THE IONIC ACTIVITY OF GELATIN

2.5 and 1.25 per cent gelatin have been titrated potentiometrically in the absence of salts and in the presence of two concentrations (0.0750 and 0.0375µ) of NaCl, MgCl₂, K₂SO₄, and MgSO₄. The data have been used to calculate values of ± S = v^z – (v – 1)^z, where v^z = v ² – (v ² – v) r_x/18. The maximum and minimum values of S with NaCl were used to calculate the mean distance (r_x) between like charges in gelatin. This is found to be 18 Å.u. or over (between acid or basic groups) which agrees with the probable value and the titration index dispersion. Thus the data with NaCl are shown to be normal and to obey the equation found to hold for simple weak electrolytes; namely, pK'' – pK = Sa See PDF for Equation where S is related to the valence and distance by the above equations. Using the NaCl data as a standard the deviations (ΔS) produced by the other salts are calculated and are found to agree quantitatively with the deviations calculated from equations derived for the simple weak electrolytes. This shows that in gelatin, as in the simple electrolytes, the deviations are related to the "apparent valences" (values which are a function of the true valence and the distance between the groups). The maximum "apparent valences" of gelatin are 2.4 for acid groups (in alkaline solution) and 1.8 for basic groups (in acid solution). These values correspond to the hypothetical condition of zero distance between the groups. They have no physical significance but have a practical utility first as mentioned above, and second in that they may be used in the unmodified Debye-Hückel equation to give the maximum effect of gelatin on the ionic strength. The true effect is probably even lower than these values would indicate. The data indicate that gelatin is a weak polyvalent ampholyte having distant groups and that the molecule has an arborescent structure with interstices permeated by molecules of the solvent and other solutes. The size and shape probably vary with the pH.     

SYNERESIS AND SWELLING OF GELATIN

1. When solid blocks of isoelectric gelatin are placed in cold distilled water or dilute buffer of pH 4.7, only those of a gelatin content of more than 10 per cent swell, while those of a lower gelatin content not only do not swell but actually lose water. 2. The final quantity of water lost by blocks of dilute gelatin is the same whether the block is immersed in a large volume of water or whether syneresis has been initiated in the gel through mechanical forces such as shaking, pressure, etc., even in the absence of any outside liquid, thus showing that syneresis is identical with the process of negative swelling of dilute gels when placed in cold water, and may be used as a convenient term for it. 3. Acid- or alkali-containing gels give rise to greater syneresis than isoelectric gels, after the acid or alkali has been removed by dialysis. 4. Salt-containing gels show greater syneresis than salt-free gels of the same pH, after the salt has been washed away. 5. The acid and alkali and also the salt effect on syneresis of gels disappears at a gelatin concentration above 8 per cent. 6. The striking similarity in the behavior of gels with respect to syneresis and of gelatin solutions with respect to viscosity suggests the probability that both are due to the same mechanism, namely the mechanism of hydration of the micellæ in gelatin by means of osmosis as brought about either by diffusible ions, as in the presence of acid or alkali, or by the soluble gelatin present in the micellæ. The greater the pressures that caused swelling of the micellæ while the gelatin was in the sol state, the greater is the loss of water from the gels when the pressures are removed. 7. A quantitative study of the loss of water by dilute gels of various gelatin content shows that the same laws which have been found by Northrop to hold for the swelling of gels of high concentrations apply also to the process of losing water by dilute gels, i.e. to the process of syneresis. The general behavior is well represented by the equations: See PDF for Equation and See PDF for Equation where P ₁ = osmotic pressure of the soluble gelatin in the gel, P ₂ = stress on the micellæ in the gelatin solution before setting, K_e = bulk modulus of elasticity, V_o = volume of water per gram of dry gelatin at setting and V_e = volume of water per gram of gelatin at equilibrium.     

HYDRATION OF GELATIN IN SOLUTION

1. It was shown that the high viscosity of gelatin solutions as well as the character of the osmotic pressure-concentration curves indicates that gelatin is hydrated even at temperatures as high as 50°C. 2. The degree of hydration of gelatin was determined by means of viscosity measurements through the application of the formula See PDF for Equation. 3. When the concentration of gelatin was corrected for the volume of water of hydration as obtained from the viscosity measurements, the relation between the osmotic pressure of various concentrations of gelatin and the corrected concentrations became linear, thus making it possible to determine the apparent molecular weight of gelatin through the application of van''t Hoff''s law. The molecular weight of gelatin at 35°C. proved to be 61,500. 4. A study was made of the mechanism of hydration of gelatin and it was shown that the experimental data agree with the theory that the hydration of gelatin is a pure osmotic pressure phenomenon brought about by the presence in gelatin of a number of insoluble micellæ containing a definite amount of a soluble ingredient of gelatin. As long as there is a difference in the osmotic pressure between the inside of the micellæ and the outside gelatin solution the micellæ swell until an equilibrium is established at which the osmotic pressure inside of the micellæ is balanced by the total osmotic pressure of the gelatin solution and by the elasticity pressure of the micellæ. 5. On addition of HCl to isoelectric gelatin the total activity of ions inside of the micellæ is greater than in the outside solution due to a greater concentration of protein in the micellæ. This brings about a further swelling of the micellæ until a Donnan equilibrium is established in the ion distribution accompanied by an equilibrium in the osmotic pressure. Through the application of the theory developed here it was possible actually to calculate the osmotic pressure difference between the inside of the micellæ and the outside solution which was brought about by the difference in the ion distribution. 6. According to the same theory the effect of pH on viscosity of gelatin should diminish with increase in concentration of gelatin, since the difference in the concentration of the protein inside and outside of the micellæ also decreases. This was confirmed experimentally. At concentrations above 8 gm. per 100 gm. of H₂O there is very little difference in the viscosity of gelatin of various pH as compared with that of isoelectric gelatin.     

FRACTIONATION OF THE EYE PIGMENTS OF DROSOPHILA MELANOGASTER

Eye pigments of normal and mutant types of D. melanogaster have been extracted with water and fractionated by chromatographic adsorption on powdered talc. Spectra of all the fractions obtainable in solution have been measured and the general chemical behavior of the pigments is described. Two chemically distinct groups of pigments are found, to be identified with the earlier designated red and brown components. The red component in the wild-type eye contains three well defined pigments, two of them capable of further subdivision so that the total number of fractions obtained is five. There is also present a brown component pigment which could not be treated quantitatively by the methods employed. All members of the wild-type red component are found in cinnabar eyes, unaccompanied by the brown component. Conversely, brown eyes contain a pigment indistinguishable from the wild-type brown component, virtually alone. In sepia eyes, one red component and two brown component pigments can be distinguished, all three pigments differing from those of wild-type eyes. Pigments apparently identical with those found in wild-type melanogaster eyes have also been found in D. virilis.     

CONDUCTIVITY TITRATION OF GELATIN SOLUTIONS WITH ACIDS

Titrations have been made, by the conductivity method, of gelatin solutions with hydrochloric and sulphuric acids. The results indicate an end-point at about 8.6 cc. of N/10 acid per gm. of gelatin, or a combining weight of about 1,160. These results are in fair agreement with those previously obtained by the hydrogen electrode method. Better agreement between the two methods was found in the case of deaminized gelatin. The data are in accord with a purely chemical conception of the combination between protein and acid.     

THE COMBINATION OF GELATIN WITH HYDROCHLORIC ACID

The amount of HCl combined with a given weight of gelatin has been determined by hydrogen electrode measurements in 1 per cent, 2.5 per cent, and 5 per cent solutions of gelatin in HCl of various concentrations, by correcting for the amount of HCl necessary to give the same pH to an equal volume of water without protein. The curve so obtained indicates that the amount of HCl combined with 1 gm. of gelatin is constant between pH 1 and 2, being about 0.00092 moles.     

THE ADSORPTION OF GELATIN BY COLLODION MEMBRANES

An experimental study has been made of the adsorption of gelatin from solution at 37°C. by collodion membranes. In the case of membranes of high permeability, very high concentrations of gelatin were required to produce maximum adsorption, and the maximum amounts adsorbed were independent of the pH values of the solutions over the range 3.8 to 4.8. With membranes of low permeability, maximum adsorption was reached at lower gelatin concentrations, and the maximum amounts adsorbed varied with the pH, being lower on either side of the isoelectric point, over the range 3.8 to 6.6. The addition of salt in such experiments raised the maximum amount adsorbed to a value equal to that obtained with solutions at the isoelectric point in the absence of salt. These experiments can be explained by, and seem to lend support to, the theory proposed by Loeb and further developed by Kunitz concerning the effects of pH and salt on the size of gelatin particles in solution.     

THE EFFECT OF SALTS ON THE IONISATION OF GELATIN

The effect of the addition of sodium chloride to gelatin solutions is shown from the Donnan relationship to increase the ionisation of the gelatin, the increase produced in acid solutions reaching a maximum at about 1/1000 molar salt concentration. This effect is attributed to the formation of complex ions. From the similar action of calcium and copper chlorides the effective combining power of gelatin for complex positive ion formation is deduced. The bearing of complex ion formation on the zwitter-ionic structure and solubility phenomena of proteins is pointed out.     

ISOLATION AND FRACTIONATION OF RAT BRAIN NUCLEI

A method for isolating pure and unaltered nuclei from rat brain by means of differential centrifugation is described. The isolated nuclei are further separated into discrete fractions of neuronal, astrocytic, and glial nuclei, with a yield amounting to 20 to 25% of the DNA of the original homogenate. Both the morphology and size of the nuclei remained unchanged. Problems concerning the composition of the isolation media, the use of detergents, as well as those raised by density gradient centrifugation in sucrose, Ficoll, and Dextran are discussed. Some values for the density of each type of brain nuclei are suggested.     

FRACTIONATION OF SPINACH CHLOROPLASTS BY FLOW SEDIMENTATION-ELECTROPHORESIS

A separation of spinach chloroplasts in vitro into fractions according to size (volume) and activity (light-dependent shrinkage and NADP reduction) has been achieved by stable-flow free boundary sedimentation-electrophoresis. The salient features of this chloroplast study are: (a) separation is achieved within 30 min; (b) only small density gradients are required, thus minimizing osmotic effects; (c) the fractions are collected continuously, with size fractionation being evidenced; and (d) particles are separated into fractions of higher and lower activities as compared with the control population.     

FRACTIONATION OF THE RNA COMPONENTS OF RAT BRAIN POLYSOMES

Abstract— The incorporation in vivo of [³H]uridine into the RNA isolated from the free polyribosomes of rat cerebral cortex was studied. Sedimentation in sucrose gradients showed that initially (at times less than 60 min after injection of precursor) the label was associated with a heterodisperse species, while at longer times there was an increased coincidence of label with stable rRNA. Further fractionation was accomplished by means of differential extraction with phenol and analysis on polyacrylamide-agarose gels. Most of the rapidly labelled RNA was concentrated in a fraction obtained at pH 8-3 and 40°C. The base composition of this fraction differed greatly from that of rRNA, preribosomal RNA and DNA. Analysis by electrophoresis on polyacrylamide-agarose gels showed it to be composed of several distinct species in addition to residual 18 and 28S rRNA. Most of the latter was concentrated in a fraction extracted at pH 60 at 0°C.     

THE SWELLING OF GELATIN AND THE VOLUME OF SURROUNDING SOLUTION

The swelling of isoelectric gelatin added to various volumes of acid of different concentration at 5°C. has been determined. The swelling is determined only by the concentration of the supernatant solution at equilibrium and is independent of the volume of acid. Similar experiments with unpurified gelatin show that in this case, owing to the presence of neutral salts the swelling is a function of the volume as well as the concentration of acid. Both results are predicted by the Procter-Wilson-Loeb theory of the swelling of gelatin.     

THE FLOCCULATION OF GELATIN AT THE ISOELECTRIC POINT

The results of this investigation show that a gelatin solution consists of a considerable number of constituents. At a particular temperature, certain gelatin constituents tend to aggregate and to flocculate from solution. When these particular gelatin constituents have completely flocculated, no further change occurs in the system and an apparent equilibrium exists. This is not a dynamic equilibrium between the gelatin flocculate as a whole and the gelatin remaining in the solution but a steady state determined for that system by the temperature. It is also shown that gelatin can be separated into fractions in which the gelatin constituents are more nearly uniform and tend to flocculate over a much narrower temperature range. It should be possible to obtain a number of fractions in which all of the gelatin would flocculate at a definite temperature. The aggregation of the various gelatin constituents is presumably due to loss of thermal energy, and the temperature at which this occurs must be some function of the mass of the constituent. It is natural to assume, then, that the constituents which flocculate at a given temperature are larger than those which remain in solution at that temperature. Recently, Krishnamurti and Svedberg (1930) have obtained evidence with the ultra-centrifuge that the constituents of a gelatin solution are heterogeneous as to mass, even at a pH value at which there is no tendency toward aggregation. There is much reason to suppose that the gelatin constituents do not differ very greatly chemically since different fractions have the same refractive index and the same isoelectric point. The data as a whole are best explained by considering the gelatin constituents to be different degrees of association of the same or very similar molecular structural units. This is in agreement with Sheppard and Houck (1930), who consider that "the molecules of gelatin are fundamentally identical with those of collagen, the difference being only in the degree of association and orientation". Meyer and Mark (1928) have interpreted the x-ray data obtained from collagen as indicating that the micelles of the collagen fiber are built up of main valency chains of anhydro-amino acids. It may be supposed that during peptization of these fibers, the amino acid chains become separated, disorientated, and partially broken up, so producing the heterogeneous system which we know as gelatin. It is evident that the manner in which this breaking-up proceeds depends upon the chemical treatment previous to the peptization process and the gelatin produced from lime-treated collagen would be expected to differ from that from acid-treated collagen. From the results herein reported it seems evident that the technique of isoelectric flocculation of electrolyte-free gelatin offers a profitable method for the study of gelatin and an extended investigation along these lines should yield much valuable information concerning the nature of gelatin. It is possible that this method may also be extended to other hydrophilic colloids.     

THE COMBINATION OF DEAMINIZED GELATIN WITH HYDROCHLORIC ACID

1. The analysis of isoelectric gelatin by the Van Slyke method indicates 0.00040 equivalents of amino N per gm. gelatin. 2. If deaminized gelatin is prepared without heating, the product contains less nitrogen than the original gelatin by an amount equal to 0.00040 equivalents N per gm. protein. 3. Deaminized gelatin, prepared either with or without heating, contains no amino nitrogen detectable with certainty by either the Van Slyke or the formol titration method. 4. The isoelectric point of deaminized gelatin prepared without heating is at pH 4.0. 5. The maximum combining capacity of this protein for HCl is 0.00044 equivalents per gm. 6. The maximum combining capacity of gelatin for HCl should be corrected to 0.00089 equivalents per gm. 7. The difference between these maximum combining capacities, 0.00045, is nearly equivalent to the loss in amino or total nitrogen occurring in the deaminizing reaction. 8. This equivalence constitutes a new indication that the combination of protein with acid is chemical combination.