AN ELECTROPHORETIC STUDY OF PLASMA AND PLASMA FRACTIONS OF NORMAL AND INJURED RATS*

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1 AN ELECTROPHORETC STUDY OF PLASMA AND PLASMA FRACTONS OF NORMAL AND NJURED RATS* BY ERLAND C. GJESSNG AND ALFRED CHANUTN WTH THE TECHNCAL ASSSTANCE OF CURTS S. FLOYD (Prom the Biochemical Laboratory, Unicersity of Virginia, Charloitesville) (Received for publication, May, 97) The development of the electrophoretic procedure has led a number of in%estigators to study the distribution of rat plasma proteins by this method. Rat serum or plasma does not yield the distinct pat,terns that are obtained with human plasma. Moore, Levin, and Leathem () could not observe the crglobulin fraction in normal rat serum dialyzed against phosphate buffer at ph 7.. After hypophysectomy () and thyroidectomy or thiouracil feeding in rats (2), the aglobulin appeared in the serum (ph 7.). When normal rat serum was examined at ph 8., the aglobulin component was seen. Deutsch and Goodloe () found no distinct component in the region of t,he aglobulin at ph 8.6 Although five components have been described (, ), the boundaries are too indistinct for accurate analysis. Experiences with the electrophoretic analysis of normal rat plasma in this laboratory were unsatisfactory and were similar to those of other investigators. n order to study the protein components more thoroughly, fractionation was undertaken according to the procedures recommended by Cohn and his associat,es (). The present, investigation is concerned with the analyses of whole and fractionated plasma obtained from normal and injured rats. Downloaded from by guest on May, 208 Methods nbred male rat,s of Wistar stock, about 70 days old and weighing between 0 and 200 gm., were used as experimental animals. About onethird of the body surface was burned by dipping t,he clipped back of the anesthetized rat in hot mater at 7 for 0 seconds. These animals had access to food and water and were sacrificed at varying periods after being scalded. The animals were anesthetized with sodium pentobarbital and were exsanguinated by drawing blood from the aorta into a tube containing heparin. Plasma was diluted with. parts of seronal buffer (r/2, 0.) at ph 8.6 * This work WRS done under contract with the Medical Division of the Chemical corps. 67

2 68 ELECTROPHORETC ANALYSS OF PLASMA and dialyzed against the buffer for or days. The electrophoretic experiments were done in a Tiselius appa,ratus at 2.0 by means of the scanning method of Longsworth (6). The concentrations of the various protein fractions mere estimated from the patterns of the ascending side. Results Diagram outlines the procedures used for fractionation of rat plasma. The extent of the fractionation was limited by the amount of plasma available (60 to 70 ml.). The four main fractions obtained were s, +, TT, and V. i + f V, ation DAGRAM Rat of Rat Plasma plasma Diluted with equal volume of 0.8% NaCl; ethanol 8%; ph 7.20; r/2, 0.2 Supernatant r.8 V All fractions were cenbifuged at for 0 minutes at 000 LP.M. Ethanol 2Oojd; ph 7.0; r/2,0.09 Ethanol 0%; ph.7; r/a, 0.09 (NaCl) Ethanol 0%; ph Discard Downloaded from by guest on May, 208 l reliminary experiments showed that the dilution of plasma with 0.8 per cent NaCl is an important initial st.ep. This dilut,ion not only yields a larger volume for manipulation but the subsequent fractionation is more satisfactory. The ionic strength maintained with NaCl is more satisfactory than with sodium acetate. f t.he ionic strength of Supernat ant + is increased above 0.09, the proteins of V become insoluble. Evidently an ionic strength greater than 0.09 at an alcohol concentration of 0 per cent causes some denaturation of one of the CXglobulins which is present, in V. Mobility DataThe mobilities of the various components are shown in Table. The data are determined in whole plasma and its fractions of control and injured rats. The results indicat e that the values for a given

3 E. C. GJESSNG AND A. CHANUTN 69 TABLE Mean Values and Standard Errors for Mob&ties OJ Proteins of Whole Plasma and s Albumin at cl* ld+lr Ascending Plasma + V V Plasma + V V Run No. 02* * iO.0.9 zko.0.0f f f i zk f f i 0.06 * Serum. t Descending 6.8 i f f f f f f 0.0. rt 0.6. i 0.0 Descending 6.2zkO.07.f0.08. ho.07.0f0.0 2.stO i 0..6 i 0.. i f t 0.0. ;fs f f & 0.. f & 0.0 TABLE Electrophoretic Analyses oj Rat Plasma and Sera 7 Days afta. Per cent distribution injury Albumin a~ a2 _ ~~ boundary. t t t f i++ Y Downloaded from by guest on May, 208 component are the same regardless of methods of fractionation. The values for the descending boundaries are lower than those of the ascending boundaries, as is to be expected.

4 660 ELECTROPHORETC ANALYSS OF PLASMA Electrophoretic AnaZysisThe electrophoretic analyses of plasma and serum and s +, N, and V are shown in Tables to.v for control and injured animals. Patterns for were impossible to obtain. The plasma of control rats is composed chiefly of albumin and CQ and Pglobulins. After the animal is burned, the percentage distribut ion of albumin decreases and t,he arzglobulin increases. A study of whole rat plasma gives a rough approximation of the distribution of the protein components Treatment (6 * Descending boundary. TABLE Electrophoretic Analyses of + Z lays after injury B Albumin a~ * Y cent per cenl be? cent (*:7 8:r is composed chiefly of fibrinogen which on standing is converted to fibrin. Extracts of this fraction wit,h barbiturate buffer were studied electrophoretically and it was found that about 60 per cent of the soluble material Gras aglobulin and the remainder was albumin and p globulin. The aglobulin present causes a marked turbidity. f this protein is not removed, the remaining fractions are frequently too turbid for electrophoresis. The analyses of + of the plasma of cont rol and burned rats are shown in Table and the electrophoretic patterns are presented in Fig.. This fraction shows outstanding changes after the animal is burned. The percentage distribution then indicat.es that the ~2 and / components increase almost immediately (2 hours) and the rglobulin concentration decreases rather markedly. These changes are reflected in the ~2 : y and /: y ratios. Downloaded from by guest on May, 208

5 NORMAL CONTROL p wa p ala PLASMA A _ lx xc BURNED WTH HOT WATER7: 0SEC. Ba2arA PLASMA ELfLL lx FG.. Typical electrophoretic patterns for the plasma and plasma fractions of control and scalded rats. Run No. Treatment ( (( * Descending boundary. TABLE Electrovhoretic Analuses of V L.z z. L Days after injury 2 6 V Albumin at az per cent per rent per cent * 9 0* Y per cent per cent [ Downloaded from by guest on May, 208

6 662 ELECTROPHORETC ANALYSS OF PLASMA The data for V (Table TT and Fig. ) show no outstanding changes in distribution of its components after burning. The concen Run No. Treatment * Descending boundary. Supernatant Vl TABLE Electrophoretic Analyses of V Days after injury V Albumin DAGRAM 2 per cenl per ted 9er cenl * 26 7* * Procedures for Removing Globulins jrom V V dissolved in 0 times its weight of Hz0 7 Ethanol %; ph.;.0 & Vl B ten: Downloaded from by guest on May, 208 Ethanol 0%; ph.8 v2 V is easily soluble in H20. t is brought to a concentration of per cent with.per cent et)hanol and ph. with acetate buffer and the resulting precipitate is immediately removed by centrifuging. The ethanol concentration of Supernatant Vl is then raised to 0 per cent with 9 per cent ethanol and the ph is raised to.8 with NaHC0. The precipitate is immediately removed by centrifuging. tration of azglobulin tends to increase somewhat during the first days after burning. No characteristic changes in the distribution of the V com

7 . C. GJESSNG md A. CHANUTN 66 ponents (Table V and Fig. ) are seen after thermal injury. This fraction is characterized by the presence of appreciable amounts of CY, and,&globulins. These globulins may be removed by treating V according to the procedures outlined in Diagram 2. The patterns for FRACTON Pl No. + V V FG. 2. Electrophoretic patterns after the subfractionation of V. 7 TARLE V Nitrogen Distribution of Plasma Components Treatment Days after injury Albumin mg N per 00 ml. plasma a a* mg. fm? B 7 mg. W Downloaded from by guest on May, 208 s Vl and V2 are superimposed on each other by using the salt effect as a point of reference (Fig. 2). t is obvious that albumin that is almost electrophoretically pure is obtained by these methods. Data for the nitrogen distribution of the components of the fractions are given in Table V. Although the data are limited, they appear to

8 66 ELECTROPiORETC AKALYSS OF PLASMA follow the general trend shown by the percentage distributions of the components in each fraction. The increase in the nitrogen concernration of CQ and &globulins and the decrease in the yglobulin are once more apparent in + after thermal injury. An increase in cw, p, and rglobulins is observed in V. The decrease in albumin in V is a characteristic finding for the rat and other species. The mean values for the nitrogen contents of three fractions obtained from control and burned rats are shown in Table V. The data for the burned rats include all values obtained during the first days after burning. After injury significant decreases are seen for s + and V. Yields of s + TABLE V in Mg. of N Per 00 Ml. of Plasma V (a) (8 rats) 9 zk 2 f 7 f.6 f 0. (8 ) 0* f 0 / 2 f 7* zk * f 0.06 * Significantly different. DSCUSSOh Electrophoret,ic patterns of normal rat plasma usually show two distinct boundaries which have considerable spreading. The two main components are classified as albumin and globulins. After fractionation of plasma, the boundaries of the components become clearly defined. These new boundaries are striking in the three fractions studied, particularly in +. t would appear that, the protein.s of rat plasma exist as complexes Tvhich are easily split by the gentle treatment used in these fractionation procedures. The factors responsible for the close association of these component s are not influenced by the electrical forces inherent in the electrophoretic method. t is planned to examine mixtures of these fractions to determine whether association of the various components will occur. The increases in the o and Pglobulins and decreases of albumin and rglobulin after thermal injury in the rat are similar t,o those obtained in the dog. The differences in the pat,terns for + of the control and injured animals are so characteristic that, it is possible to classify them by visual inspection. These characteristic injury patterns of + are also observed after administration of turpentine and of adrenal cortical horm0nes.l (b) V (b) 7 Downloaded from by guest on May, 208 Gjessing, E. C., and Chanutin, A., unpublished data.

9 E. C. GJESSNG ND A. CHANUTN 66 SUMMARY A procedure is outlined for fractionat.ing rat plasma into four portions by means of ethanol at low temperatures. ation makes it possible to obtain reasonably accurate analyses for the distribution of the protein components. Mobility data obt,ained from electrophoretic patterns of the comljonents of rat plasma and its fractions are presented for the ascending and descending boundaries. The electrophoretic analyses for the plasma and fract,ions of control and burned rats are given. Thermal injury gives rise to an increase in the concentrations of Q and &globulins and a decrease in the yglobulin and albumin. BBLOGRAPHY. Moore, D. H., Levin, L., and Leathem, J. H., J. Biol. Chem., 6, 9 (9). 2. Moore, D. H., Levin, L., and Smelser, G. K., J. Biol. Chem., 67, 72 (9).. Deutsch, H. F., and Goodloe, M. B., J. Biol. Chem., 6, (9).. Li, C. H., and Reinhardt, W. O., J. Biol. Chem., (97).. Cohn, E. J., Strong, L. E., Hughes, W. L., Jr., Mulford, D. J., Ashworth, J. N., Melin, M., and Taylor, H. L., J. Am. Chem. Xoc., 68, 9 (9). 6. Longsworth, L. G., J. Am. Chem. Sot., 6, 29 (99). Downloaded from by guest on May, 208

10 AN ELECTROPHORETC STUDY OF PLASMA AND PLASMA FRACTONS NORMAL AND NJURED RATS OF Erland C. Gjessing, Alfred Chanutin and With the technical assistance of Curtis S. Floyd J. Biol. Chem. 97, 69:66. 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#reflist Downloaded from by guest on May, 208