THE BEHAVIOR O F DEAMINIZED GELATIN* BY
z. c. LOEBEL**
According to the modern view, a protein is an amphoteric electrolyte capable of combining with hydrogen ion a t the terminal amino groups and a t the polypeptid linkages; and capable of neutralizing hydroxyl ion a t the terminal carboxyl groups and at the polypeptid linkages. Recent investigations, 1,*9 have pointed to the close relationship between the properties of the proteins and their state of combination with acid and base. The study of many of the properties of gelatin as a function of hydrogen ion concentration indicates a point of abrupt change occurs at its iso-electric point, pH 4.7. Thus at pH 4.7 there is a minimum of solubility, osmotic pressure, swelling, viscosity a t 2 5 O C . , poiential difference, optical rotatory power a t and above 27i°C,and absorption of light; a maximum is found a t this point for foaming and optical rotation below 27+OC. In 1 9 2 2 Wilson and Kern*found that the swelling of buffered gelatin solutions gave a swelling-pH curve with a second minimum a t p H 7.7. h second point*** of abrupt change has been found for other properties too; and the work of Davis and Oakes4 indicates a shift of the minimum for viscosity a t 4o°C from 4.7 a t 2 5 O C to about 8.0. It was thought that an investigation of the behavior of gelatin, on whose molecular structure the free amino groups had been replaced by less reactive hydroxy groups might throw further light on the physical chemistry of the proteins. Such an alteration of the structure of the gelatin molecule should change its chemical and physical properties in a definite and measurable manner. Materzals Used I n all the experiments “Putmann’s Silver Label” gelatin was used as a source of ordinary and deaminized gelatin. The sodium nitrite, glacial acetic acid, ammonium sulphate, sodium hydroxide and hydrochloric acid used were of the ordinary C. P. variety and the two dyes for the isoelectric point experiment were used as received from the manufacturer. The Preparation of Deaminized Gelatin**** Introductory When a protein such as gelatin is treated with nitrous acid thereisanevolution of nitrogen, presumably from the reaction of the nitrous acid with the *Contribution from the Depai tment of Chemistry, Columbia University, S o . 565. Premeeting of the American Chemical Society. sented a t the Philadelphia (1926) **This communication is an abstract of a thesis submitted by the author in partial fulfillment of the requirements for the degree of Doctor of Philosophy, Columbia University. *** The two points always occur as geminated points: either both are maxima o r both are minima. *I* The * term “deaminized gelatin” as hereinafter used, refers to the gelatin which has undergone the nitrous acid treatment.
764
2. C. LOEBEL
amion groups of the protein, the amino nitrogen being replaced by hydroxy groups. This reaction forms the basis of the Van Slyke method for the quantitative estimation of amino nitrogen. Nitrous acid was first used as a deaminizing agent for proteins in 1885 by Loew.6 Skraup’ and his co-workers have been largely responsible for perfecting the methods used in preparing deaminized proteins. In 1914 Blase1 and Matulas deaminized gelatin according to Skraup’s method and showed by hydrogen electrode.measurements that the deaminized protein was still capable of combining with hydrochloric acid. Recently Hitchcocke has published a quantitative study of the acid-combining capacity of ordinary and deaminized gelatin. He prepared the deaminized product by Skraup’s method, but omitted the heating on the water-bath, a preliminary study indicating that the higher temperature induced a slight hydrolysis. Analysis of Hitchcock’s preparation showed an exact agreement between the difference of the total nitrogen for ordinary and deaminized gelatin, and the amino nitrogen removed in the Van Slyke analysis; in each case o.oo040 equivalents of nitrogen per gram. Furthermore, the difference between the maximum combining capacity of the ordinary and the deaminized products for hydrochloric acid was nearly equivalent to the loss in amino or total nitrogen in the dearninization.
Procedure The deaminized gelatin was prepared after Hitchcock’s m e t h ~ dbrought ,~ to pH 4.0, dehydrated with alcohol, ground in a pebble mill, and passed through a 60-mesh sieve. The final product was left exposed to the atmosphere for a day to allow it to reach equilibrium with atmospheric moisture. I t was a canary yellow, fibrous, solid and swelled in cold water like gelatin. Analysis of the final product for total nitrogen by the Kjeldahl method gave 17.38 per cent while the original gelatin contained 17.96 per cent; a loss of 0.58 per cent or 4.1 X IO-^ equivalents per gram absolutely dry weight. Ordinary iso-electric ash-free gelatin was prepared according to the method of Loeb,’ ground in a pebble mill for 2 4 hours, passed through a 60 mesh sieve and left exposed to the atmosphere for a day to reach equilibrium with the atmospheric moisture. The moisture in all samples of ordinary and deaminized gelatin was determined by heating in a 105’ oven over night and all weights are reported on this moisture-free basis. Determination of the Iso-electric Point of Deaminized Gelatin by the Dye Technique Loeb’ has shown that protein can combine with cations only on the alkaline side of its iso-electric point, and with anions only on the acid side. Hence deaminized gelatin should combine with the colored cation of a basic dye on the alkaline side, and the colored anion of an acid dye on the acid side of its iso-electric point. The iso-electric point as determined by the dye technique for gelatin, collagen and deaminized collagen shows good agreement with the values obtained by other methods.
765
THE BEHAVIOR O F DEAMINIZED GELATIN
TABLE I Determination of Iso-Electric Point of Deaminized Gelatin Acid Black
Fuchsin
Deep Blue "Green "Green (slight) 'Yellow
'Yellow 'Yellow Reddish Red (deep)
pH of solution
3.6 3.8 4.0
4.2
'-color of deaminized gelatin. "-due to yellow of deaminized gelatin plus the blue of the dye.
Employing the dye technique as used by Thomas and Kellyz8for collagen, the results given in Table I were obtained with Acid Black as acid dye and Fuchsin as basic dye. These results indicate the iso-electric point to be a t pH 4.0, checking the value obtained by Hitchcockg for minimum of osmotic pressure. Method of eflecting Solutions It was found that, because of the color and the turbidity of the solutions, a concentration below 0.5 per cent would be necessary in order to make optical rotation readings. It was planned to compare the different properties a t a constant concentration and after a preliminary study, 0.4760 per cent was found most satisfactory. The solutions were made up as follows: T o 0.4760 gms. of sample in a I O O cc. standard flask, I O cc. of distilled water were added, then varying amounts of acid or alkali and finally enough distilled water to reach the IOO cc. mark. After remaining at room temperature for one hour to allow the sample to or a t 75°C for five swell, the flask was placed in a water-bath a t either SOT minutes, removed, inverted slowly five times and replaced in the same bath for fifteen minutes. The flask was then kept in a 2s0C water-bath for ten minutes and was ready for use. All pH values recorded in this report were obtained with the aid of a Raturated KCI-calomel half cell junction with the solution being effected through a saturated salt bridge. pH was calculated from the equation, pH = (E 0.2466) 1'0.000198T. Measurements were made a t room temperature, which was recorded in each case. Viscosity of Deaminized Gelatin I t will be recalled that Loeb's' viscosity-pH curves of gelatin a t 2 j"C indicate a minimum of viscosity a t the iso-electric point, pH 4.7. The viscositypH curves of Davis, Oakes and Brown3 a t zg°C indicate no minimum a t pH 4.7 while the viscosity-pH curves of Davis and Oakes4 a t 40% give no minimum at 4.7 but give one a t about 8.0. Loeb's solutions were effected by heating to 45'C for ten minutes. HitchcockI4 found the viscosity-pH curve of gelatin a t 40°C (solution effected by heating to 4oOC) to be similar to that obtained by Loeb, with a minimum a t pH 4.7. Blase1 and Matulas measured the viscosity of deaminized gelatin but no valid conclusions can be drawn from their results because their observations were made at such wide intervals of pH.
766
2. C. LOEBEL
Two groups of experiments on viscosity of deaminized gelatin were performed; in the first the solution was effected at 5o°C and in the second the solution was effected at 75°C. In the first group, j cc. of each solution was pipetted into three Ostwald viscosimeters which were placed, respectively, into water-baths regulated a t IO'C, 2s0C and 50°C ( &O.I"C), and after fifteen minutes the viscosities were measured. In the second the same procedure was followed except that the determinations at 10°C were omitted.
TABLE I1 Viscosity of Deaminized Gelatin Solutions at Varying Temperatures and Hydrogen Ion Concentrations Concentration of deaminized gelatin--o.4760% Solution effected at 50°C pH
V~PC
VIUOC
4.6
2.69; 2.746 3.222 3.316 3.199 2.871 2.692 Turbid 2.866 3.104
5.0
5.7 6.2
1.4 1.9 2.7
2.9 3.3 3.6 3.9 4.0
4.3
~janc
0.i91j 1.542 0.9123 1.791 1.036 1.836 1.050 1,654 0.9538 1.452 0.8578 1.312
1.217
0.7251
Turbid Turbid 1.240
0.7275
1.427
0.8282
3,457
1.775
1.055
3.765 3.800
2.309 2,396
1.456
1.322
pH
VZS'C
VIO'C
6.5 6.9 7.3 7.6 7.9 8.4
3.849 3.801 3.770 3.771 3.774 3.698
8.8
3.511
9.1 3.365 9.6 3.207 10.4 3.055 10.7 2.968 I I .6 2.384
2.448
VSOOC
1.506 1.436 1.403
2.435 2.406 2.418 1 , 4 1 7 2.442
1.421
2.397 2.239
1.405
2.127
2.045
1.334 1.256 1.175
1.970 1 . 1 2 0 1.9j8 1 . 1 1 8 I ,440 0.8863
TABLE I11 Viscosity of Deaminized Gelatin Solutions at Varying Temperatures and Hydrogen Ion Concentrations Concentration of deaminized gelatin-0.47607~ Solution effected at 7 5 " PH
VZSOC
VSOOC
PH
V?SCC
1.5
I. 198
0.7251
6.j
2.002
2.3
1,357
0.8128
7.0
2.9 3.3
1.411
0.8389 0.8199 0.7465 0.7038 0.7276 0.7998 I ,066
7.3 7.4
1.978 1.965 1,995
10,3
I . 113
11.0
3.7 4.0 4.4
1.367 1.264 1 . I79 1.269
5.5
I ,361 I . 787
5.9 6.2
1.987
4.7
I
,872
I.
190
7.9 8.3 9.2 9.9
2,000
v500c
I ' I99 I. 185
I . 169 I . 182 I . 185
1.933 I ,861 1.754
1.175
1 .7 5 2 I .520
1.051
I . 118 1.050
0.9123
767
T H E BEHAVIOR OF DEAMINIZED GELATIN
The results are given in Tables I1 and 111 and plotted in Fig. is reported as "Viscosity Ratio" Vt:
Vt =
I.
The viscosity
Time in seconds at temperature t for deaminized gelatin Time in seconds at 2 5 T for water
33 3.6
3.4 3.2 3.0
2.8
.-
2.6
i-
\ -4
i2
2 2.4
.-*x g 2.2 8
5 2.0 1.8 I .6
1.4
I .2 1.0
.8 .6
o
1
2
3
4
5
6 PH
7
8
g 1 o 1 1 1 2
FIG.I 0.4760% solutions of: .Deaminized Gelatin-Solution effected at SOT. Vicosity at (a) I O T , (b) 25"C, ( c ) S O T *Deaminized Gelatin-Solution effected a t 75°C.Viscosity at (d) 25t C, (e) 50°C oGelatin-solutioh effected at 75°C. Viscosity a t ( f ) 25OC, (9)SOT
Although turbidity a t pH 4.0 of solutions effected a t so°C permitted no viscosity measurements a t this point, the direction of the curves on both sides shows a distinct tendency to locate the minimum a t this point. It will be noted further that: (a) Each curve, whether the solutions were effected a t 50°C or 75"C, or whether the viscosities were determined at IOT, ns°CJ or so"C, is similar in shape.
Z. C. LOEBEL
(b) Each has a minimum at its iso-electric point, pH 4.0 (c) I n each the rise on the alkaline side is greater than on the acid side. (d) Each has a second minimum a t pH 7.3. (e) Each has maximums a t pH 2.9,6.5and 7.9. In Table IV and Fig. 2 are given the results of a series of experiments with gelatin solutions a t 2 5°C and jo"C,which were of the same concentration as those of the deaminized gelatin described above (0.4760%). The solutions were effected a t the same technique as used for the viscosities described being employed. These curves and the curves found by Loeb a t 2 5 O C and by Hitchcock at 40°C show many points of similarity with those of deaminized gelatin: (a) Each of the gelatin curves is similar in shape, independent of temperature of effecting the solution. (b) Each has a minimum a t the iso-electric point. I n the deaminizing reaction amino groups are replaced by less basic hydroxy groups, and it is thus expected that with deaminization there should be a shift of the isoelectric point, to the acid side, as does occur.
TABLE Is' Viscosity of Gelatin Solutions at Varying Temperatures and Hydrogen Ion Concentrations 0.4760% gelatin solutions Solutions effected a t j 5 O C PH
VSOOC
VZSOC
2.2
1.653
2.8
I.
3.3 3.6 4.4 4.6 4.7
I . 820
759 741)
I
'
I
,361
PH
0.9348
5.0
,041
VSO'C
0.7204
5.7
1.446
0.8152
I . jr7
0.8898
,005
7.0 8.8
1.549
0,9135
0.8057
9.6
565 1.612 I . 646
0.9240
I
I. 062
r
I . 282
0.7500
10.2
I57
0.6718
10.9
I,
VZS'C
I . 226
I'
0.9502 0.9739
(c) With gelatin the rise on the acid side of the iso-electric point is greater than that on the alkaline side; the reverse is the case with the deaminized gelatin. Comparing the 25'C and 5o°C curves, it is seen that deaminization does not affect the viscosity a t the iso-electric point, but lowers it on the acid side and raises it on the alkaline side; the rise on the alkaline side being very steep. Thomas and Foster15 report a similar steeper rise of swelling with deaminization of collagen. They suggest as a possible explanation that the hydroxy groups which have substituted the amino groups are acidic in character. This would result in the formation of a greater amount of sodium salt on addition of sodium hydroxide and thus cause greater swelling. The same explanation may be applied to account for the anomalous behavior of. viscosity with deaminization of gelatin. Einstein's theory' holds that the viscosity is a linear function of the relative volume occupied by the
THE BEHAVIOR O F DEAMINIZED GELATIN
769
solute in the solution. The volume size of the protein solute, according to Loeb, would be dependent upon the swelling and osmotic forces which are governed by the Donnan Theory. It has been shown by Blase1 and Matulaj and by H i t c h c o ~ k that , ~ deaminized gelatin combines with less hydrochloric acid than does gelatin. Then, following Loeb's theory, deaminized gelatin should have a lower viscosity than gelatin on the acid side, as is actually the case. I n a later section a greater base-combining capacity is shown for the deaminized product, thus accounting for the greater viscosity on the alkaline side. (d) The maximum on the acid side of the iso-electric point for gelatin is a t pH 3.2, while that for the deaminized is a t pH 2.9, indicating again a shift to the acid side. (e) The second minimum for deaminized gelatinis a t pH 7.3 ; that of gelatin is a t 7 . 7 ; showing still another shift towards the acid side. A more complete discussioxi of the second point of abrupt change must be left to a later section. Optical Rotation of Deaminized Gelatin C . R. Smith postulated that there is a close relationship between the mutarotation of gelatin and its power to jellify. Prolonged heating on a waterbath causes a gelatin solution to lose both its power to jellify and to mutarotate. Kraenier and Fanselow'* studied the optical rotation of gelatin solutions of varying hydrogen ion concentration at different temperatures, using the mercury light as a source of illumination. Their results of optical rotation as a function of pH show a minimum both a t pH 4.7 and a t about pH 8.0 for temperatures of 27ioC and above, and show a maximum a t the same points for temperatures below z i + " C . I t was planned to study the optical properties of deaminized gelatin and to compare them with those of ordinary gelatin. TABLE V Optical Rotation of Deaminized Gelatin a t Varying pH and Temperatures PH 1.4
Kegative Ventzke reading a t 10°C 2fC 50°C 7.1
1.9
7.1
2.7
6.2
2.9
3.3 3.6 3.9 4.0
4.3 4.6 j .O
5.7 6.2
6.2 6.7 7.3
4.3 4.5 4.8 4.8 4.6 4.7
6.5
Segative Ventzke reading a t 10°C 2joC jo'C
6.9
5.3 5.4
7.3
5.1
4.5 4.6 4.6
7.6 7.9
5.1
4.7
5.3
4.6 4.6
8.4 8.8
5.2
9.1
5.3
4.8 4.8
4.8 4.8
9.6 10.4
5.0
4.6
5.3
4.8 4.7
10.7
5.0
4.5 4.6
11.6
5 . 1
4.5
turbid turbid turbid turbid 7.7 7.6 6.5 5.4 5.3
PH
4.5
5.2
4'4 4 4
4.3 4'3 4.3 4'3 4.3 4 3 4.1 4.4
4.3 4'4
Z.
770
C. LOEBEL
Procedure The solutions for the optical rotation experiments were prepared as described above, the solution being effected a t 5o°C. The solutions were polarized in 2 . 2 dcm. jacketed tubes, water respectively a t I o O C , z 5 O C and jo°C ( i 0 . 3 C ) being pumped through the jackets. After 15 minutes a t the proper temperatures, readings were taken. The instrument used was a Schmidt and Haensch saccharimeter, with a dichromate filter. White light was used as a source of illumination. The direct readings (negative degrees Ventzke) of IoOC, 25°C and jO°C are tabulated in Table V and plotted as a function a t pH in Fig. 2 .
-a -7
-4
0
I
2
3
4
5
6
7
8
9
1 0 1 1 1 2
PH FIG.2 Effect of temperature and p H on the optical rotation of 0.4760 per cent solution of deaminized gelatin. (a) = 10°C tb] =Zj"C (c) = j0"C
Because of the turbidity and color no readings could be made at pH 4.0. The curve for 10°C readings shows a minimum a t pH 2.9 and a tendency to form a maximum as the pH approaches 4.0. On the alkaline side of pH 4.0 the rotation falls off with increasing pH. At zs0C and 50°C the rotation is almost constant for each respective temperature. Unfortunately, the color and turbidity do not permit the use of higher concentrations which might magnify any deviations, and detect any mutarotation.
Surface Tension and Foaming of Deaminized Gelatin Like gelatin, deaminized gelatin solutions foam on shaking. The adsorption film theory advanced by Bancroft is founded on Gibbs' statement that any substance which lowers the surface tension of a liquid must concentrate in the surface, thus forming a film and preventing the coalescence of the gas bubblcs. Accordingly, gelatin and deaminized gelatin should foam most
THE BEHAVIOR OF D E A M I S I Z E D GELATIS
771
a t the points where the surface tension is least. BogueZnreports that the foaming of gelatin is a maximum a t its iso-electric point. JohlinZ6finds its surface tension is a minimum a t this point.
Surface tension Surface tension measurements were made with the Du X0iiyZ7tensiometer. The solutions, of 0.4j60 per cent concentration, were made up by the same
o
I
2
3
4
5
6
7
8
g 1 0 1 1 1 z
PH FIG.3 Effect of pH on the surface tension of 04760 per cent solution of deaminized gelatin and effect of pH on the foaming of 0.4760 per cent solutions of gelatin and deaminized gelatin.
technique as described above, solution effected a t 5o°C. Two cc. of each solution were pipetted into respective 4 cm. watch crystals and left a t room temperature for one hour. During this hour it was covered with an inverted Petri dish. Because of the exposed surface of the alkaline solutions it can not be claimed that the p H values are accurate. However, they are not sufficiently far removed to prevent the indication of the general direction of the surface tension change with pH change. The usual precautions of flaming the pipette, watch crystals and platinum ring were taken. The apparatus was calibrated against water at 23OC giving the value of 73 dynes per cm. The results, given in Table VI and plotted in Fig. 3 , indicate a minimum a t p H 4.0, a maximum a t pH 2.9, which remains constant with further decrease of pH; and a maximum a t about pH 6.5, which remains constant with further increase of pH.
2 . C. LOEBEL
772
TABLE VI Surface Tension and Foaming of Deaminized Gelatin a t Varying Hydrogen Ion Concentration PH 1.5 2.3 2.9
3.3 4.0
Surface Tension a t 23' C Dynes/cm. 61.8 61.8 61.8 61.5 57.2
Foaming a t 23' C cm. 1.4 1.9 I .6 1.8 2.3
PH
Surface Tension a t 23' C dynes/cm.
4.7
58.7
5.5
62. I 66.3 65.2
6.j 7.4 10.3
The foaming experiments were performed with the same solutions. Ten cc of each solution was added to respective 15 cc graduated test tubes and shaken all together, and the volume of foam read off. The results are recorded in Table VI and plotted in Fig. 3. They show a maximum a t pH 4.0 and a minimum a t pH 2.9. The curve obtained by Bogue is inserted for comparison. Although absolute values cannot be compared here, the general trend of the two cuwes show several interesting differences. Both curves show a maximum a t their respective iso-electric points, but the gelatin curve has a second maximum on the alkline side, while the deaminized gelatin has the corresponding maximum on the acid side. It will be noted that the surface tension in each case is lower than that of water and that the greatest lowering point of the surface tension curve is a t the iso-electric point and corresponds with the highest point of foaming. These results are in conformity with the adsorption film theory, and are similar to those of Bogue and of Johlin for gelatin. Titration of Gelatin and Deaminized Gelatin with Sodium Hydroxide I n a preceding section it was pointed out that the increase of viscosity on the alkaline side of the iso-electric point with deaminization is very likely due to the acidic nature of the hydroxy groups'j that replaced the amino groups. If this is true, the base-combining capacity of deaminized gelatin should be greater than that of gelatin by an amount equivalent to the replacing hydroxy groups. To obtain comparable results both gelatin and deaminized gelatin were titrated. Samples equal to 0.4617 gm. absolutely dry weight were used; varying aniounts of O.IOIZNsodium hydroxide added and the pH determined electronietrically. A single sample was employed for each titration curve, the original volume being 25.30 cc. Preliminary results indicated that to adjust the p H of gelatin and deaminized gelatin to p H 7.0 required respectively 1.43 cc and 3.01 cc of the sodium hydroxide used. To keep the volumes at pH values above 7 . 0 more nearly constant, 1 . 5 8 cc (the difference between 3.01 and 1.43) of water was added to the gelatin sample. The results are given in Table VI1 and plotted in Fig. 4.
773
THE BEHAVIOR O F DEAMINIZED GELATIS
TABLE VI1 Titration of Gelatin with NaOH Weight of gelatin sample 0.4617 gm. (dry weight). cc. O . I O I 2 N cc. 0.1s Volts NaOH NaOH E.1R.I.F. added required'
pH
0.j1g1
4.58
0.15
0.0
0.5216
4.70
0.55
0.40
0.545j
5.11
0.95
0.81
0.j721
5.56
0.0
cc. O . I O I 2 S cc. 0 . I X NaOH KaOH added required* 1.75 1.62 1.90 1.77 2.45 2.33 3.25 3.14 3.95 3.85
Volts E.M.F. 0.8722
0,8737
0.8755 0.9231 0.9412 0.9joo 0.9543
pH 8.98 9.69 10.j4 11.56 11.86
0.5843 5.j7 0.627'$ 6 . 5 1 5.15 5.06 12.01 1.55 1.42 0.6996 j.74 8.15 8.10 12.09 1.52 0.7406 8 . 4 4 1.65 'cc of exactly o.rT TaOH required to adjust pH from 4.70 to that recorded in the last column. 1.15
1.01
1.35
1.21
TABLE VI11 Titration of Deaminized Gelatin with NaOH Weight of deaminized gelatin sample 0.461 j gm. (dry weight). cc. 0.1012s cc. 0 . I N NaOH XaOH added required'
cc. O . I O 1 2 X cc. 0 I K XaOH Na,OH added required' 0.4806 4.00 - 0 . IO** 0.0 3.20 3.35 0.0 3.69 0.10 0.4834 3.55 4.05 4.15 0.20 4.09 0. I O 4.00 0.4860 0.j0 0.81 0.5052 4.42 4.20 4.35 1.51 4.6; 1.63 0.5340 4.91 4.50 2.00 5.15 2.12 0.5533 5.24 4.99 0.sj4j 5.67 2.51 2.64 5.60 5.50 2.70 2.83 0.6010 6.06 7.50 j.69 2.80 2.93 0.6065 6.15 10.50 10.73 3.10 3.24 0.6718 7.45 14.50 14.77 *CCof exactly 0 . 1 s S a O H required to adjust pH from 4.00to that column. * * 0.10 cc 0 . 1 s HCl required. Volts E.M.F.
pH
Volts E.M.F.
0,7540 0.8330 0.8912 0.8993 0.9100 0.9202 0,9311 0.9421 0.9jOI 0.9561 recorded in
pH 8.67 10.02 11.02
11.15
11.34 11.51 11.70
11.89 12.02
12.13 the last
Examining the slopes of the first part of each curve, rising from their respective iso-electric points, it is seen that the slope of the curve for the deaminized sample is steeper. This first portion of the curve for the deaminized gelatin rises to a higher value than that of the gelatin, indicating that the hydroxy groups begin to combine with sodium hydroxide directly beyond the iso-electric point. The sudden rise in viscosity directly beyond the iso-electric point is thus accounted for. There is a change of slope a t p H 7.5 for the deaminized gelatin which corresponds to a similar point for the gelatin a t pH j . j ; it is interesting that pH 7 . j corresponds to the second point of abrupt change of the properties of gelatin.
774
2. C. LOEBEL
At pH 7.5, 0.4617gm of deaminized gelatin combines with 3 . 2 4 cc 0.1X sodium hydroxide. A t pH 7.7, 0.4617gm of gelatin combines with 1.40cc O.INsodium hydroxide. The difference is 1.84cc O.IXsodium hydroxide. For I gm this difference is 4.0 cc O.INsodium hydroxide or 4.0 X I O - ~ equivalents.
0
1
2
3
4
5
6
7
8
9
1
0
1
1
1
2
1
3
PH FIQ.4
Titration Curves for 0.4617 gms of originally isoelectric: a) 'Gelatin and b) "Deaminized Gelatin with N/IO NaOH
The Kjeldahl nitrogen of the gelatin deaminized gelatin difference equivalents nitrogen per gm. showing very good agreement with the difference of their base-combining capacity.
THE BEHAVIOR O F DEAMINIZED GELATIN
775
Examining the slope of the two curves beyond pH of I I , it may be seen that there exists a constant difference of about 4.0 X IO-^ equivalents of sodium hydroxide per gram dry substance. This may be construed to mean that the base-combining capacity for the deaminized gelatin is equal t o that of gelatin plus 4.o X 104, Taking the value of the base-combining capacity of gelatin as found by Loebll and Hitchcock10 a t 5.7 X IO-^, that for deaminized gelatin would be 9.7 X IO-^ equivalents per gram.
The Second Point of Abrupt Change Several properties of gelatin, studied as a function of the hydrogen ion concentration, give curves of the same type as the viscosity curves. Some of these properties have been studied in the unbuffered range. Wilson and Kern2 found a second minimum for swelling of buffered gelatin at pH 7 . 7 . Higley and Mathews found a second minimum a t the same point for absorption of light. Kraemer and Fanselow12 and also Velles and VellingeP found a second point of abrupt change for optical rotation. There seems to be only four proteins which show these double geminated points. They are collagen, deaminized collagen, gelatin and deaminized gelatin. C. R. Smith7 postulated two forms of gelatin in solution, a “sol’’ form stable a t temperatures above 3 j°C and a “gel” form stable a t temperatures below I ~ O C ,while at temperatures between, the existence of both in equilibrium. The work of Smith and Lloydi7 shows that the change from “gel” to “sol” form takes place with increase of Sorensen value of the solution. Wilson and Kern suggested that the two forms had different iso-electric points, the “sol” form a t pH 7 . j and the “gel” form at 4.7; and that a preponderance of either one would determine the behavior. Support for this idea is found in the work of Thomas and Kellyi8~ 19. In studies of the rate of fixation of tannin by hide substance a t Toom temperature’* as a function of hydrogen ion concentration of the solutions, they showed that the degree of fixation at a maximum at pH dropped to a minimum a t pH 5 ; (the iso-electric point) rose again when a pH of about 8 was reached and then abruptly fell off with further increase in alkalinity. Since the tannin particles are negative, the decrease in rate of fixation from pH 3 to j was readily explained by the Procter-Wilson theory as due to the decreasing positive charge of the collagen. The rise in rate of fixation from pH j to 8 was unexplainable except on the basis of a shift of the iso-electric point. Thomas and Kelly1Qrepeating the same work a t 40°C obtained no minimum at pH 5 and conclude, therefore, that the collagen exists in two modifications analogous to the “sol” and “gel” forms of gelatin; the iso-electric points of the collagen being a t pH j and at 8. With gelatin the evidence is more conflicting. To begin with, the exact location of the transition temperature, “gel” to “sol,” is uncertain. C. R. Smith says it is at 3 j°C. Davis and Oakes place it a t 38.03”C and Kraemer and Fanselow place it a t 271’C. Bogue,20on the other hand, says it depends on the concentration, heat treatment, pH and previous history.
776
Z. C.
LOEBEL
Although Davis and Oalces report no minimum for the viscosity-pH curves of gelatin a t pH 4.7 a t 4ooC and do report one at pH 8.0, it cannot be said to represent a shift of the minimum due to the temperature. Davis, Oakes and Browne using the same technique as Davis and Oakes, found the viscosity-pH curves of gelatin a t 25’C gave no minimum a t 4.7 either. It was a t first thought that the difference in method of effecting solution employed by Davis and his co-workers might be the cause for the difference in results. The matter was gone over with Dr. Davis and he determined the viscosities of two series of gelatin solutions, one series being treated as in his two papers-by heating on a hot plate for 2 0 minutes with continuous stirring till the solution reached 75’; the other series being treated in a manner similar to that employed in this research. His results indicated that the same qualitative results were obtained by either treatment. The interpretation that has been placed on the work of Davis et al. has been that the niinimum of viscosity a t pH 4.7 a t zs0C shifts to pH 8.0 a t 4 0 T . Apparently the gelatin used by them did not have its iso-electric point a t pH 4.7 as did that of Loeb, Hitchcock and that used in this research. Hitchcock’s results for gelatin a t 4ooC and those reported in this investigation for gelatin and deaminized gelatin a t various temperatures indicate no change of minimum with change of “gel” to “sol” form. Loeb‘ postulated that the large changes of viscosity with pH of gelatin solutions at 2 5 T were due to two factors, the great magnitude of viscosity of gelatin solutions and the swollen micellae present. While such a theory is tenable enough for lower temperatures, it can hardly hold for higher temperatures] like jo°C. However, as a matter of pure speculation, it might be suggested the viscosity of gelatin is dependent upon a solbtion structurez1 which in turn is dependent upon swelling forces, and therefore, according to the Donnan Theory, dependent upon osmotic force. I n the viscbsity experiments of gelatin and deaminized gelatin, the solutions of which were effected a t 7j°C, no shift of minimum was observed. A preliminary experiment with gelatin solutions a t different pH, but in which the solutions were effected by heating a t 80°C for one hour, indicated no shift of the minimum. Although the above evidence indicates no shift of the minimum of gelatin and deaminized gelatin, there is every indication of the existence of the second point of abrupt change of properties. Wilson and Kern’s idea of this point as a possible iso-electric point of the “sol” form finds further support in the work of Miss Lloyd.]’ She found that the “gel”-“sol” equation, brought about with increase of pH is reversible if sufficient acid is added. She offers the following explanation : “Brailsford Robertson2* has suggested that acids and bases attached themselves to protein molecules a t the -COHN- linkage. He points out that this linkage may exist as an enol linkage
