ELECTROPHORETIC STUDIES ON FAST MOVING COMPONENTS OF HUMAN SERUM*

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1 ELECTROPHORETIC STUDIES ON FAST MOVING COMPONENTS OF HUMAN SERUM* BY HANS HOCH AND ALFRED CHANUTIN (From the Biochemical Laboratory, Medical School, University of Virginia, Charlottesville, Virginia) (Received for publication, May 26, 1952) Electrophoretic patterns of dialyzed human serum or plasma usually do not show components that migrate faster than the albumin. Seibert and Nelson (1) described the occurrence of a fast moving component in sera of tuberculous subjects. Hoch-Ligeti and Hoch (2) observed a fast moving component in seven out of eight sera from healthy subjects when electrophoresis was continued beyond the time needed for spreading the ascending pattern over the entire length of the cell. In view of the lack of information concerning trace component,s of greater anodic mobility than albumin in human serum, experiments were undertaken to establish the conditions for their differentiation. This paper presents data for the distribution and characteristics of some of these fast moving components in human serum. EXPERIMENTAL Blood was obtained from blood bank donors, staff members, and patients of the University Hospital and the Veterans Administration Hospital, Richmond, Virginia. In the procedure adopted, 4 ml. of serum or plasma were diluted with 5 ml. of 0.6 per cent NaCl, and 3 ml. of water were added to reduce the stationary salt boundaries (3). The ph of these solutions ranged between 7.8 and 8.0. It was raised to ph 8.0 to 8.3 by addition of N NaOH, and the solutions were examined by electrophoresis in 0.6 per cent NaCl in an 11 ml. cell without dialysis. The Pearson model of the Tiselius electrophoresis apparatus was used. Maximum resolution in the ascending pattern was achieved by sharpening the initial boundary with a glass capillary (4), the tip of which was bent at a right angle. The capillary was connected to narrow bore polyethylene * This investigation was supported by a research grant from the National Cancer Institute of the National Institutes of Health, United States Public Health Service. 1 Since it was essential to avoid convection currents which arise from unstable salt boundaries, electrophoresis without dialysis necessitated the study of the moving salt boundaries of the major dialyzable ion constituents of serum. Experiments showed that Na2HP04 and NaHC03 in 0.6 per cent NaCl gave gravitationally stable boundaries in both limbs. The boundaries of HPOI- appeared as positive and the boundaries of HC03- as negative peaks in the patterns. 241

2 242 COMPONENTS OF SERUM tubing and the flow was regulated by adjusting the level of the free end of the tubing. In preparative runs, fractions were removed by means of this capillary. A current of 20 ma. was used which corresponded to a potential gradient in the supernatant of 4.3 volts per cm. The optical system was adapted for the recording of patterns by the Rayleigh-Calvet-Philpot interference method (5-7). A mask with a vertical slit (0.07 mm. wide) was placed against the horizontal slit opened to 1.3 mm., and the diaphragm which masked the channel of the cell was adjusted to include the light passing through the channel and the optical flats of the by-pass. Patterns were obtained with both the interference and the diagonal edge methods. The refractive index change at a boundary was measured by the number of fringes that crossed an undeviated fringe, which had been recorded in the absence of boundaries. Small gradients were established by the lateral displacement of a fringe and expressed in terms of the distance between two adjacent fringes. A value of 12 mg. of protein per 100 ml. for a displacement equal to the distance between two adjacent fringes was obtained with crystalline bovine plasma albumin and serum solutions of different prot.ein concentrations. The migration velocities were measured from the position of the original boundary. In some experiments patterns were recorded at intervals after the current had been stopped at the end of the run in order to check for boundary shifts due to liquid leaks. Shifts of between 0.1 and 0.5 mm. per hour, frequently observed, contributed to the variations in the migration velocity of albumin. In dialysis experiments, the cellophane (Visking) bag, enclosing about 2 ml. of air, was tilted about the horizontal position at a rate of 60 to 90 times per minute for 2 hours at 25 (8). The absorption spectra of the fractions were measured in a micro cell (9) with a Beckman DU spectrophotometer. An electrophoretic pattern of a serum from a healthy individual is shown in Fig. 1. The regions in the interferometric record in which the fringes deviate from the horizontal direction correspond to the elevations in the diagonal edge record. The number of major elevations is the same as is obtained in Verona1 buffer, except for the y- and &peaks, which do not separate from each other or from the peaks due to glucose and urea. The fastest component is the bicarbonate ion. Its boundaries are seen as a negative peak in the ascending and as a positive peak in the descending pattern.2 When serum is acidified with HCl and the COZ is removed under 2 Svensson s (10) Equation 27, Acij = -Ci+l/Bj(nj) X (c+;j)l(uii - oi), was used to obtain an estimate of the change in refractive index at the bicarbonate

3 H. HOCH AND A. CHANUTIN 243 reduced pressure, the bicarbonate boundaries are no longer observed. A small component that has a mobility about 18 per cent (range 12 to 23 per cent) greater than albumin is noted in the ascending pattern and is designated as the fast component (FC). Normally the concentration of phosphate is too low and the mobility at ph 8 is too close to that of the bicarbonate ion for a separate boundary to be discerned, i j It was noted that the difference between the migration velocities of the albumin (V,) and of the tip of the slowest peak (V,+S) was more reproduc- FIG. 1. Ascending electrophoretic patterns of human serum in 0.6 per cent NaCl by (1) the diagonal edge and (3) interference methods with (2) the base-line. Fast component, f.c. ible and more constant than the absolute velocity of the albumin and that this difference did not change between ph 7.9 and 8.5. Therefore, the mobility of the fast component was also characterized by VFC - v,,, VA - v,+& boundary. Using c& = -cct = 0.1, cnco$ = cj+r UN:~ = 22.2 (II), UC; = (II), and Di = (la), one finds Ac:,/C,+r = Ac&/cE&~ = Below the bicarbonate boundary there is a deficit of 1.4 equivalents in Cl- per equivalent of HCO,. Since the refractive indices of 0.1 N NaCl and 0.1 N NaHCOs are similar, the refractive index of the layer below the bicarbonate boundary is smaller than that of the supernatant. Such a change in refractive index at the bicarbonate boundary corresponds to an inverse, negative peak in the pattern. On electrophoresis of serum or plasma the negative refractive index gradient in the descending limb due to the bicarbonate boundary is superimposed upon a larger, positive, protein concentration gradient. Consequently, the bicarbonate boundary appears as a positive peak on the descending pattern.

4 244 COMPONENTS OF SERUM where v,, is the migration velocity of the fast component; an average value of 1.20 for twenty-one sera was obtained, with variations between 1.16 and The values for V,/(V, - V,+,) ranged between 1.05 and The error involved by assuming a constant mobility for r-globulin was less than the variation in the boundary displacements, owing to leaks and other causes in routine analysis. Fast Component Characteristics-This component was observed in the electrophoretic patterns of thirty-one out of thirty-two sera from healthy individuals. The apparent concentrations (mg. per 100 ml. of serum) together with the percentage distribution are grouped as follows: 10 to 14 (10 per cent), 14 to 28 (67 per cent), 28 to 42 (20 per cent) and 49 (3 per cent). It was necessary to correct these values for the changes in concen tration that occur at the albumin and at the d-boundary. The concentration of the fast component below the ascending albumin boundary was estimat)ed with the aid of an equation previously derived (2). c= c 1 + ; K(Ct - a where C = the concentration of the fast component below the ascending albumin boundary; C = apparent concentration of the fast component above the ascending albumin, V = the migration velocity of albumin; S = separation of the boundaries of the fast component and of albumin in unit time; C, = the total concentration in the region immediately below the ascending albumin boundary, and K = l/c X (V - Vo)/Vo; I/o = the migration velocity of albumin at infinitely low albumin concentration. K was assumed to be the same for the fast component and albumin and it was determined from a plot of migration velocity of the ascending albumin boundary at different dilutions of a serum versus albumin concentration. By substituting the experimental values of K = 0.07, V/S = 6, and Ct - C = 1.5 in the equation, C becomes l/1.6 X C. This calculation is applicable if it is assumed that there is no interaction betlveen the fast component and albumin. Data on the effect of dilution on the apparent proportions of the components are shown in Table I. From these data an estimate of the concentration change at the d-boundary can be obtained. If the relative concentration of the slowest component (20 per cent) at the lowest protein concentration is subtracted from the apparent relative concentration of the slowest component (31 per cent) at the highest protein concentration in the ascending limb, a lower limit of 11 per cent is obtained for the &boundary. 3 The value for K, at 1.1 per cent protein, computed from Dole s (13) theory is for both albumin and the fast component.

5 H. HOCH AND A. CHANUTIN 245 Furthermore, if the lowest value for the apparent percentage of the y- globulin, glucose, etc., is arbitrarily set at 10 per cent, t,he upper limit of the d-boundary would be 21 per cent. Since it is not possible to determine experimentally the magnitude of the c-boundary, the dilution factor at the b-boundary cannot be calculated from the formula Dilution factor = total area minus 6 total area minus E suggested by Longsworth (3), but it is certainly greater t han 0.8, the value obtained when E = 0. Assuming 0.9 for the dilution factor and l/1.6 for C/C, t,he concentrations of the fast component in the diluted sera are about two-thirds of the TABLE ApparenCConcentrations of Serum Components in 0.6 Per Cent NaCl I I I SWUIII, diluted to 12 ml. HZ0 Total protein ml. ml. gm. *er 100 ml t * Includes a-boundary, r-globulin, glucose, urea, etc. t Conditions of the standard procedure. I Per cent of total fringe count Slowest peak* Albumin Ascending Descending Descending apparent concentrations. The range for the corrected concentrations was 7 to 33 mg. per 100 ml. of serum. The fast component was demonstrated in fresh undiluted serum, which had been analyzed without dialysis. By comparing concentrations of the fast component in seven sera before and after dialysis, it was shown that the fast component did not pass through cellophane. The fast component was not adsorbed on a Seitz filter. Xo difference in concentration of fast component was found between serum and plasma and fresh and stored sera of the same bloods in experiments in 0.6 per cent NaCl at ph 7.8 or above. The fast component was not observed under the following conditions: (a) in 0.6 per cent NaCl at ph 7.5 or below, (b) in phosphate buffer at ph 8.0 and ionic strengths of 0.1 and 0.05, or (c) in Verona1 buffer at ph 8.6 and ionic strength of 0.1. In these experiments the patterns were spread over about 6 cm. The solution removed after electrophoresis of a 1: 1 non-dialyzed diluted

6 246 COMPONENTS OF SERUM serum from the region ahead of the ascending albumin boundary had an absorption spectrum similar to that of uric acid as regards the position of X,,,. (291 mp) and Xmin. (261 mp). The value of 3.16 for the ratio Emax./Emin. was smaller than 5.5 for uric acid, but greater than 2.1 for serum ultrafiltrate (14), whose absorption at 290 rnp is principally due to uric acid. Absorption by ascorbic acid was negligible, since it was presumably present in its oxidized form (15). In order to prepare a urate-free solution of fast component for spectroscopic examination, serum was exhaustively dialyzed against 0.6 per cent NaCl containing M Na2HP04 and the solution was subjected to electrophoresis in 0.6 per cent NaCl. Two fractions (Nos. I and II) were separated between the boundaries of the ascending albumin and the fast FIG. 2. Absorption spectra of two fractions of fast component (Curves 1 and 2) and of albumin (Fraction III, Curve 3) in 0.15 M acetic acid. component, and one fraction (No. III) was obtained from the region immediately below the albumin boundary. At about ph 7, the absorption spectra of Fractions I and II were similar to those of both serum albumin (16) and protein in Fraction III. The curves differed so litt le in the region at 260 rnp that the conclusion seemed justified that the fast component contained less than 1 per cent of a nucleic acid, with a specific extinction coefficient at X,,. (260 mp) that is 20 times that of albumin at X,,. (278 mp). In 0.15 M acetic acid, the absorption of the fast component in the 250 to 260 mp region was greater than that of albumin (Fig. 2). None of the purines, pyrimidines, or their derivatives tested by Hotchkiss (17) showed a rise in this region when t,he reaction was changed from neutral to acid. The fast component gave a biuret color 0.43 times as strong as was given by serum albumin if the absorption at 278 rnp was taken as a basis for comparison. The sediment.ation rate of the fast component was compared wibh that of

7 H. HOCH AND A. CHANUTIN 247 albumin by analyzing four fractions obtained with the.preparative ultracentrifuge. Serum was centrifuged for 34 hours at 200,000 X g and the top lipide layer and three bottom layers were separated. The fast component was absent in the top layer. Comparison of the concentrations of the fast component in the two bottom layers with that in the remaining layer, consisting mainly of albumin, indicated that the fast component sedimented as fast or slightly faster than albumin. Electrophoresis of undiluted serum ultrafiltrate under conditions similar to those used for serum revealed a large, almost stationary boundary (glucose, urea, 6-, or E-), the bicarbonate boundary, and a very small boundary of material migrating with the mobility of albumin. The concentration of this material, calculated from the fringe displacement, was less than 10 mg. per 100 ml. of serum. Pathological Xera-Twenty-seven of forty-three sera from patients with a variety of diseases had concentrations of 10 to 24 mg. per 100 ml. of serum of fast component, a range observed for most of the healthy individuals. These diseases included La&neck cirrhosis, infectious hepatitis, chronic hepatitis, diabetes mellitus, pernicious anemia, and cardiac and pulmonary involvements. Mere traces of fast component (4 mg. per 100 ml. or less) were observed in five cases of Laennec s cirrhosis and in six cases which included obstructive jaundice, myocardial insufficiency, multiple myeloma, typhoid fever, and adenoma of the thyroid. The fast component was entirely absent in sera from two patients with La&neck cirrhosis. The relative migration velocity of fast component with respect to albumin of the same sample ranged between 1.ll and 1.25, with an average value of The values for the ratio VFC - v,,, VA - Vy+6 varied between 1.13 and 1.25, with an average value of Sera from four patients (chronic glomerulonephritis, hepatitis and nephrosis associated with CC14 poisoning, disseminated myelitis, and myocardial infarction) each contained two fast components whose mobilities are given in Table II. The mobility of FC-1 was close to that of the urate ion. Two sera, from cases of myocardial infarction (Table II) and arteriosclerotic heart disease, contained components with relative velocities of 1.26 and 1.33 which were intermediate between FC and FC-1. The fast component observed in the present experiments appeared to be different from that reported by Hoch-Ligeti and Hoch (2) on account of its greater migration velocity relative to that of albumin. However, the difference in the relative migration velocity may be a consequence of the

8 248 COMPONENTS OF SERUM ionic environment. They noted that this component was absent in the sera of a number of diseased individuals. Further work is necessary to establish the relation between the findings with 0.6 per cent NaCl and with phosphate after prolonged electrophoresis. TABLE II Apparent Concentrations (Mg. Per Cent) and Relative Migration Velocities of Components Faster than Fast Component Diagnosis Chronic glomer- II.16 ulonephritis Disseminated 1.21 myelitis Hepatitis, ne- i phrosis (I ) I I I Relative velocity FC (l-17-51) Myocardial in- 1.17, farction Serum + 30 mg. urate per 100 ml. serum 4pparen1 concentration 35 v 1 FC Rela- ;;y;+ ity FC-2 I Apparen concentrs tion VFC - VA , 28* 1.19, 1.29* (~7 * Fast component with mobility between FC and Fe-l. SUMMARY FC / FC-11 FC-2 v,,a v7+a A protein component, that migrates 18 per cent faster than albumin, has been demonstrated in human serum and plasma by electrophoresis at ph 7.8 to 8.3 against 0.6 per cent NaCl. Thirty-one of thirty-two sera from healthy individuals contained this component in concentrations varying bebween 7 and 33 mg. per cent. The component was absent or its concentration was low (4 mg. per cent or less) in sera of thirteen of forty-three patients with a variety of diseases. Most of these low values were observed in cases with liver pathology. Fast components which migrated 26, 33, 50, and 300 per cent faster than albumin were noted in the sera of four patients. The bicarbonate boundary appeared as a negative peak in the ascending and as a positive peak in the descending patterns of undialyzed serum.

9 H. KOCH AND A. CHANUTIN 249 The authors wish to express t,heir gratitude to Mr. Charles F. Farnsworth, Jr., and to Mr. Morris H. Clarke for their technical assistance. BIBLIOGRAPHY 1. Seibert, F. B., and Nelson, J. W., J. Biol. Chem., 143, 29 (1942). 2. Hoch-Ligeti, C., and Hoch, H., Biochem. J., 43, 556 (1948). 3. Longsworth, L. G., Chem. Rev., 30, 323 (1942). 4. Polson, A., Joubert, F. J., and Haig, D. A., Biochem. J., 40, 265 (1946). 5. Svensson, H., Acta them. &and., 3, 1170 (1949). 6. Svensson, H., Actu them. Stand., 4, 399 (1950). 7. Longsworth, L. G., Anal. Chem., 23,346 (1951). 8. Reiner, M., and Fenichel, R. L., Science, 108, 164 (1948). 9. Lowry, 0. H., and Bessey, 0. A., J. Biol. Chem., 163, 633 (1946). 10. Svensson, H., Ark. Kemi, Mineral o. Geol., 22 A, No. 10 (1946). 11. Nichol, J. C., J. Am. Chem. Sot., 72, 2367 (1950). 12. International critical tables of numerical data, physics, chemistry and technology, New York, 6, 248 (1929). 13. Dole, V. P., J. Am. Chem. Sot., 67, 1119 (1945). 14. Smith, F. C., Biochem. J., 22, 1499 (1928). 15. Sophian, L. H., and Connolly, V. J., J. Phys. and Colloid Chem., 66, 712 (1951). 16. Gitlin, D., J. Immunol., 62, 437 (1949). 17. Hotchkiss, R. D., J. Biol. Chem., 176, 315 (1948).

10 ELECTROPHORETIC STUDIES ON FAST MOVING COMPONENTS OF HUMAN SERUM Hans Hoch and Alfred Chanutin J. Biol. Chem. 1953, 200: Access the most updated version of this article at Alerts: When this article is cited When a correction for this article is posted Click here to choose from all of JBC's alerts This article cites 0 references, 0 of which can be accessed free at tml#ref-list-1