126
CHARLES A.
ZITTLE AND
Hygromycin Mercapto1.-A mixture of 10 g. of hygromyciti, 15 ml. of 1 N hydrochloric acid and 20 ml. of ethyl mercaptan was shaken a t room temperature for 2.5 hr. During this period three phases appeared. The oily bottom layer mas separated, washed thoroughly with water and dissolved in ethanol. On concentrating the alcohol solution t o dryness 7 g. of a white amorphous powder was obtained. The material had a pK,' of 11.2 in 667, dimethylformamide, and its infrared spectrum was very similar to that of hygromycin, with the exception that the carbonyl band a t 5.84 + was no longer present. Microbiologically active hygromycin was regenerated from the mercaptol. One gram of the mercaptol was dissolved in 20 ml. of ethanol. To the solution was added 900 mg. of mercuric chloride in 10 ml. of ethanol. The mixture wits warmed on a steam-bath for 15 minutes and then was allowed to stand 1 hr. a t room temperature. The insoluble ethyl mercaptide was removed by filtration, and the filtrate was saturated with hydrogen sulfide t o remove excess mercuric chloride. Immediately after filtration the solution was neutralized with anion-exchange resin (Amberlite IK-45). The solution was concentrated in vacuo to a white powder having a pK,' of 8.9 and ultraviolet and infrared spectra identical with those of hygromycin. Preparation of Dihydrodeoxyhygromycin and its Degradation to 5,6-Dideoxy-~-arabohexose.-A mixture of 20 g. of hygromycin mercaptol and 250 g. of Raney nickel in 400 ml. of ethanol mas refluxed with stirring for 4 hr. The suspension mas filtered, and the Raney nickel was washed twice with 100-nil. portions of ethanol. The filtrate and washings were combined and concentrated i n vacuo to
[COSTRIBUTION FROM THE
s.
EDWARD DELLAMONICA
Vol. 79
dryness. Nine grams of amorphous dihydrodeoxyhygromycin was obtained. There was no absorptLn a t 5.84 p in the infrared, otherwise the spectrum was similar to t h a t of hygromycin. A mixture of 8 g. of dihydrodeoxyhygromycin, 15 ml. of 6 N hydrochloric acid and 25 ml. of ethyl mercaptan was shaken for 3.5 hr. The ethyl mercaptan phase was separated and concentrated to dryness in F U C U O . The oily, partly crystalline residue was dissolved in hot xmter and was allowed to crystallize. Two recrystallizations from water yielded 1.3 g. of 5,6-dideoxy-~-araboliexose, m.p. 10s- 109O . Anal. Calcd. for C ~ O H ~ ~ SC, Z O47.21; ~: H , 8.72; S , 25.20. Found: C, 47.40; H,8.68; S, 24.98. Periodate Oxidation Product of 5,6-Dideoxy-~-arabohexose.-One hundred mg. of 5,6-dideoxy-~-arabohexose was dissolved in 50 ml. of water, and 1 g. of sodium metaperiodate was added. The solution was allowed to stand a t room temperature for 1.5 hr. a t which time the maximum consumption of 6 moles of periodate had been attained. The reaction mixture was distilled into a flask containing 50 mg. of 2,4-dinitrophenylhpdrazine in 50 ml. of 2 LL' hydrochloric acid. The crystalline 2,4-dinitrophenylhydrazone which formed was recrystallized from 9570 ethanol; yield A8 mg. (72%). The compound was shown to be identical with a n authentic sample of propionaldehyde-2.4-dinitrophenylh) drazone by comparison of their X-ray diffraction patterns.
INDIAXAPOLIS 6, INDIANA
EASTERN REGIONAL RESEARCH LABORATORY']
The Viscosity and Opacity of Heated p-Lactoglobulin Solutions : The Effect of Salts, and Oxidizing and Reducing Reagents BY CHARLES A. ZITTLE AND EDWARD S. DELLAMOKIC.I RECEIVED JULY 16, 1956 The viscosity of heated B-lactoglobulin solutions is pH-dependent. When solutions are salt-free the increases in viscosity on heating in the pH range 6.2 to 7.5 are relatively slight with a maximum a t pH 6.8. In general, the viscosities are increased by salts with the greatest increases a t the low pH values. There are, however, specific salt effects; sodium phosphate and citrate prevent the aggregation and concomitant opacity in the low pH region and bring about regular increases in viscosity. Sodium chloride, on the other hand, increases the viscosity but does not prevent opacity a t low pH values, and there is a viscosity maximum a t pH 6.7 with 0.025 JI sodium chloride. I n general, however, increases in viscosity and opacity are parallel. The increases in viscosity in the presence of salts are greatest with high concentrations of protein indicating a high degree of cross-reaction. The more viscous solutions also show structural viscosity. Treatment of 0lactoglobulin with iodine leads to some increase in the viscosity and clearing of the solutions which is consistent with sulfhydryl, disulfide participation in formation of opaque gels. Very large increases in viscosity are obtained in the presence of excess sulfhydryl reagents. The probable explanation of this increase is the opening of loops formed by disulfide bridges with consequent elongation of the molecule.
Protein solutions denatured by heat or urea become highly viscous or gel, a property which is strongly pH-dependent2*3with maximal gelling near the isoelectric point. The diminished gelling tendency a t more remote pH values is viewed as a consequence of the increased electrostatic repulsion, and thereby decreased interaction, between the like-charged r n o l e c ~ l e s . ~I n~ ~view of this interpretaiion of the pH dependence, the presence of salts would be expected to increase the viscosity by reduction of the electrostatic repulsion. The present studies with p-lactoglobulin and chiefly with the salts sodium phosphate and chloride, revealed in general the expected increase in viscosity (1) A laboratory of t h e Eastern Utilization Research Branch, Agricultural Research Service, U . S. D e p a r t m e n t of Agriculture. (2) (a) E . V. Jensen, V. D. Hospeihorn, D. F. Tapley a n d C. Huggins, .I. B i d . C h e m . , 189, 411 (1950); (b) V. D.Hospelhorn a n d E. V. Jensen, TEIISJ O U R N A L , 76, 2830 (1951). (3) H. I;. Frensdorff, A I , T. Watson a n d \V. K a u z m a n n , ibid., 76, 6157 (1953).
and aggregation with increase in salt concentration. There were, however, exceptions to this generalization, apparent a t low salt concentrations and low pH values, which indicated that there were also specific salt effects probably due to binding of the salt ions to the protein. The effects of treating plactoglobulin with iodine, and mercaptoethanol and other reducing agents before heating, were also studied because of the participation of sulfhydryl and disulfide groups in gel f o r m a t i ~ n . ~ ~ ~ Materials p-Lactoglobulin.-This protein was prepared from raw milk by the method of Palmer4 and recrystallized once. It was electrophoretically homogeneous a t PH 8.6. This material was dried from the frozen state and stored as described previously.6 0-Lactoglobulin, Iodine-treated.-Ten milliliters 3 .6vo p-lactoglobulin in water a t pH 7.5 was mixed with 1.0 ml. (4) A. H . Palmer, J . B i d . Chcm., 104, 359 (1931).
(5)
C. A. Zittle a n d E. S. DellaMonica, J . Daivy Sci., 39, 514
(1956).
VISCOSITY AND OPACITYOF
Jan. 5, 1957
127
HEATED@-LACTOGLOBULIN
of 0.1 M sodium phosphate buffer, pH 7.5, and treated with 0.6 ml. of 0.1 N iodine in 0.15 N KI a t 30". The PH dropped 0.5 unit and was adjusted to 7.5 with NaOH. The molar ratio of iodine to protein is 7, or approximately 2 per sulfhydryl group. I n the studies of Jensen, et a1.,2with serum albumin the molar ratio was 2.6.
I
Methods Viscosity.-The viscosity and other properties of the heated @-lactoglobulin solutions were measured without further adjustment of protein concentration, PH or ionic composition. The viscosity was measured in a Bingham type6 viscometer with a variable pressure head. The characteristics of this viscometer have been described previously.6 The results are recorded as relative viscosities, compared with water a t 30". The viscosities were determined with pressures of 15 to 50 cm. of water. The precision is &0.5%, with the principal error in the pressure reading. With relative viscosities up to about 3 the product of pressure (centimeters of water) and time (seconds) for a particular sample was constant. For higher viscosities the pressure-time product was not constant, indicating a yield point or structural viscosity. Solutions showing structural viscosity are indicated and viscosities are given for a pressure of 30 cm. of water. Heating.-The @-lactoglobulinsolutions were heated in a constant-level water-bath a t 90°, in 18 X 150 mm. Pyrex rimless test-tubes. Small-flanged test-tubes containing icewater were placed in the top of the large tubes to prevent the loss of water by evaporation. The volume of solution heated was 6.0 ml. and the time of heating 30 minutes. pH Values.-The pH values of the @-lactoglobulin solutions with all reagents present were measured a t 25", before and after heating. Light Absorbance.-Spectrophotometer readings were made in the same tubes used for heating, with a Beckman model B photometer, a t 600 mp. The apparent absorbance readings serve to measure the opacity or gross visible aggregation of the heated solutions.
6.0
6.5
7.0
7.5
8.0
PH.
Fig. 1.-Influence of PH on the viscosity of heated @lactoglobulin solutions, salt-free and with sodium phosphate and chloride present. The results given are for 1.8% 0lactoglobulin. The solutions containing 0.05 p NaC1 show slight structural viscosity a t low PH values. For example, at pH 6.4 the viscosity a t 30 cm. pressure is 3.26, a t 50 cm. pressure it is 5% less.
cosity in salt-free solutions below p H 6.8 shown in Fig. 1 is accompanied by a sharp increase in apparent absorbance, that is, aggregation, as shown in Fig. 3. The presence of phosphate reduces the Results Effect of p H and Salts on Viscosity of Heated aggregation in this p H range. Sodium citrate p-Lactoglobulin Solutions.-The effect of p H tested a t PH 6.2 kept the heated solutions as clear as on the viscosity of heated 1.8% @-lactoglobulin did phosphate. With sodium chloride, on the solutions was determined in the p H range of 6.2 to other hand, the aggregation a t low p H values is al7.7. The results are shown in Fig. 1 for systems most as great as in salt-free solutions, as is also true containing sodium chloride or phosphate. The of sodium sulfate. The aggregation, measured by the absorbance, phosphate data are presented a t constant ionic strength ( p ) rather than molarity, for comparison in heated 1.8% P-lactoglobulin solutions with a with the sodium chloride results. The viscosity range of phosphate concentrations is shown in Fig. increases sharply a t low pH values when phosphate 4,as well as the results with sodium citrate a t p H is present. Sodium chloride on the other hand a t 7.5. At low p H values the aggregation, compared low concentrations increases the viscosity at all p H to salt-free solutions, is decreased by small convalues to about the same extent, but a t higher con- centrations of phosphate, although the viscosity centrations the greatest increase in viscosity is increases (Fig. 1). Sodium citrate also has a like found a t the lower p H values. Sodium sulfate be- effect. Effect of Oxidizing and Reducing Reagents on haved similarly; a t a concentration of 0.01 M the (0.03 p ) the viscosity curve closely paralleled the Heated ,&Lactoglobulin Solutions.-Treating /3-lactoglobulin with iodine did not change its be0.025 p sodium chloride curve. The effect of higher concentration of phosphate havior greatly. Solutions heated a t p H 7.5 in on viscosity with several concentrations of p-lacto- 0.2 p phosphate, however, had a relative viscosity globulin is shown in Fig. 2. In this case reduced about 2570 greater than the untreated, and were viscosity (7 rel. l/concn. of protein) is plotted considerably clearer (Fig. 4). The presence of low against concentration of ,&lactoglobulin for pH 7.5 concentrations (0.01 M ) of the sulfhydryl comand 6.2. It is apparent that the viscosity is not only pound mercaptoethanol when p-lactoglobulin soludependent on phosphate concentration and p H but tions were heated had no influence on the viscosity is also sharply dependent on the protein concen- or clarity of the solutions. With high concentratration, particularly a t high salt concentration and tions of this and several related compounds, howlow PH values. The viscosity a t p H 7.5 is de- ever, there were striking increases in the viscosity pressed slightly by low concentrations of phosphate, as shown in Fig. 5. Most of these solutions show structural viscosity, that is, the apparent viscosity an effect that is also shown by citrate. Effect of pH and Salts on Absorbance of Heated changes with the pressure, and in these instances @-Lactoglobulin Solutions.-The decline in vis- apparent viscosities are given for pressures of 30 cm. The magnitude of this effect was evident from (6) E. C. Bingham a n d R. F. Jackson, Bur. Stds. Sc. Paper h'o. 298, 1917. a plot of volume/time against pressure for the solu-
-
CHARLES A. ZITTLE AND EDWARD S.DELLAMONICA
128 I .o
0.8
1
Vol. 70
I I I
I I I I
I
I I
I
/
I I
'
0.4
n
i
1
W
0
1 0
I I
2
3
A - L A C T O G L O B U L I N%,.
IONIC
Fig. %.-Reduced viscosity of heated /3-lactoglobulin wlutions as a function of protein concentration, sodium phosphate and pH The sodium phosphate concentration is indicated on the graph (at pH 7.5, p = 2.8 X Jf; at pH 6.2, p = 1.7 X 31). Data given by the solid lines are for p H 7.5, the dashed lines are for pH 6.9.
STRENGTH
()I).
Fig. 4.-Influence of ionic strength on absorbance of heated &lactoglobulin solutions at several pH values Concentration of P-lactoglobulin is 1.87,. n'here not indieated the solutions contain sodium phosphate.
M E R CAPTOETHA NOL
MERCAPTOACETIC
ACID
1.8 % / j
CY S T E I N E
0 6.0
6.5
7.0
7.5
PH, Fig 3 -Influence of PH 011 absorbance of heated p-lactoglobulin solutions, salt-free and with sodium phosphate and chloride present.
tion containing 0.24 -Idmercaptoethanol which gave a yield point (intercept of the pressure ordinate) of 15 cm. 9 t GO ctii. the value for volume/tinic wa5 0.04. Comparison of the viscosity of 0.9 ant1 I .h$$ $-lxtoglobuliii solutiou sliows ail extreme tic1 ) v i i t l r i i c . c . 011 the concciitrcitioii of the protein.
0.I REAGENT,
0.2 MOLES/LITER.
Fig. 5.--Viscosity a t 30 cni. pressure of heated @-lactoglobulin solutions containing various reducing agents at p H 7 . 5 . All the solutions, except those containing cysteine and Na2SO8, show structural viscosity. This is marked for the more viscous solutions. For example, the apparent viscosity of 1.8%&lactoglobulin containing 0.24 ,W mercaptoethanol is 33% less a t 50 ciii. pressurc.
'l'liese solutiolis are clew, with absorb:tnce readings of 0.1 or less. 'They also becoinc iiiorc viscous wlieri sodiuin pliosph;ttt* is present; ?(,,A p-lacto-
Jan. 5 , 1957
VISCOSITYAND OPACITY OF HEATED P-LACTOGLOBULIN
129
phosphate (Figs. 1 and 2) and citrate is not clear. I t too may be due t o the binding of the polyvalent anions for all concentrations of sodium chloride a t this p H increase the viscosity. Heated salt-free solutions of p-lactoglobulin aggregate a t the lower pH values, as shown by the great increase in absorbTABLE I ance (Fig. 3 ) . Simultaneously a reduction in visISFLUESCE OF MERCAPTOETHASOL AND SODIUM PHOSPHATEcosity occurs which may indicate that the aggreON VISCOSITYOF 0.9% ~-LACTOCLOBULIS SOLUTIONS AT pH gates are compact and symmetrical. Aggregation 7.5 HEATED WITH THE REAGENTS PRESENT of 6-lactoglobulin heated in the presence of low conMercaptoSodium phosphate centrations of calcium chloride also is accompanied ethanol, 41 'Sane 0.025 M 0 050 .M Relative viscosity (30 cm. pressure) by a reduction in viscosity5 and calcium chloride Sone 1.07 1 13 1.07 both aggregates and reduces the viscosity of un3.1 3 1 0.12 2.1 heated casein solutions (unpublished studies). 3.1 4.7 0.24 2.6 The use of reagents that oxidize or combine with the sulfhydryl groups of proteins has shown that Discussion these groups participate in protein gel for ma ti or^.^^^ The viscosities of heated protein solutions, in In the case of heated proteins2 the gels are clearer general, are very much greater than the viscosities and firmer after treatment with sulfhydryl reagents, of native protein solutions, particularly when salts leading to the conclusion that the sulfhydryl groups are present (compare Fig. 2 ) . The heat not only participate in lateral association with opaque gel alters the shape of the protein molecule by de- formation. Other groups, perhaps principally hynaturation with concomitant viscosity increases, drogen-bond forming, form the three-dimensional but cross reactions between the protein molecules clear gels. The influence of iodine treatment on affected by the heat are increased by the presence p-lactoglobulin gels fits this picture, although the of salts and are greatest near the isoelectric point. effects appear to be less striking than with heated These cross reactions increase the viscosity. The plasma albumin.? present studies of the effect of $H with heated pTreatment of p-lactoglobulin with a sulfhydryl lactoglobulin solutions containing phosphate are compound like mercaptoethanol, in slight excess of in general agreement with the behavior of human equivalence, had no detectable effect on the visand bovine plasma albumin reported by Jensen, cosity or appearance of heated p-lactoglobulin s o h et al.? The extent of the cross reactions which tions. When excess amounts of these and similar lead to viscosity increases is viewed2 as limited by reagents were added, as shown in Fig. 5, large inthe like electric charges on the molecule since these creases in viscosity resulted and the solutions relead t o electrostatic repulsion between molecules. mained clear. Presumably with excess of the reThe present results show that, as would be ex- agent, loops of the p-lactoglobulin molecule formed pected, salts increase the viscosity, presumably by by disulfide (four disulfide and four sulfhydryl reduction of electrostatic repulsion with concomi- groups are present in ~-lactoglobulin7) were opened tant increase in cross reaction. This is observed to give a greatly elongated molecule, as Kauzmann, with sodium phosphate, but only above concen- et aZ.,* have postulated for the effect of sulfhydryl trations of 0.02 X (Fig. 2 ) , and for sodium chloride compounds on serum albumin in urea. The effect a t all concentrations (Fig. 1). The maximal effect with heated p-lactoglobulin is much more striking of salts is exerted where the charge is smallest, that than for serum albumin in urea, and the viscosity is, toward the isoelectric region. There are, how- is sharply dependent on the concentration of pever, specific salt effects particularly apparent a t lactoglobulin. Greater increases in viscosity might low concentrations (
