the isoelectric point of insulin · 25”, by the method of rosebury (5), with a film glass...

THE ISOELECTRIC POINT OF INSULIN

ELECTRICAL PROPERTIES OF ADSORBED AND CRYSTALLINE INSULIN*

BY OSKAR WINTERSTEINER AND HAROLD A. ABRAMSON

(From the Department of Biological Chemistry, College of Physicians and Surgeons, Columbia University, New York)

(Received for publication, December 6, 1932)

Chemical researches on the crystalline protein first obtained from commercial insulin preparations by Abel (1) in 1926 have left no doubt that this substance is identical with the hormone in its purest stable form and represents a definite chemical entity, though it may be well to remember that this term should be applied in protein chemistry with certain reservations. The re- cent experiences of Sorensen (2) with crystalline egg albumin, as well as certain suggestions made by Freudenberg and Dirscherl (3) in regard to insulin itself, are of interest in this connection. The isoelectric point of crystalline insulin has not been determined hitherto in more than an approximate fashion, though the literature contains many references to measurements on more or less purified amorphous insulin preparations. Abel and coworkers (4) noted that the pH of a solution from which insulin crystallized in satisfactory yields was 5.60 to 5.65. These solutions contained ammonia, pyridine, brucine, and acetic acid in high concentration. Since such a medium is highly instrumental in holding insulin in supersaturat.ed solution and in favoring the slow deposition of crystals, the influence of these different constituents on the solubility of insulin must be considerable. The conditions in the presence of materials like pyridine and brucine can hardly be compared to those prevailing in dilute univalent buffer systems. It seemed of theoretical as well as of practical interest to determine the iso-

* This work was aided by the Research Grant to this department by the Chemical Foundation, Inc.

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742 Isoelectric Point of Insulin

electric point of crystalline insulin by electrophoresis and to com- pare the results with those obtained by solubility measurements and nephelometric determination of the point of maximum precipitation.

Methods

Insulin-The insulin used in the experiments was obtained in crystallized form by the brucine method of Abel. For the electrophoresis experiments and the solubility measurements a sample was used which had been recrystallized three times without addition of brucine. The preparation used for the flocculation experiment was from the same source but recrystallized only once.

pH Measurements-The pH measurements were carried out at 25”, by the method of Rosebury (5), with a film glass electrode of the type described by MacInnes (6).

Electrophoresis-The electrophoretic method employed in this work is based on direct measurement of t,he electric mobility of microscopically visible quart,z particles coated with an adsorbed film of protein (7). The justification for the employment of this method depends upon the experimental fact that the electric mobility of quartz particles covered with a film of egg albumin or of serum albumin is, within the limits of error, practically the same as that of the same proteins studied under the same conditions by the moving boundary method of Tiselius (8). It would perhaps be better to use the moving boundary method on insulin, but be- cause of its low solubility in the isoelectric zone and unavailability in large quantities in the pure state, the microscopic method is preferable. It is assumed that the quartz particles covered with a film of adsorbed insulin possess the electric mobility of the protein in the dissolved state, as is the case with egg albumin and serum albumin. It was possible by this method to study not only the mobilities of insulin-covered quartz particles but also those of amorphous particles of insulin and of insulin crystals.

The quartz used in the experiment was boiled with hydrochloric acid, then treated with hot chromic acid-sulfuric acid mixture, and finally washed with distilled water for several days.

For the preparation of the insulin-coated quartz suspensions a suitable amount of insulin was dissolved in 0.4 M sodium acetate solution and a small volume of quartz suspension was added. In


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0. Wintersteiner and H. A. Abramson 743

this way the quartz surfaces remained in contact with a relatively concentrated insulin solution for about 10 minutes. Then the appropriate amounts of acetic acid and water were added to bring the pH to the required value and to make the final concentration of acetate ion ~/30. The measurements were carried out at room temperatures varying between 24-27”. No preservat.ive was needed.

Solubility Measurements-Insulin crystals and ~/30 acetate buffer of varying pH were placed in Pyrex bottles, sealed with paraffin, and rotated or shaken in a thermostat at 25” f 0.01”. The amount of insulin and the volume of the solution were varied according to the expected solubility. A small amount of toluene was added as a preserving agent. The time of shaking was 48 hours. Before the solution was filtered, care was taken to assure, by microscopic inspection, the presence of well defined crystals in the solid phase, for it was occasionally observed that some amorphous material (probably formed by slight denaturation under the combined action of shaking and of toluene) was suspended in the solution at the end of the shaking period. Solubility values in the neighborhood of the isoelectric point did not increase between 24 and 48 hours. Filtration was effected through a very small (8 mm.) asbestos filter, in which losses due to adsorption were probably negligible compared with adsorption by the glassware. Suit- able volumes of the clear filtrate were analyzed in duplicate for nitrogen by the micro-Kjeldahl method of Pregl. Blank determinations were carried out on large volumes of the buffer solutions used, after they had been subjected to the same procedure as the solutions containing insulin. No nitrogen could be detected in the buffer solutions.

Nephelometric Determinations of Point of Maximum Floccula- tion-Insulin was dissolved in 0.4 M acetate and precipitated at varying activities of hydrogen ion by the addition of the appropriate amount of acetic acid and water. The insulin concentration in respect to the final volume of the solution was 1: 10,000. The buffer concentration was ~/30 in respect to sodium acetate. At an insulin concentration of 1: 10,000 the suspensions obtained in the neighborhood of the isoelectric point remained stable for about 2 hours. Doubling this concentration resulted in microscopic flocculation within 5 to 7 minutes. A measure of the rela-


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tive turbidities of the insulin suspensions was obtained by com- paring them in a Kober nephelometer with standards of isoelectric casein suspensions stabilized with gum arabic. The relative turbidities of the standards were found to conform with their relative concentration within 5 per cent. In order to test the experimental procedure measurements were made on non-stabilized casein suspensions obtained by precipitating casein dissolved in sodium acetate at varying values of pH by the addition of acetic acid. At a casein concentration of 1: 10,000 the turbidity values were constant between pH 4.4 and 5.0 with a sharp drop on either side of that range, suggesting an isoelectric point for casein in the neighborhood of 4.7, in agreement with the data in the literature. With a lower casein concentration (1: 50,000) the zone of constant turbidity around the isoelectric point became narrower, but the reproducibility of the turbidity values became less satisfactory. With higher casein concentration (1: 5,000) macroscopic flocculation took place within a very short time.

Results

Adsorption of Insulin on Quartz-In common with most proteins insulin is adsorbed by quartz particles, with profound alteration of their surfaces, and consequently of their electrokinetic properties. A certain minimum concentration of insulin in the solution in which the quartz particles are suspended is required to reach a limiting value of the effect of the protein on the electric mobility, which is taken to represent complete coating of the particles with the protein. In order to obtain the exact value of this minimum concentration two series of experiments were carried out on either side of the isoelectric point; in these, quartz particles were suspended in solutions of insulin in 0.4 M acetate of varying insulin concentrations, and the pH brought to 4.0 and 6.0 respectively by the addition of acetic acid. The final sodium acetate concentration was ~/30. Fig. 1 gives the relation between the electric mobilities of the particles and the insulin concentration. The ordinate values are in p per second per volt per cm. ; the abscissa? in weight fractions of insulin in the aqueous medium referring to the final concentration of insulin in the solution after addition of acetic acid and water. That is to say, the concentration of insulin to which the quartz particles were originally exposed was higher


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than the final concentration and can be obtained by multiplying the figures on the abscissa scale by 12.

The quartz particles change their surface charge in the presence of the merest traces of insulin. The saturation point is practically reached when the insulin concentration is about 1: 20,000. This experiment yielded the information that between pH 4.0 and pH

TABLE I

Interpolated Values of Electric Mobility of Insulin (Adsorbed) in S/SO Sodium Acetate Buffer (V,,, in Cm. per Second per Volt per Cm. X 104)

pH ..I 4.0 / 4.4 j 4.6 / 4.8 1 5.0 j 5.2 / 5.4 j 5.6 / 5.8 / 6.0

V, . . . . +1.58+1.48+1.29+0.93+0.56+0.19-0.16-0.51-0.83-1.12

A 3 pH+.O

FIG. 1. The adsorption of insulin by quartz. The ordinate units are in g per second per volt per cm.; the abscissa units are in weight fraction of insulin in buffer. C indicates concentration. The quartz particles change their surfaces in the presence of the merest trace of insulin, saturation being practically reached when the insulin concentration is about 1:20,000. This experiment yielded the information that between pH 4.0 and pH 6.0, adsorbed insulin probably completely covered the quartz particles.

6.0 adsorbed insulin probably completely covered the quartz particles under the conditions adopted.

Electric Mobility of Adsorbed and Amorphous Insulin-Curve I in Fig. 2, A shows the influence of pH on the mobility of quartz particles presumably completely covered with insulin. The suspensions were prepared as indicated under “Methods.” The final concentration of insulin in these experiments was 1: 10,000. In the neighborhood of the isoelectric point a part of the insulin was


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precipitated under these conditions in the form of amorphous aggregates which could be distinguished under the microscope from the quartz particles by the difference in their refractive power. These amorphous aggregates move in the electric field with the same velocities as the quartz particles. From these measurements it appears that the point of zero mobility lies between pH 5.3 and pH 5.35, and according to the assumptions stated in the beginning of the paper the isoelectric point of dissolved insulin is probably to

FIG. 2, A. The electric mobility of quartz particles covered with insulin in 430 acetate buffers. The isoelectric point is between pH 5.30 and pH 5.35. The same data were obtained with particles of amorphous insulin. The ordinate units are in p per second per volt per cm.

FIG. 2, B. The smooth curve (Curve I) gives the electric mobilities of adsorbed or of amorphous insulin. The lower curve (Curve II) gives the mobilities of insulin crystals or crystal fragments in the same medium. Curve II has been roughly fitted to the open circles (mobilities of crystals suspended in ~/30 acetate buffer). For significance of the open and solid circles consult the text. The ordinate units are in I( per second per volt per cm.

The readings below the zero line denote negative mobilities.

be found in that range. For convenience, the interpolated values of the motilities of insulin adsorbed on quartz are given in Table I.

Electric Mobility of Insulin Crystals-The electric mobilities of insulin crystals suspended in ~/30 acetate buffers of varying pH were also studied. The crystals were kept in contact with the buffers for about 10 minutes before the measurement was made. Crystal fragments obtained by grinding the crystals in an agate mortar showed essentially the same mobility as the intact crystals. The mobilities observed are represented by the open circles in Fig. 2, B. The smooth curve (Curve II) drawn through these


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points cuts the pH scale on a definitely lower level, in the neighborhood of pH 5.0. Curve I is given for comparison and represents the mobilities of adsorbed or amorphous insulin. The differences between the two curves is well outside of the range of experimental error. Measurements were also made on insulin crystals suspended in insulin-containing acetat,e buffers which had been made up in the same way as the insulin-coated quartz suspension used in the measurements described above, and brought to the required pH before addition of the crystals. The mobilities found in these measurements are given by the solid circles in Fig. 2, B. In the neighborhood of the isoelectric point a part of the insulin, as mentioned above, is precipitated under t,hese conditions, but such solutions probably remain in a state of supersaturation with respect to insulin for considerable time after the precipitation, as will be pointed out later. It is therefore to be expected that a certain amount of insulin will be deposited on the surface of crystals left in contact with such solutions. As a matter of fact, it was found that the mobilities of crystals under t,hese conditions do not deviate appreciably from the values for crystals suspended in the pure buffer solutions presumably incompletely saturated with insulin. If deposition of insulin from the solutions considered as super- saturated has taken place on the surface of the crystals, it does not seem to have influenced their surface charge to any greater extent than those of crystals not in equilibrium with a saturated solution, as can be seen from Fig. 2, B.

Volubility of Insulin in M/SO Acetate Buffers-The results of the solubility determinations are represented by Curve I in Fig. 3. Over the broad range pH 4.6 to 6.5 the solubility is very low. The solubility values between pH 4.8 and 6.2 fluctuate between 3.1 and 4.0 mg. per liter. At pH 6.0 a value of 5.3 mg. was found, probably due to experimental error. With increasing solubilities at both sides of this pH range the reproducibility was not very satisfactory. We have not tested the possibility that the solubilities are dependent on the amount of solid phase present, nor that at pH values where the solubility increases the solution had failed to attain equilibrium with the solid phase within the time allowed for saturation. Our limited supply of highly purified material prevented further inquiry into these points. It may be mentioned in this connection that t’he recovery of crystalline material from


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those parts of the solution which were not used for the nitrogen determination was not as good as expected, so that possibly some denaturation may have taken place. It is difficult to decide from the present data whether the shape of the curve is strictly symmetrical, as is the case with ideal ampholytes. These circumstances, in conjunction with the uncertainty of the absolute solubility values in the regions where the solubility increases, throw doubt on the application of the mass law to the evaluation of the isoelectric point. If a smooth curve be drawn through the experimental points, and the isoelectric point be taken as the mean of

FIG. 3. The open circles (Curve I) represent the solubilities of crystalline insulin in ~/30 acetate buffers in mg. per liter. The dotted part of the solubility curve indicates uncertain course. The solid circles (Curve II) represent relative turbidities.

two points of equal solubility on that curve, values between 5.55 and 5.60 are obtained. However, the positions of this so called isoelectric point would, when the solubility curve is not symmetrical, depend on the level of solubility chosen.

Point of Maximum Flocculation-Readings on insulin suspensions prepared as indicated under “Methods” were taken from the 3rd to the 12th minute after the addition of the acid to the solution of insulin in sodium acetate. In the neighborhood of the isoelectric point (pH 4.8 to 6.0) the turbidity values remained practically constant after the 5th minute. At higher or lower pH values a slight increase could be noted during the period of observation. The (relative) turbidity values determined between the 10th and the


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12th minutes were used for constructing Curve II in Fig. 3. No aggregation of particles visible to the naked eye was observed during this period. The turbidity values obtained for suspensions prepared under identical conditions were reproducible within about 10 per cent, though in most cases the agreement was much better. The comparatively large limits of error make it difficult to ascribe a very definite value to the point of optimum flocculation. A smooth symmetrical curve drawn through the experimental points indicates a maximum of flocculation at approximately pH 5.4, which is in fair agreement with the value pH 5.3 to 5.35 found for the isoelectric point by mobility measurements.

DISCUSSION

Besides giving the required information as to the isoelect.ric point of insulin in ~/30 acetate buffer the experiments reported contribute a few observations of general interest. The difference observed between the mobilities of adsorbed or amorphous insulin and of crystals suspended in the same medium shows that the surface charge of the amorphous particle differs from that of the surface composed of the limits of the crystal lattice. The isoelectric point of the crystal deviates by 0.3 pH units from that of insulin- coated quartz particles or amorphous insulin. Since the behavior of the latter reflects in all probability that of the dissolved protein, a smaller number of positively charged groups is available on the crystal surface than on the freely dispersed molecule over the pH range under consideration. In this respect the behavior of insulin crystals resembles the behavior of crystals of the less soluble amino acids, tyrosine, cystine, and aspartic acid. One of us (9) has pre- viously observed that the isoelectric point determined by electric mobility of crystals of these amino acids suspended in dilute hydrochloric acid and in buffers is the same, namely about pH 2.3, and bears no relationship to the isoelectric point of these amino acids in the dissolved state as determined by the magnitude of their dissociation constants. The behavior of crystals of the above amino acids in protein solutions has been compared with that of protein-covered quartz particles and other inert surfaces. The surfaces of the amino acid crystals adsorbed gelatin just as inert surfaces do, the elect,ric mobilities of both being identical.1

1 Unpublished data.


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The insulin crystals have here been examined in dilute solutions of insulin. The surface of the insulin crystals could be (1) entirely made up of the limits of the crystal lattice, (2) a surface completely covered by adsorbed protein, (3) a combination of (1) and (2), the surface being partially made up of crystal lattice and of adsorbed protein. Fig. 1 demonstrates that very minute traces of insulin changed the electrical mobility of quartz particles. If this fact be taken, together with the fact that the ampholyte surfaces of the amino acids mentioned before seem to adsorb protein quite readily, it follows that only when every molecule of insulin collid- ing with the lattice enters the lattice at precisely the same rate as the rate of collision, can case (1) be realized. It seems unlikely that this ideal ca.se can occur, for in the most acid of the solutions studied (at, about pH 4) where insulin becomes soluble, the mobilities of both amorphous and crystal surfaces approach one another. In an analogous fashion a charge reversal of the amino acid crystals is observed in a region where these amino acids become soluble. In ot.her words, here, the crystal surface loses its integrity and acts as an amorphous surface. The simplest explanation would be that the insulin crystals adsorbed sufficient insulin from the solution to cover their surfaces in just the same way as the surfaces of inert particles are covered, the area involved being dependent on the amount of insulin in solution (see Fig. 1). Variations in the proportion of crystal surface to amorphous surface would thus ac- count for the differences between the mobilities of adsorbed insulin and insulin crystals in the isoelectric region. We admit that the experimental evidence adduced in this work is insufficient to permit any far reaching conclusions to be drawn with regard to the differences between the surface charges of crystals and of amorphous particles of amphoteric substances. A careful study of these phe- nomena on a crystalline protein of relatively low solubility in the isoelectric range, but more easily available than crystalline insulin, seems highly desirable.

The possibility has also to be considered that the crystals used in the electrophoresis experiments did not actually represent the isoelectric protein in a strict sense. The pH of the solutions from which the crystals of insulin employed in these experiments were obtained was not measured. However, it could not have been very far removed from the pH of the solution containing ammonia,


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pyridine, and acetic acid in the proportions employed for the re- crystallization. The pH of such solutions was found to lie between 6.0 and 6.1. The insulin crystals may therefore have contained small amounts of base, presumably bound to the acidic groups of the protein. If these groups on the surface of the crystals were fully dissociated at the true isoelectric point of the protein, then the number of positively and negatively charged groups on the surface would be determined only by the pH of the solution; in other words the crystals would show the same isoelectric point as freely dispersed protein molecules. If, on the other hand, the crystal surface retains the bound base, t,hen the number of groups able to carry a negative charge would be diminished; that is, the crystal should assume a positive charge at the (true) isoelectric point. Since it was found that the crystals are highly negatively charged at this point, the discrepancy discussed above is probably not caused by differences in the chemical composition of the amorphous and the crystalline surface.

Another point of interest is the difference between the isoelectric point as determined from the solubility data and from the turbidity determinations. Comparison of the solubility and turbidity curves in Fig. 3 shows at once that their mid-points do not exactly coincide. It is doubtful whether the point of equal solubility on either side of the isoelectric point can be considered as related to the position of the isoelectric point in the case of a highly polyvalent ion like that of protein, in the sense of the theory derived for ideal ampholytes. On the other hand, the nephelometrically determined turbidity value is determined by the number of particles of such a size as to be able to display the Tyndall effect, leaving aside the question of particle size which determines the color of the Tyndall light. At a given total protein concentration the number of particles will depend upon the solubility of the protein at the pH under consideration as well as upon the rate of aggregation to such particles. In the case of insulin we may consider the solubility as constant over practically the whole pH range under consideration. The turbidity values obtained at different levels of hydrogen ion activity at any given point of time may then be taken as an approximate measure of the rate of flocculation for that point of time. Further, the rate of flocculation is related to the electrokinetic potential of the protein at that pH. From this


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point of view it is not surprising that the turbidity curve shows a fairly well developed maximum, whereas the minimum of solubility extends over a very broad range. No simple relationship between solubility and surface potential can yet be envisaged without as- suming constant composition of the solid phase with varying pH. It is, however, justifiable to relate the position of the maximum flocculation point to the point of zero potential of the particle. We, therefore, consider the approximate value (pH 5.4) for the isoelectric point,, as derived from the turbidity measurements, to be in essential agreement with the result of the electrophoretic measurement on adsorbed or amorphous insulin; and we are in- clined to disregard as less significant the value of pH 5.55 to 5.60 derived from the solubility measurements.

SUMMARY

1. The isoelectric point of crystalline insulin, as determined from measurement in ~/30 acetate buffers of the electric mobility of insulin adsorbed on quartz or precipitated in amorphous form, is found to be between pH 5.3 and pH 5.35. Insulin crystals suspended in ~/30 acetate buffers are isoelectric at pH 5.0. The significance of this difference between the behavior of the surfaces of adsorbed and crystalline insulin is discussed.

2. Insulin is slightly soluble in ~/30 acetate buffers over a broad pH range extending from pH 4.8 to 6.5. The solubility in that range is constant, and of the magnitude of approximately 4 mg. per liter. The mid-point of the range of comparative insolubility lies at pH 5.55 to 5.60. This point is not considered to be a significant indication of the isoelectric point.

3. The maximum flocculation point of crystalline insulin determined nephelometrically in ~/30 acetate buffers lies at about pH 5.4, which is in essential agreement with the result of the electric mobility measurements.

Addendum-After the conclusion of this work a paper by Howitt and Prideaux (lo), which is in essential agreement with the foregoing, appeared, giving measurements of the electrical mobility of gold particles coated with insulin, by the moving boundary method. The authors find the isoelectric point of insulin at pH 5.4, which is in excellent agreement with our results. Their figure was obtained by interpolation over about 2 pH units (between pH 4.2 and 6.8) in the region of insolubility. Though amorphous insulin of about the same activity as crystalline insulin was used in their


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work, evidence was adduced showing that no fractionation of physiological activity had occurred during electrophoresis.

BIBLIOGRAPHY

1. Abel, J. J., Proc. Nat. Acad. SC., 12, 132 (1926). 2. Sorensen, S. P. L., Compt.-rend. trav. Lab. Carkberg, 18, No. 5 (1930). 3. Freudenberg, K., and Dirscherl, W., Z. physiol. Chem., 202, 192 (1931). 4. Abel, J. J., Geiling, E. M. K., Rouiller, C. A., Bell, F. K., and Winter-

Steiner, O., J. Pharmacol. and Exp. Therap., 31, 65 (1927). 5. Rosebury, F., Ind. and Eng. Chem., Anal. Ed., 4, 398 (1932). 6. MacInnes, D. A., and Dole, M., Ind. and Eng. Chem., Anal. Ed., 1, 57

(1929). 7. Abramson, H. A., and Grossman, E. B., J. Gen. Physiol., 16,575 (1932). 8. Tiselius, A., Dissertation, Upsala (1930). 9. Abramson, H. A., Physic. Rev., series 2, 37, 1714 (1931).

10. Howitt, F. O., and Prideaux, E. B. R., Proc. Roy. Sot. London, Series B, 112, 13 (1932).


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Oskar Wintersteiner and Harold A. AbramsonINSULIN

OF ADSORBED AND CRYSTALLINEINSULIN: ELECTRICAL PROPERTIES

THE ISOELECTRIC POINT OF

1933, 99:741-753.J. Biol. Chem.

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the isoelectric point of insulin · 25”, by the method of rosebury (5), with a film glass...

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