SeparationJ Biol. Chem. 1940 Kingsley 731 5

8/6/2019 SeparationJ Biol. Chem. 1940 Kingsley 731 5

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A RAPID METHOD FOR THE SEPARATION OF SERUMALBUMIN AND GLOBULINBY GEORGE R. KINGSLEY

(From the Division of Biochemistry, Laboratories, Philadelphia GeneralHospital, Philadelphia)(Received for publication, February 17, 1940)

Many attempts have been made to shorten and improve theHowe (1) method for separation of serum globulin from albumin.Howe (2) and Goettsch and Kendall (3) found that errors werecaused by the adsorption of albumin by filter paper. These errorswere shown by Robinson, Price, and Hogden (4) to vary with theamounts of solution filtered and the kind and size of filter paperused. The loss was greatest in the first portions of the filtrateand it was recommended that these be discarded. Harris (5)evaluated statistically the loss of albumin due to adsorption byfilter paper and found it to be 0.05 gm. per cent. To avoid lossof time and errors of filtration, Robinson, Price, and Hogden (6)employed the angle centrifuge to separate the globulin precipitate.They found that globulin precipitated by 1.5 M sodium sulfatesolution could be separated quantitatively after standing 4 ormore hours by centrifuging at 4200 R.P.M. for 1 hour. Theseauthors (7) also showed that globulin could be salted-out at 25as well as at 38.Because of the limitations of existing methods, especially whenlarge numbers of determinations are made, a further simplificationof the procedure for globulin precipitation was made. The modi-fied method is based on the use of ether to decrease the density ofglobulin precipitated by sodium sulfate. After ether extractionand brief centrifugation, globulin separates in a compact layer atthe bottom of the ether phase above the sodium sulfate solution.No preliminary period of standing is required.

MethodAdd 0.5 cc. of serum to 7.5 cc. of 23 per cent sodium sulfatein a test-tube (15 cc. capacity and at least 15 mm. diameter),

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732 Serum Albumin and Globulin SeparationTABLE I

Determinations of Serum Album in by New Centrifugation Tech niqueCompared with Two Methods of Filtration. Evaluation by

Biuret and Kjeld ahl MethodsThe values, given in per cent, represent sing le determinations.

Filtrates

5.4 5.1 5.2 5.1 5.2 5.04.5 4.7 4.6 4.5 4.9 4.74.4 4.6 4.5 4.9 4.8 4.74.4 4.4 4.2 4.4 4.1 4.54.3 4.5 4.7 4.7 4.7 4.44.1 4.1 4.4 4.4 4.3 4.53.9 3.8 3.8 4.1 4.0 4.03.9 3.5 3.8 3.7 4.0 3.83.8 3.7 3.9 3.7 4.0 3.73.8 3.7 4.0 3.7 3.7 3.73.8 3.7 3.8 3.5 3.7 3.83.8* 3.8 3.8 3.8 3.8 3.83.7 3.8 4.0 3.8 3.8 3.83.6 3.7 3.6 3.7 3.3 3.73.6 3.2 3.6 3.5 3.3 3.43.5 3.7 4.0 4.1 3.9 4.03.3 3.6 3.9 3.3 3.6 3.63.2 3.0 3.1 3.2 3.3 3.02.9 3.1 3.1 3.3 3.2 3.62.5* 2.5 2.7 2.6 2.7 2.62.4 2.4 2.4 2.3 2.5 2.32.3* 2.3 2.4 2.3 2.4 2.32.3 1.9 2.1 2.2 2.3 2.11.9 2.3 2.3 2.3 2.5 2.31.6* 2.0 1.9 2.0 1.9 2.0

Average. .3.48 3.48

BicYt-

- _- 3.59

Biuret(4)

3.56Standard error of difference between averages o

Columns 3 and 5 and 4 and 6..* Lactescent.

1 iltratea obtained by theHarris (6) technique

3.66

3~0.246

.-

_---

-

Biuret(6)

3.57

f0.249

and mix thoroughly by inverting. Add about 3 cc. of ether(u.s.P. quality is satisfactory), and shake vigorously for 20 to30 seconds. Cap the tube toavoid loss of the ether and centrifuge

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G. R. Kingsley5 to 10 minutes at about 2200 R.P.M. After centrifugation, slantthe tube so that the tightly packed globulin precipitate floatingon the sodium sulfate solution separates from the walls of thetube. Insert a pipette through the ether layer preferably alongthe lower wall, and withdraw the desired volume of the centrifu-gate for albumin determination. For analysis by the authorscalorimetric biuret method (8), use a 2 cc. sample. If a greatervolume is desired, as for example 10 cc. for a macro-Kjeldahldetermination, 1 cc. of serum may be used with proportionatelyincreased amounts of reagents in a test-tube 17 to 20 mm. indiameter. Total globulin values are calculated by the differencebetween serum total protein and albumin. If the sodium sulfateis stored at 37.5, no difficulty is encountered with crystal forma-tion at room temperatures above 21.

Comparison with Other Procedures-Filtrates of serum were pre-pared by precipitating globulin at 37.5 from 1 cc. of serum with15 cc. of 23 per cent sodium sulfate solution and after standingovernight were filtered at 37.5 through one sheet of 11 cm.Schleicher and Schtill No. 602eh paper. Filtrates were alsoprepared by the technique of Harris (5). Centrifugates wereprepared from the same samples of serum by the new methoddescribed. The protein of the filtrates and centrifugates wasdetermined both by the macro-Kjeldahl and the biuret methods.The data in Table I show that the albumin values of centrifugatesagreed well with those of filtrates prepared by the technique ofHarris, while the albumin in filtrates prepared by the Howe tech-nique was 0.09 and 0.12 gm. per cent lower. This loss is inharmony with the 0.05 gm. per cent loss by adsorption on one7.0 cm. sheet of Whatman No. 50 filter paper reported by Harris.The standard error of the difference between the means of thealbumin of centrifugates and of filtrates prepared by the Harrismethod was not significant. The standard error of measurement(S.E.m = &!d2/2n) of the difference of single Kjeldahl and biuretdeterminations in each group was ~0.128 for filtrates, ho.107for centrifugates, and fO.ll l for Harris filtrates (9).

DISCUSSIONIn a preliminary investigation it was found that a quantitative

separation of globulin was effected by mixing 0.5 cc. of serum with

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734 Serum Albumin and Globulin Separation6.5 cc. of 27 per cent sodium sulfate and immediately adding 1 cc.of 15 per cent barium chloride and again mixing. The suspendedprecipitate of barium sulfate and globulin was separated by cen-trifugation or by gravity on standing 2 to 4 hours at 37.5. Usu-ally either procedure gave a clear supernatant solution. However,this method could not be applied to sera generally because acol loidal suspension formed in the presence of an abnormal amountof lip id. Formation of such suspensions could be prevented byextraction of the mixture with ether. It was discovered subse-quently that when ether was used barium sulfate was no longernecessary for the rapid separation of the globulin precipitate.

The following experiment offers a possible explanation of theaction of ether in accelerating the separation of globulin by sodiumsulfate. Globulin was salted-out from 1 cc. of serum by additionof sodium sulfate and after shaking with ether was separatedby centrifugation. The precipitate was redissolved in 1 cc. of3 per cent sodium chloride and evacuated until no odor of etherremained. Centrifugation after addition of sodium sulfate causedno separation of the suspended globulin. However, centrifugationafter the addition of ether gave a sharp separation. When aheavier solvent such as carbon tetrachloride was used instead ofether, the globulin separated between the two liquid phasesbeneath the sodium sulfate solution. Thus the globulin orglobulin- lipid complex adsorbed enough of the lip id solvent tochange its density sufficiently for separation by centrifugation.However, carbon tetrachloride and many other fat solvents, suchas carbon disulfide, xylene, bromobenzene, 1,2,4-trichloroben-zene, dichloroethylene, tetrachloroethane, and benzene, could notbe used in the place of ether because the separation of globulinwas not complete or a portion of the albumin was precipitated.However, globulin entered and was retained within the abovesolvents regardless of their density.

Aside from accelerating the separation of globulin, ether didnot appear to modify the action of sodium sulfate. Close agree-ment between centrifugates and Harris filtrates indicated thatalbumin was not altered. Globulin likewise could not have beenaltered greatly, since it redissolved in saline as shown above.No difficulties were encountered in obtaining clear centrifugates.The data presented in Table I represent the albumin concentra-

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G. R. Kingsley 735tions of many types of pathological sera, including several speci-mens from patients exhibiting hyperlipemia associated with thenephrosis syndrome or with obstruction of the bi le ducts. Robin-son, Price, and Hogden (6) were unable to obtain clear supernatantsolutions from some pathological sera, such as those from patientswith lipoidal nephrosis. Many workers have reported similardifficulties with the Howe (1) method.In the new method the delay of several hours required for floc-culation of globulin by previous methods is dispensed with, andin 5 to 10 minutes of centrifugation a clear solution is provided foralbumin determination. If this method is used in connectionwith the authors quantitative biuret method for protein deter-mination (8), total protein, albumin, and globulin can be deter-mined in about 45 minutes. Further studies on the possibleapplication of the method to spinal fluid and effusions are inprogress.

SUMMARYA rapid centrifugal method for the separation of globulin pre-cipitate is presented, based on the use of sodium sulfate in combina-tion with ether. Certain advantages over filtration and previouscentrifugal methods are described. The results were found toagree with those of accurate filtration techniques. The newmethod is superior to previously reported methods for analysisof certain types of pathological sera. The accuracy of the biuretmethod for the determination of serum proteins has beenconfirmed.

BIBLIOGRAPHY1. Howe, P. E., J. Biol. Chem., 49, 109 (1921).2. Howe, P. E., J. Biol. Chem., 49, 93 (1921).3. Goettsch, E., and Kendall, F. E., J. Biol. Chem., 109, 221 (1935).4. Robinson, H. W., Price, J. W., and Hogden, C. G., J. Biol. Chem., 120,481 (1937).5. Harris, R. C., J. BioZ. Chem., 137,751 (1939).6. Robinson, H. W., Price, J. W., and Hogden, C. G., J. Biol. Chem., 126,207 (1938).7. Robinson, H. W., Price, J. W., and Hogden, C. G., J. BioZ. Chem., 126,

213 (1938).8. Kingsley, G. R., J. BioZ. Chem., 131, 197 (1939).9. Troloar, A. E., An outline of biometric analysis, Minneapolis, 139 (1935).

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