71,s - 70.5
0.73 0.67 0.27 0.55 0.73 0.50 0.86 0.30 0.47
-69.1 -64.4 - T-1.3 -67.1 - il.2 -63.0
Reduwd riscosity
0.115 0. 130 0.11’1 0.0s; 0 I)<%$ 0. 109 0.07x 0 106 0 OY5
0.159 0.2% 0.15; 0.067
4. Optical Rotation and Viscosity of :~lbumirr and y-Globuli/l Denatured with Guanirlinr Hydrochloride The results are compiled in Tables III and IV. The lr\-orotatioll a11c1 reduced viscosity both increase in the presence of the guanidinr~ salt, as denaturation occurs. Contrary to the acid or alkaline buffers, whic*h rcwt instantly with the protein, the guanidinc acts more or less slow1~. ‘l’hc reaction rate depends on the concentration of the components and on temperature (1,2,3,13). In order to obtain comparable results, the esperiments therefore were carreid out at as constant, a temperature as possible (about 25”, the viscosity measured at 28.4”). The concentration of guanidine hydrochloride with the albumin solutions was 2.0 M, and with the r-globulin fraction it was 1.5 M. The data reported in thr tables nrr finnl, constant values. Although the drterminations could IIO~ tw matlt> with t,he same protein concentrations, the dificrenws in flax propertiw and optical activity of the various spccimrns a.w cwnspiwous. ‘I’hc \.ariations are especially remarkable in the albumins (‘1’al)k 1111. III malt> albumins had lower viscwity instances the denatured “pathologkal” than the normal ones, although in somp c*ascs the opposite is true. ‘l‘ht~ same can be said about t,he viscosity of various spwimelrs of tlut i\fts albumin (Table II). ;3. Optical Rotation of the Fractions ITT-.; ant1 I\‘-7 of Human I’lasmtr It is well known that the albumin frncbions obtained by (‘01~11’s mc4hocl So. 10 are not 100 % pure. They contain small amounts of other protcaills
SATIVE
AND DENATURED TABLE
Optical
kotation
and Viscosity Hydrochloride,
Specimen
i.
Normal,
Harvard Pregnancy, 1 Pregnancy, 2 Pregnancy, 3 Cancer, uterus Cancer, cervix, Cancer, breast
1
S. Cancer, aast,rointestinsl 0. I,gmphosnrcoma 10. Hodgkins disease
40!)
IV
IV
of Y-Globulin Denatured i .6 M; pH 5.4-5.8
Concentration of protein g/100
1. 2. 2. 4. j. 6.
PROTEINS.
0.50 0.33 0.33 0.43 0.47 0.50 0.4i 0.46 1.10 1.25
ml.
with
Guanidinc
Specific rotation
Reduced viscosity
deg.
-55.2 -49.2 -60.4 -57.0 -43.8 -40.1 -53.3 -53.x -53.4 -54.3
0 .1020 0.0748 0.0755 0 0763 0.0640 0.0660 0.0653 0.0729 0.0780 0 0760
and traces of nonprotein material. It would be of int,erest to l;no\v ho\\ much an extrarleous protein might change the optical rot’ation of pure albumin. For this purpose the optical rotation of C’ohll’s fractions IIT:md IV-7 was det,ermined.s The fraction IV-4 is composed largely of cdl-, a?-, and &globulins. The optical rotation of t,his fraction was determined in glycine buffer of pH 8.7 (ionic strength O.l), the protein concenIrat,ion being 1.0 %. The specific rotation was found to be -50” (for moisture-free material); i.e., it is of about the same magnitude as that of the r-globulin. The fraction IV-7 is composedlargely of the ,@I-globulin. The specific rot’ation of this fraction was found to he -45” (for moist,urcfree material, other conditions being the same as for II’-4). We caould not, find any trace of the y-globulins in our albumin fractions, but it is possible t’hey contain someof the IV-4 components. The calculat’ed values for an albumin, containing 10 % impurity with a specific rotation of - 40”. would be -59.8” if -62” be the value for pure albumin. We a&ally found - 60.1” for a mixture composedof 80 % of pure crystalline albumin and 20% of the fraction IV-4. This proves that extraneous protein can influence the specific rotation but very little. DISCUSSION
The results of t,his study indicate that specimens of the same kind of proteins differ considerably. The differences are quitJe conspicuous in the 3We are grateful to Laboratories for their protein preparations.
Dr. J. L. Oncley and the staff of the kind, manifold assistance in sending
Harvard us these
ITniversit> and other
410
B.
JIRGENSONS
AND
S.
SIROTZKY
albumins; the y-globulin fraction obtained from various sources, however, cannot be distinguished so easily by the methods applied in this study. Noteworthy is the dependence of the specific rotation of the albumin fractions on pH, and the differences existing between various specimens of albumin in this respect. As Cohn’s method No. 10 renders nondenatured proteins, the differences cannot be explained as caused by denaturation. Moreover, the procedure in all cases was the same. We repeatedly found that the specific rotation is not constant in the pH limits of 4-10, as stated formerly (11, 14), but there is a flat maximum of rotation (minimum in the negative plot) at about pH 6-7. This is, however, the only common feature of the curves for the various specimens; otherwise the curves differ considerably. The curves for the “pathological” albumins usually lie closer to the abscissa than the curves for the normal or “pregnancy” albumins. The shape of the curves differs also. Yang and Foster in a recent paper reported no change in rotation of bovine albumin in the pH limits 4-7 (15). We found in many instances that the specific rotation begins to increase on acidifying, not at pH 4 but in less acidic solutions, usually at pH 4.5, and in some cases even at pH 5. It is generally assumed that the high increase in rotation observed in solutions with pH below 4 and above 10 is due to denaturation. Yang and Foster concluded (15) that the albumin molecule undergoes an isotropic swelling in acid solution. The small rotational changes with pH in the pH limits between 4.5 and 10 may be due to changes in ionization of the -COOH and --NH2 groups attached to the asymmetric carbon atoms of the protein molecules. This conclusion is based on the fact that the optical activity of the amino acids also depends on this ionization (16). However, these effects cannot be very pronounced, as these active groups comprise only a small portion of the large protein molecule. The differences in the optical activity and viscosity of the various albumin specimens might be suspected as being caused by contamination with some extraneous material. As the carbohydrate test was positive, it is possible that the albumin fractions contain some carbohydratecontaining protein. According to Schmid (17), one of these proteins, the so-called acid glycoprotein, showed a specific rotation of only -24” (measured with a wavelength of X = 5461 A.). However, it is doubtful that all differences could be explained as caused by contamination. A liberal estimate of having about 5 % of a constituent of specific rotation -20” and 5 % of an optically inactive impurity still should give - 56.8”
NATIVE
AND
DEN.4TURED
PROTEIKS.
IV
411
for such impure albumin if -62” be the value for pure albumin. We had, however, several preparations which possessed a specific rot8at’ion of only -55” or even lower. Moreover, all specimens were handled similarly, and denaturation was avoided. It seems likely t’hat there may be individual differences in the albumin itself, e.g., in the ratio of the various components constituting it (l&21), or even differences in chrmjcal composition and constitution of these components themselves can be considered. The various human albumin specimens from Harvard University are pooled samples, and the specific rotation (Na light,) at pH 5 for them repeatedly was found to be 59-61”. The same values are obtained with bovine albumin, i.e., 60-62” for several samples of t,he A4rmour productas. However, the pooling itself, or the presence of many individual species of albumin, may be the chief reason for rather constant values of rotat’ion. Several authors have found differences in the same kind of protein isolated from various sources. For example, Pedersen showed that t,here may be variations in sedimentation constants of various specimens of bovine carboxylhemoglobin and fetuin (22). The Bence-.Jones proteins, too, may differ greatly, e.g., in electrophoretir mobility (23), molecular size (2-l)) viscosity, and optical rotation (3). hnot her important example is that of sickle-cell anemia hemoglobin which difiers from normal hemoglobin electrophoretically (25). Variations in chemical composit’ion have been found also in specimens of serum albumin isolated from various sources (20)) as well as in insulin (26). The curves giving an account about t’he dependence of t’he specif-ic rotation on pH seem to be very good characteristics for protein specimens, provided the measurements are made with a sufficiently large amount, of material in order to secure a high degree of precision. The optical rotation tests might, also be of some diagnostic value. We hare tested until now about 35 albumin preparations from t,he blood of persons with tumors. In 32 cases of t’hese 35, the specific rot,at’ion was lower than -58” (at pH 5), and only in 3 cases was it about 59-60. These were compared with 15 specimens of normal albumins isolat,ed in our laboratory, and with several other samples from other laborat,ories. From t’he 15 normal (including pregnancy) specimens, three had low values. There seem to be other fact’ors besides the disease which may influence the properties, and the latter fact complicates t,he problem of diagnostic application. Further work may clarify this \.ery importSant problem.
412
B. JIRGENSONS
AND S. SIROTZKY
ACKNOWLEDGMENTS We are glad Jorge Awapara,
to acknowledge the friendly cooperation Clifton D. Howe, and C. C. Shullenberger.
of our
colleagues,
Drs.
SUMMARY
Serum albumin and y-globulin fractions were separated by the method of Cohn (No. 10) from human blood specimens,and the optical rotation and viscosity of the proteins were studied. The dependence of the specific rotation on pH was determined. Considerable differences were found in both the optical activity and viscosity of the various specimensin cases of pregnancy, and in some diseases. 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. and Biophys. 39, 261 (1952). JIRGENSONS, B., Arch. Biochem. and Biophys. 41, 333 (1952). JIRGENSONS, B., Arch. Biochem. and Biophys. 48, 154 (1954). KAUZMANN, W., AND SIMPSON, R. B., J. Am. Chem. Sot. 76, 5154 (1953). SIMPSON, R. B., AND KAUZMANN, W., J. Am. Chem. Sot. 76, 5139 (1953). FRENSDORFF, H. K., WATSON, M. T., AND KAUZMANN, W., J. Am. Chem. Sot. 76, 5167 (1953). FRENSDORFF, H. K., WATSON, M. T., AND KAUZMANN, W., J. Am. Chem. Sot. 76, 5157 (1953). JIRGENSONS, B., AND SIROTZKY, S., J. Am. Chem. Sot. 78, 1367 (1954). COHN, E. J., GURD, F. R. N., S~RGENOR, D. M., BARNES, B. A., BROWN, R. Ii., DERO’UAUX, G., GILLESPIE, J. M., KAKNT, F. W., LEVER, W. F., LIU, C. H., MITTELMAN, D., MOUTON, R. F., SCHMID, K., AND UROMA, E., J. Am. Chem. Sot. 72, 465 (1950). LEVER, W. F., CURD, F. R. N., UROMA, E., BROWN, R. Ii., BARNES, B. A., SCHMID, K.? AND SCHULTZ, E. I,., J. Gin. Invest. 30,99 (1951). ALMQUIST, H. J., AND GREENBERG, D. M., J. Biol. Chem. 106, 519 (1934). ONCLEY, J. L., SCATCHARD, G., AND BROWN, A., J. Phys. & Colloid Chem. 61, 194 (1947). SCHELLMAN, J., SIMPSON, R. B., AND KAUZMANN, W., J. Am. Chem. Sot. 76, 5152 (1953). BULANKIN, I. N., NAGORNAYA, N. A., AND PARINA, E. V., Biokhimiya 14, 517 (1949). YANG, J. T., AND FOSTER, J. F., J. Am. Chem. Sot. 78,1588 (1954). LUTZ, O., AND JIRGENSONS, B., Ber. 63,448 (1930); ibid. 64, 1221 (1931). SCHMID, K., J. Am. Chem. Sot. 76, 60 (1953). MCMEEKIN, T. L., J. Am. Chem. Sot. 61, 2884 (1939). LUETSCHER, J. A., J. Am. Chem. Sot. 61, 2888 (1939). BRAND, IX., KASSELL, B., AND SAIDEL, L. J., J. Clin. Invest. 23, 437 (1944).
21. 22. 33. 2-l. 25. 26.
HUGHES, W. L., JR., Cold Spring Harbor Symposia Quant. Biol. 14, 79 (1950). PEI~ERSEN, B. O., (‘old Spring Harbor Symposia Qua-nt. Biol. 14, 1-M (19501. GUTMAS, h. 13.. ddoances in Protein (‘hem. 4, 172 (1948). PUTNAM, F. W., AXD STELOS, P., J. Biol. Chem. 203, 347 (1963). PAULIXG, L., 1~~1x0, H. A., SINGER, S. J., ASD \VEI.LS, I. C., Science 110, 5% jlW), ~LIRFENIST, I*:., J. .lrtr. (‘hem. Sot. 76, 5X% (1953).
and S. Sirotzky2
of Texas, M. D. Anderson Hospital for Cancer Houston, Texas
Received February 16,
Research,
1954
INTRODUCTION
Optical rotation gives valuable information about possible small structural differences and changes in the protein molecule. The viscosity, on the other hand, may give hints on possible changes in molecular shape, as well as about the interactions of the protein with the solvent. We used lately both the optical rotation and viscosity method to test the stability of various proteins toward denaturing agents (1)) to check the reversibility of denaturation (a), as well as to characterize specimensof the BenceJones protein (3). Important papers on kinetics of protein denaturation, using both the optical rotation and viscosity method, were recently published by Kauzmann and his associates (4-7). The main purpose of this study was to find possible differences in the same kind of proteins which were isolated from various sources, e.g., serum albumin fractions from normal serum and from sera of sick individuals. Some preliminary data on the optical rotation of albumin and y-globulin isolated from blood of cancer patients were reported recently (8). The specific rotation of the serum albumin fractions from cancer patients was found to be a little lower than that of normal subjects, especially at pH 5.0-5.2. The work has now been extended to other casesof cancer, and to some diseasesrelated to cancer. Several specimens of the proteins were isolated from blood of pregnant women and tested for comparison. As single data of rotation are of little value, the meas1 Supported in part by grants from the National U. S. Public Health Service, and from the American 2 With the technical assistance of Elouise Oliver. 400
Institutes Cancer
of Health Society.
(C-1785))
h-:\TIVE
.\SD
DENATURED
PROTEINS.
IV
401
urementa were made on series of solutions lvhich had difierent pH. The A-incosity of the solutions of both native proteins and proteins denatured with guanidine hydrochloride was also determined. EXPERIMENTAL
Materials
and ill ethods
The proteins were isolated by Cohn’s method No. 10 (9. 10) from human blood serum. Since individual specimens of blood which can be drawn are too small to obtain sufficient amounts of proteins, the sera of two or three individuals usually were pooled. (The specimens of the proteins from patients with lymphosarcoma and Hodgkin’s disease were not pooled.) Only sera from patients with t,he same condition were pooled, such as those obtained from three individuals with cancer of the lung. All the procedures of precipitation, washing, and centrifugation were performed at -5”. The treatment of the albumin fraction with Xalcitc and the dialysis were carried out in the refrigerator at j-8“. The completeness of dialysis was tested 4-\: means of conductivity measurements. The pure solutions were finally lyophlhzed and stored in the refrigerator. All samples of sera were handled in the same fashion. The purity of the protein preparations ~v:ts tested by means of electrophoresis on paper, and the proteins were found to 1)~ wlatively pure by this criterion. Small amounts of a- and &globulins could 1~ detected (Fig. 1). The ~-globulins were found to be completely free of :tn~- fast-moving components (Fig. 1). No differences were found bctxvcen thrl rlcctr~~phorrtic patterns of our
FIG. 1. The paper-electrophoresis fractions. AH. the albumin from and SP1 are the albumin fractions other A-fractions are those from globulin fraction.
patterns of some albumin and r-globulin Harvard University (WY0 albumin) ; APl. A1’2, isolated from the blood of pregnant women; the patients with tumors; ??: is an example of a y-
402
B.
JIRGENSONS
AND
S.
SIROTZKY
albumin specimens and a 97% serum albumin obtained from Harvard University. The moisture and ash content of the specimens was determined, and the specific rotation values were calculated for moisture-free material. The ash content in all cases was found to be very low ((rO.5%). The specimen of the crystalline bovine serum albumin was obtained from Armour & Co. (lot N.66706). All the chemicals used were reagent grade. The Molisch tests revealed that all of the seven albumin fractions tested contained some carbohydrate. The specimens showing high optical rotation, however, gave a color of the same intensity as those which had low rotation values of 50-55”. A 97T0 pure albumin from Harvard (the fraction IV-8) showed color of about the same intensity as our samples. Furthermore, we tested some of our albumin fractions for the presence of lipides by extracting them with ether. Only traces of lipides were found. The concentration of the protein was determined by evaporating the aqueous solutions and drying at lOb108”, or by determining the moisture of the lyophilized specimens (by drying at the above mentioned temperature), and dissolving a weighed amount of the not-dried material either in water or in a buffer solution. All specimens were dried in the same fashion. The pH was varied with glycine buffers, the ionic strength in the mixtures being 0.05. The final pH was measured with a Beckman glass-electrode pH meter. PH.
4
5
6
FIG. 2. The dependence of specific albumin isolated from sera of pregnant AP3, 1.10%; AP4, 1.00%.
a
rotation women.
9
10
on pH for various API, 0.7201 albumin;
specimens of AP2,0.9201,;
N.lTIVE
AND
DEN.kTCRED
PROTEINS.
403
IV
The optical rotation was measured with a Schmidt and Haensch precision polarimeter (maximum accuracy of readings 0.002”) in a thermostatted room at, 25°C:. Sodium light was used for all determinations. The values reported are mean values from 10 to 26 readings; in each case the total range of variation is indicated by the vertical line through the appropriate point. In most cases the accuracy of the hnally calculated specific rotation was 0.5”. The viscosity was measured with an Ostwald capillary viscometer at BX.S”C.. as described in a previous paper (3).
1. Difiwvaccs
in the Dependence of Levorotation on pH for Various Specimensof Serum Albumin Fractions
Figure 2 shows this dependence for four specimensof albumin isolated from blood of pregnant women. Each albumin specimen was obtained from t,he pooled sera of two individuals. In all instances the pregnancy PH 3
4
5
6
1
9
9
10
t:
FIG. 3. The dependence of specific rotation on pH for serum albumin from various sources. Curve 1: Serum albumin from patients with multiple myeloma, 0.470/c; Curve 2: Albumin of Hodgkin’s disease, 0.7OoJo; Curve 3: Normal human albumin, 0.61%; Curve 4: Normal bovine albumin (Armour), 0.80%.
404
B.
JIRGENSONS
AND
S.
SIROTZKT
was in the latest stages, and the persons were in good health. The curves reveal definite differences in the optical activity of these albumin specimens. The specific rotation is not constant in the pH limits of 4-10, as assumed formerly (ll), but the curves in most cases are saddleshaped [see also Ref. (S)]. We did not investigate the optical activity of solutions possessing very high or very low pH because of lack of material. However, it is well known that the levorotation of such solutions is always greater than near the neutrality point (dotted continuation of the curves). In Fig. 3 are presented data about albumin fractions isolated from blood of patients with multiple myeloma (two patients), and Hodgkin’s disease (two specimens at different times from the same individual). Curves for normal human albumin (pooled from three persons) and
FIG. 4. The dependence of specific rotation on pH for serum albumin from cancer patients. Curve 1: Albumin, 0.73%, cancei of lung; Curve 2: Albumin, l.OS%, gastrointestinal cancer; Curve 3: Albumin, 0.8401,, cancer of cervix; Curve 4: Albumin, 0.869& cancer of face; Curve 5: Albumin, 0.46?&, cancer of mouth; Curve 6: Normal albumin, 1.06%.
SITIVE
.4ND
DENATURED
PROTEIKS.
405
IV
bovine albumin (Armour) also are presented for comparison. The specific rotation values of the “pathological” albumins are considerably smaller than those of the “normals.” Figure 4 gives information on the behavior of albumins isolated from blood of rawer patients. Another pooled normal human albumin is included again for comparison. Most of the readings in t,he cases of malignancy are lower (the points in the negative plot higher) t,han for the normal albumin or t,he “pregnancy albumin” in Fig. 2. However, in some instIantw the albumin fraction from the serum of canc’er patient’s has about the same specific rot,ation values as the normal albumin. .Igain, the dift’erences in t,he various cwrves in this figure are remarkable. C~omparison of Figs. 3 and 4 shows that, the albumin from normal subjects (from various sources) also differs considerably, as the various “pregnancy albumins” do. Several experiments with various buffer concentrat)ions were made (at the same pH) to clarify the importanre of ionir strength. The result) was t’hat the buffw cunrentration has no significant, influence on the spwitiv
Spa hen
Concentration of the protrin
Solvent
pH
Buffer Water Buffer Buffer Buffer Buffer Buffer Buffer Water Buffer Buffer Wat,er Buffer Water Water Buffer Water Buffer Buffer Buffer Buffer
7.Y
1 .o
5.5 4.0 s.5 8.6 4.9 x.3 8.8 5.5 6.0 x.4 5.6 8.X 5.7 5.9 8.6 5.9 4.0 4.1 6.4 8.7
0.66 0.49 0.49 0.49 0.43 0.43 0.43 0.66 0.43 0 .43 0 (93 0.i-l 0.71 2.2 1.1 2.5 1.25 1.25 1.25 1.25
g.,
1. iX ormal , Harvard 2. Pregnancy, 2 Do. Do.
(Run
DO.
3. Pregnancy, 3 Do. Ih. 1. Cnnrer, breast 5. Canwr, Ih.
6. 7. 8. 9.
wrvis
Cancer, gastrointest~inal Cancer, face Cancer, mouth Lymphosarcoma Do. 10. Hodgkins disease DO. DO. DO.
I)o.
Goli)
JO0
ml.
iprcilic rotation deg.
-51.2 -53.3 -52.X -32.3 -52.8 -51.6 -53.6 -51.6 ---I!).$ -- 50, :i -49.1 -50.4 -50.2 -49.1 -51.4 -51.3 -51.6 -51.9 -52.1 -.51 .fi -51 .-I
406
B.
JIRGENSONS
AND
S.
SIROTZKY
rotation of albumin. Additional measurements in the presence of 0.1-0.4 M barium chloride or 0.5-1.0 M sodium bromide showed that even such high salt concentrations did not influence significantly the optical activity of serum albumin. The influence of the protein concentration on the rotation (at constant pH and ionic strength) also is very slight and has no bearing on the differences between the samples. It seemed necessary also to check the reproducibility of the measurements. For example, specimens were taken from a protein which was lyophilized in various degrees, the rotation was measured, and the protein concentration was determined by evaporation and weighing. The data were plotted, and fitted well on the curves. Several solutions were preserved in the refrigerator, and, after several days, the optical activity was measured. No changes were observed with time. This pertains also to albumin solutions which were kept in a frozen state at - 5°C. Moreover, we checked the reproducibility of the fractionation procedure. Three albumin fractions, which were isolated from three batches of the same normal serum, gave almost identical rotation curves. All this supports the conclusion that the differences in the optical activity between the various samples are independent of the experimental procedure but that they are inherent to the protein itself.
FIG. 5. The dependence of reduced viscosity on concentration mens of human serum albumin. AP3, albumin in pregnancy; ACU, of uterus; ACL, albumin, cancer of lung.
for three albumin,
specicancer
NATIVE
2. The Optical Activity
AND
DENATCRED
of Various
PROTEINS.
407
IV
Specimens of y-Globulin
Fractions
The small amounts of the globulin fract’ions available made it impossible to invest,igate the dependence of the opt,ical rot’ation on pH as completely as was done with the albumin specimens. Some data are presented in Table I. The data indicate that the opt’ical activity of the various samples is not quite the same. 3. The Viscosity
of 1’arious Specimens of Native Albumin
In Fig. 5 is shown the change of the reduced viscosity (specific viscosity divided by concentration of the albumin in grams/l00 ml.) as caused by change of concentration. Extrapolation to zero concentration gives the so-called intrinsic viscosity value, which for the presented cases of “cancer albumin” is only about 0.0364I.037. The “pregnancy albumin” gave the intrinsic viscosity of 0.0404.041, which is the same as for pooled samples of normal albumin (12). However, considerable variations were found in the viscosity readings for different specimens, as indicated in Table II. TABLE l’iscosities
of Various
Specimen
Specimens
II
of
Human
Serum
Solvent
PII
Water Water Buffer
5.1 4.9 6.4 6.4 6.4
Albumin
Concentration of albumin gJ100
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. l-4. 15. 16.
Normal, Xormal, Normal, Pregnancy, Do. Pregnancy, Do. Pregnancy, Do. Cancer, Cancer, Cancer, Do. Cancer, Cancer, Cancer, Cancer, Cancer, Cancer, Multiple
Harvard 1 2 1
Buffer Buffer
2 3 prostate breast cervix,
1
cervix, 2 uterus lung, 1 lung, 2 gastrointestinal face myeloma
Buffer Buffer Buffer Buffer Buffer Buffer Buffer Water Water Buffer Buffer Buffer Buffer
6.i
6.i 6.9 6.9 7.i 6.4 4.2 4.2 6.0 5.0 4.9 7.5 6.7 7.6 4.6
0.47 0.275 0.63 0.54 0.41 0.92 0.69 0.83 0.62 0.50 1.0 1.25 0.62 0.67 0.48 0.66 0.n 0.50 0.86 0.47
Reduced viscosity
ml. 0.044 0.044
0.039 0.053 0.051 0.052 0.048 0.0-u 0.045 0.030 0.030 0.040 0.032 0.043 0.039 0.039 0.052 0.041 O.Oi8 0.032
108
R.
JIRGENSOSS
ASD
TABLE Optical
Rotation
and Viscosity Specimen
1. 2. 3. 1. 5. 6. 7. 8. 9. 10. 11. 12. 13.
Normal Pregnancy, Pregnancy, Cancer, Cancer, Cancer, Cancer, Cancer, Cancer, Cancer, Cancer, Lymphatic Multiple
1 2 breast., 1 breast, 2 breast, 3 cervix, 1 lung, 1 lung, 2 gastrointestinal face leukemia myeloma
S.
SIROTZliY
III
of Albumin Denatured 2.0 M; PH 5.2-5.6 Concentration of the albumin .$.:I00 ml.
IrithGun,bidineH!Jtl~(~chl~~i(~(,. Specific rotat1on dq.
0. 63
-74.6
0.36 0. 92
-TJ.-l - ii.8
O.%Tl
-64.2 -61.5 - 71,s - 70.5
0.73 0.67 0.27 0.55 0.73 0.50 0.86 0.30 0.47
-69.1 -64.4 - T-1.3 -67.1 - il.2 -63.0
Reduwd riscosity
0.115 0. 130 0.11’1 0.0s; 0 I)<%$ 0. 109 0.07x 0 106 0 OY5
0.159 0.2% 0.15; 0.067
4. Optical Rotation and Viscosity of :~lbumirr and y-Globuli/l Denatured with Guanirlinr Hydrochloride The results are compiled in Tables III and IV. The lr\-orotatioll a11c1 reduced viscosity both increase in the presence of the guanidinr~ salt, as denaturation occurs. Contrary to the acid or alkaline buffers, whic*h rcwt instantly with the protein, the guanidinc acts more or less slow1~. ‘l’hc reaction rate depends on the concentration of the components and on temperature (1,2,3,13). In order to obtain comparable results, the esperiments therefore were carreid out at as constant, a temperature as possible (about 25”, the viscosity measured at 28.4”). The concentration of guanidine hydrochloride with the albumin solutions was 2.0 M, and with the r-globulin fraction it was 1.5 M. The data reported in thr tables nrr finnl, constant values. Although the drterminations could IIO~ tw matlt> with t,he same protein concentrations, the dificrenws in flax propertiw and optical activity of the various spccimrns a.w cwnspiwous. ‘I’hc \.ariations are especially remarkable in the albumins (‘1’al)k 1111. III malt> albumins had lower viscwity instances the denatured “pathologkal” than the normal ones, although in somp c*ascs the opposite is true. ‘l‘ht~ same can be said about t,he viscosity of various spwimelrs of tlut i\fts albumin (Table II). ;3. Optical Rotation of the Fractions ITT-.; ant1 I\‘-7 of Human I’lasmtr It is well known that the albumin frncbions obtained by (‘01~11’s mc4hocl So. 10 are not 100 % pure. They contain small amounts of other protcaills
SATIVE
AND DENATURED TABLE
Optical
kotation
and Viscosity Hydrochloride,
Specimen
i.
Normal,
Harvard Pregnancy, 1 Pregnancy, 2 Pregnancy, 3 Cancer, uterus Cancer, cervix, Cancer, breast
1
S. Cancer, aast,rointestinsl 0. I,gmphosnrcoma 10. Hodgkins disease
40!)
IV
IV
of Y-Globulin Denatured i .6 M; pH 5.4-5.8
Concentration of protein g/100
1. 2. 2. 4. j. 6.
PROTEINS.
0.50 0.33 0.33 0.43 0.47 0.50 0.4i 0.46 1.10 1.25
ml.
with
Guanidinc
Specific rotation
Reduced viscosity
deg.
-55.2 -49.2 -60.4 -57.0 -43.8 -40.1 -53.3 -53.x -53.4 -54.3
0 .1020 0.0748 0.0755 0 0763 0.0640 0.0660 0.0653 0.0729 0.0780 0 0760
and traces of nonprotein material. It would be of int,erest to l;no\v ho\\ much an extrarleous protein might change the optical rot’ation of pure albumin. For this purpose the optical rotation of C’ohll’s fractions IIT:md IV-7 was det,ermined.s The fraction IV-4 is composed largely of cdl-, a?-, and &globulins. The optical rotation of t,his fraction was determined in glycine buffer of pH 8.7 (ionic strength O.l), the protein concenIrat,ion being 1.0 %. The specific rotation was found to be -50” (for moisture-free material); i.e., it is of about the same magnitude as that of the r-globulin. The fraction IV-7 is composedlargely of the ,@I-globulin. The specific rot’ation of this fraction was found to he -45” (for moist,urcfree material, other conditions being the same as for II’-4). We caould not, find any trace of the y-globulins in our albumin fractions, but it is possible t’hey contain someof the IV-4 components. The calculat’ed values for an albumin, containing 10 % impurity with a specific rotation of - 40”. would be -59.8” if -62” be the value for pure albumin. We a&ally found - 60.1” for a mixture composedof 80 % of pure crystalline albumin and 20% of the fraction IV-4. This proves that extraneous protein can influence the specific rotation but very little. DISCUSSION
The results of t,his study indicate that specimens of the same kind of proteins differ considerably. The differences are quitJe conspicuous in the 3We are grateful to Laboratories for their protein preparations.
Dr. J. L. Oncley and the staff of the kind, manifold assistance in sending
Harvard us these
ITniversit> and other
410
B.
JIRGENSONS
AND
S.
SIROTZKY
albumins; the y-globulin fraction obtained from various sources, however, cannot be distinguished so easily by the methods applied in this study. Noteworthy is the dependence of the specific rotation of the albumin fractions on pH, and the differences existing between various specimens of albumin in this respect. As Cohn’s method No. 10 renders nondenatured proteins, the differences cannot be explained as caused by denaturation. Moreover, the procedure in all cases was the same. We repeatedly found that the specific rotation is not constant in the pH limits of 4-10, as stated formerly (11, 14), but there is a flat maximum of rotation (minimum in the negative plot) at about pH 6-7. This is, however, the only common feature of the curves for the various specimens; otherwise the curves differ considerably. The curves for the “pathological” albumins usually lie closer to the abscissa than the curves for the normal or “pregnancy” albumins. The shape of the curves differs also. Yang and Foster in a recent paper reported no change in rotation of bovine albumin in the pH limits 4-7 (15). We found in many instances that the specific rotation begins to increase on acidifying, not at pH 4 but in less acidic solutions, usually at pH 4.5, and in some cases even at pH 5. It is generally assumed that the high increase in rotation observed in solutions with pH below 4 and above 10 is due to denaturation. Yang and Foster concluded (15) that the albumin molecule undergoes an isotropic swelling in acid solution. The small rotational changes with pH in the pH limits between 4.5 and 10 may be due to changes in ionization of the -COOH and --NH2 groups attached to the asymmetric carbon atoms of the protein molecules. This conclusion is based on the fact that the optical activity of the amino acids also depends on this ionization (16). However, these effects cannot be very pronounced, as these active groups comprise only a small portion of the large protein molecule. The differences in the optical activity and viscosity of the various albumin specimens might be suspected as being caused by contamination with some extraneous material. As the carbohydrate test was positive, it is possible that the albumin fractions contain some carbohydratecontaining protein. According to Schmid (17), one of these proteins, the so-called acid glycoprotein, showed a specific rotation of only -24” (measured with a wavelength of X = 5461 A.). However, it is doubtful that all differences could be explained as caused by contamination. A liberal estimate of having about 5 % of a constituent of specific rotation -20” and 5 % of an optically inactive impurity still should give - 56.8”
NATIVE
AND
DEN.4TURED
PROTEIKS.
IV
411
for such impure albumin if -62” be the value for pure albumin. We had, however, several preparations which possessed a specific rot8at’ion of only -55” or even lower. Moreover, all specimens were handled similarly, and denaturation was avoided. It seems likely t’hat there may be individual differences in the albumin itself, e.g., in the ratio of the various components constituting it (l&21), or even differences in chrmjcal composition and constitution of these components themselves can be considered. The various human albumin specimens from Harvard University are pooled samples, and the specific rotation (Na light,) at pH 5 for them repeatedly was found to be 59-61”. The same values are obtained with bovine albumin, i.e., 60-62” for several samples of t,he A4rmour productas. However, the pooling itself, or the presence of many individual species of albumin, may be the chief reason for rather constant values of rotat’ion. Several authors have found differences in the same kind of protein isolated from various sources. For example, Pedersen showed that t,here may be variations in sedimentation constants of various specimens of bovine carboxylhemoglobin and fetuin (22). The Bence-.Jones proteins, too, may differ greatly, e.g., in electrophoretir mobility (23), molecular size (2-l)) viscosity, and optical rotation (3). hnot her important example is that of sickle-cell anemia hemoglobin which difiers from normal hemoglobin electrophoretically (25). Variations in chemical composit’ion have been found also in specimens of serum albumin isolated from various sources (20)) as well as in insulin (26). The curves giving an account about t’he dependence of t’he specif-ic rotation on pH seem to be very good characteristics for protein specimens, provided the measurements are made with a sufficiently large amount, of material in order to secure a high degree of precision. The optical rotation tests might, also be of some diagnostic value. We hare tested until now about 35 albumin preparations from t,he blood of persons with tumors. In 32 cases of t’hese 35, the specific rot,at’ion was lower than -58” (at pH 5), and only in 3 cases was it about 59-60. These were compared with 15 specimens of normal albumins isolat,ed in our laboratory, and with several other samples from other laborat,ories. From t’he 15 normal (including pregnancy) specimens, three had low values. There seem to be other fact’ors besides the disease which may influence the properties, and the latter fact complicates t,he problem of diagnostic application. Further work may clarify this \.ery importSant problem.
412
B. JIRGENSONS
AND S. SIROTZKY
ACKNOWLEDGMENTS We are glad Jorge Awapara,
to acknowledge the friendly cooperation Clifton D. Howe, and C. C. Shullenberger.
of our
colleagues,
Drs.
SUMMARY
Serum albumin and y-globulin fractions were separated by the method of Cohn (No. 10) from human blood specimens,and the optical rotation and viscosity of the proteins were studied. The dependence of the specific rotation on pH was determined. Considerable differences were found in both the optical activity and viscosity of the various specimensin cases of pregnancy, and in some diseases. 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. and Biophys. 39, 261 (1952). JIRGENSONS, B., Arch. Biochem. and Biophys. 41, 333 (1952). JIRGENSONS, B., Arch. Biochem. and Biophys. 48, 154 (1954). KAUZMANN, W., AND SIMPSON, R. B., J. Am. Chem. Sot. 76, 5154 (1953). SIMPSON, R. B., AND KAUZMANN, W., J. Am. Chem. Sot. 76, 5139 (1953). FRENSDORFF, H. K., WATSON, M. T., AND KAUZMANN, W., J. Am. Chem. Sot. 76, 5167 (1953). FRENSDORFF, H. K., WATSON, M. T., AND KAUZMANN, W., J. Am. Chem. Sot. 76, 5157 (1953). JIRGENSONS, B., AND SIROTZKY, S., J. Am. Chem. Sot. 78, 1367 (1954). COHN, E. J., GURD, F. R. N., S~RGENOR, D. M., BARNES, B. A., BROWN, R. Ii., DERO’UAUX, G., GILLESPIE, J. M., KAKNT, F. W., LEVER, W. F., LIU, C. H., MITTELMAN, D., MOUTON, R. F., SCHMID, K., AND UROMA, E., J. Am. Chem. Sot. 72, 465 (1950). LEVER, W. F., CURD, F. R. N., UROMA, E., BROWN, R. Ii., BARNES, B. A., SCHMID, K.? AND SCHULTZ, E. I,., J. Gin. Invest. 30,99 (1951). ALMQUIST, H. J., AND GREENBERG, D. M., J. Biol. Chem. 106, 519 (1934). ONCLEY, J. L., SCATCHARD, G., AND BROWN, A., J. Phys. & Colloid Chem. 61, 194 (1947). SCHELLMAN, J., SIMPSON, R. B., AND KAUZMANN, W., J. Am. Chem. Sot. 76, 5152 (1953). BULANKIN, I. N., NAGORNAYA, N. A., AND PARINA, E. V., Biokhimiya 14, 517 (1949). YANG, J. T., AND FOSTER, J. F., J. Am. Chem. Sot. 78,1588 (1954). LUTZ, O., AND JIRGENSONS, B., Ber. 63,448 (1930); ibid. 64, 1221 (1931). SCHMID, K., J. Am. Chem. Sot. 76, 60 (1953). MCMEEKIN, T. L., J. Am. Chem. Sot. 61, 2884 (1939). LUETSCHER, J. A., J. Am. Chem. Sot. 61, 2888 (1939). BRAND, IX., KASSELL, B., AND SAIDEL, L. J., J. Clin. Invest. 23, 437 (1944).
21. 22. 33. 2-l. 25. 26.
HUGHES, W. L., JR., Cold Spring Harbor Symposia Quant. Biol. 14, 79 (1950). PEI~ERSEN, B. O., (‘old Spring Harbor Symposia Qua-nt. Biol. 14, 1-M (19501. GUTMAS, h. 13.. ddoances in Protein (‘hem. 4, 172 (1948). PUTNAM, F. W., AXD STELOS, P., J. Biol. Chem. 203, 347 (1963). PAULIXG, L., 1~~1x0, H. A., SINGER, S. J., ASD \VEI.LS, I. C., Science 110, 5% jlW), ~LIRFENIST, I*:., J. .lrtr. (‘hem. Sot. 76, 5X% (1953).