Isolation from plasma of an α1-glycoprotein resembling “orosomucoid”

ARCHIVES

OF

BIOCHEMISTRY

AND

Isolation

BIOPHYSICS

from

Plasma

Resembling A Simple Method LUDWIG

KORNEL’,

Departments

RALPH

of Biochemistry,

280-288 (1967)

122,

of an cu,-Glycoprotein

“Orosomucoid”

for Its Estimation

in Blood

E. SCHROHENLOHER,

AND ROBERT

Medicine, and Oral Biology, University Birmingham, Alabama, 35203

Received

January

26, 1967; accepted

and Tissues*

May

of Alabama

C. CALDWELL Medical

Center,

19, 1967

An acid aY1-glycoprotein has been isolated from human plasma, and closely resembles “orosomucoid” as to its ultracentrifugal and electrophoretic behavior and chemical composition. However, the two preparations differed from each other immunologically. The data obtained suggest that the isolated protein is actually “orosomucoid,” but probably less denatured than the preparat,ions obtained by other procedures. A method is described for the isolation and estimation of this cyl-glycoprotein from plasma and tissues, which, owing to its simplicity, is suitable for use in routine clinical laboratories. The procedure described is also highly specific, as indicated by over 90% purity of the isolated product.

In the course of work concerned with the development of a technique for deproteinization and defatting of plasma, suitable for use in the method for the estimation of conjugated corticosteroids in blood (l), it was found that one of the separated fractions consisted of a protein with electrophoretic mobility of an cY1-globulin. This protein proved to be homogeneous on ultracentrifugation and to have properties of an acid (Alglycoprotein. Further investigation demonstrated a high degree of purity of the isolated protein and established its chemical composition and some of its immunological characteristics. Although it closely resembled “orosomucoid” (2), it differed from it immunologically. The simplicity of the technique used for * This work was supported in part by grants HE-04751, AM-03555, and DE-02110 from the National Institutes of Health, Bethesda, Maryland. 1 Present address: Steroid Unit, PresbyterianSt. Luke’s Hospital, 1753 W. Congress Parkway, Chicago, Illinois 60612. Please send requests for reprints to this address.

the isolation of this oil-glycoprotein and the high degree of purity of the isolated product prompted us to report this method. The method described in this paper is suitable for use in routine clinical laboratories. EXPERIMENTAL

PROCEDURE

n/IATERIALs All solvents used for the protein isolation procedure were J. T. Baker, analytical grade, were redistilled without further purificat,ion, and were of chloroform, stored at 4”, with the exception which was freshly distilled from anhydrous K&O,.

TECHNIQUE FOR THE ISOLATION OF THE al-GLYCOPROTEIN FROM PLASMA A lo-ml aliquot of hepariniaed plasma, separated immediately after the withdrawal of blood, was placed in a 90.ml glass-stoppered test tube and extracted with 5 volumes of chloroform by vigorous shaking for 30 seconds. After centrifugation, the extract was separated,2 and 20 ml 2 The separation is conveniently achieved by means of a pressure-operated liquid separator (Cat,. No. JE-4225X) obtained from Scientific Glass Apparatus Co., Bloomfield, New Jersey.

280

ISOLATION

FROM

PLASMA

of methanol was added from a burette to the chloroform-extracted plasma. To assure formation of a fine precipitate the tube was simultaneously agitated. It was then stoppered, the contents were thoroughly mixed by inversion, and were refrigerated for 10 minutes (O-4”). Forty ml of carbon tetrachloride was subsequently added to the tube, and the mixture was vigorously shaken for 90 seconds. After standing for 20 minutes at room temperature, the tube was centrifuged for 15 minutes at 2500 rpm, and the upper alcoholic layer was carefully decanted into another 90.ml glassstoppered test tube. The precipitated proteins formed a strong barrier, stuck to the walls of the test tube, between the CC14 and the methanolic and prevented the former from being layers, poured off during the decantation of the alcoholic supernat,ant fluid. The aqueous met,hanolic extract was then acidified to pH 4.5-5.0 (indicator paper) by addition of 25-30 drops of 2 M acetate buffer (pH 4.5). The buffered alcoholic extract was refrigerated (O-4”) for 30 minutes, and was subseqtiently washed twice witn 40 ml of n-hexane by shaking each time for one minute and centrifuging for 10 minutes at 2000 rpm. The hexane layers were carefully aspirated off, and the aeqrtoits methanolic layer was decanted into another clean test, tube to be used for further fractionation.3 The remaining white gelatinous sediment in the tube was the separated al-glycoprotein. This sediment is readily soluble in a small volume (0.2-0.5 ml) of 0.05 M sodium barbiturate buffer, pH 8.6.

TECHNIQUE FOR THE ISOLATION OF THE o(l-G~~~~~~~~~~~ FROM TISSUES Tissues were homogenized, and the homogenate was extracted twice with distilled water (1 ml H,O/20 mg of tissue weight prior to homogenization) by vigorous shaking for 20 minutes each time, followed by centrifugation (10 minutes at 2000 rpm) and decantation of the aqueous extract. The extracts were combined and divided into g-ml aliquots, each of which was extracted with 50 ml of chloroform (or dichloromethane) in the same manner as in t’he method for plasma. The extract was removed, and to the remaining aqueous phase 20 ml of methanol was added from burette. The procedure followed is identical with that described for plasma, with the exception that the aqueous methanolic layer is pipetted off, not decanted, since the homogenized t,issue proteins do not always form a strong barrier between the Ccl, and the alcoholic layers. Tissue residues left after the extraction with water were resuspended in HZ0 3 Isolation and characterization of further fractions will be described in a subsequent communication.

OF AN oli-GLYCOPROTEIN

251

(with a volume of water equal to that with which the extraction was previously carried out), and this suspension was subjected to the same fractionation procedure as that described for the aqiieous extracts.

NETHODS USED FOR THE CHARACTERIZATION OF THE ISOLATED PROTEIN Electrophoretic studies. Conventional paper electrophoresis was carried out in a Beckman model R electrophoretic apparatus; runs were performed at pH 8.6 in 0.05 M Verona1 buffer and at, pH 6.4 in pyridine-acetic acid buffer (3), at 250 V and 1.5 mA for 6 hours. For the isoelectric point estimation, electrophoretic runs were performed High Voltage Electrophoretic in an “Ensco Chamber” at 0.9 mA/cm paper width (400-GO0 V per paper strip, depending on the composition of the buffer used), on Whatman No. 3MM paper as supporting medium, at, ten different pH values: 1.9, 2.2, 2.4, 2.8, 3.0, 3.3, 3.7, 5.3, 6.4, and 8.6. The buffers used were those described by us previously (3) only without any alcohol content,? formicacetic acid for pH 1.9; formic-acetic acid-pyridine for pH 2.2-3.4; pyridine-acetic acid for pH 3.7G.4; and sodium barbital buffer for pH 8.G. Electrophoretograms were stained with 0.1% bromophenol blue in methanol. Disc-gel electrophoresis was performed according to the method of Davis (4) on a 7.5y0 polyacrylamide gel, in Tris-glycine buffer, 0.1 p, pH 8.3, at, 5 mA per column. After the completion of the rim, the gel columns were stained with amidoSchwartz, followed by destaining of the “backwith 7.5y0 acetic acid (soaking, with grotmd” frequent changes of the acid). Ultracentrifugal analysis. Studies of homogeneity on ult,racentrifugation and of the sedimentation coefficient, were performed in a Spinco model E ultracentrifuge at. 20” with 12.mm single or double sector cells. Samples were dissolved in 0.97;, saline or pH 8.6 sodium barbital buffer, 0.1 p. Measurements on t,he resulting patterns were made by using a Nikon microcomparator. Sedimentation rates were calculated and corrected to standard conditions as described by Schachman (5). The partial specific volume of the (~1 preparations was assumed to be 0.73. Sedimentation values at infinite dilution (si,.m) were estimated by extrapolation of the SZO.~ values determined at four different concentrations over the range of l-6 mg/ml. The concentrations of the samples were estimated from the area under the boundary, and 4 Alcohol was necessary for high voltage electrophoretic runs in some buffers to increase their volatility and improve dissipation of heat generated (3).

282

KORNEL,

SCHROHENLOHER,

the appropriate correction for radial dilution was applied. Chemical analysis. Chemical analyses for protein, hexose, hexosamine, sialic acid, and fucose were performed as previously described (6). Nitrogen was determined by a micro-Kjeldahl method (7). Since sialic acid and hexosamine also contain nitrogen, these nitrogen values were subtracted from the total to give the protein nitrogen value which was multiplied by 6.25 to give the protein concentration. Hexose was determined by Dische’s method (8) using a 1:l mixture of galactose and mannose as a standard. Dische and Shettles’ (9) cysteine-sulfuric acid method (CyR 10) was used to measure fucose concentration. Hexosamine was determined by Boas’ modification (10) of the Elson-Morgan method, but the ion-exchange step was omit,ted. Prior hydrolysis of the al-glycoprotein was performed in 4 N HCI at 100” for 3 hours, and the glucosamine-HCl standard was treated in the same way. Sialic acid was determined by the direct Ehrlich method (11) ; a standard of chromatographically homogenous N-acetylneuraminic acid (Sigma, St. Louis) was used that contained 4.527, nitrogen. The concentration of the al-glycoprotein in plasma was calculated by adding the concentrat,ion (mg/lOO) of protein to the total carbohydrate concentration. Immunological studies. The method for immunoelectrophoresis was based on the micromethod of Scheidegger as described by Grabar (12). Double diffusion in agar was performed by the technique described by Ouchterlony (13). Two y0 agar in pH 8.6 sodium barbital buffer, 0.05 ionic strength, was employed for these studies. Antisera to pooled normal human serum, prepared in the horse, were generollsly supplied by Dr. William Hammack of this institution, as well as by Institut Pasteur, Paris, France. Antisera prepared in the rabbit were the product of Mann Research Laboratories, Inc., New York. For certain experiments the antisera were absorbed with slight excess of human serum albumin. The presence of excess albumin was confirmed by Ouchterlony agar diffusion studies.

AND

CALDWELL

of the tube. This material, whether in dry or wet form, was only scarcely soluble in water or in weak acids, but it was readily dissolved in a few drops of saturated solution of NaHC03 or of a 0.05 M sodium barbital buffer, pH 8.6. The isolated protein, when in an aqueous solution, was not precipitated by boiling, by 0.6 M perchloric, 5 % trichloroacetic, or 0.2 M sulfosalicylic acids, or by ammonium sulfate concentrations below 70% of saturation, but it was readily precipitated with phosphotungstic acid. Eleclrophorelic studies. h conventional paper electrophoresis of the isolated protein at pH 8.6 and 6.4 revealed that it had the mobility of an cY1-globulin. Free electrophoresis (Tiselius) of this protein gave a single symmetrical peak. Disc-gel electrophoresis revealed, in addition to the main protein component, 3-4 very faint bands of mobility slower than that of the al-glycoprotein (Fig. 1). The concentration of these minor components, estimated by densitometry, and after the elution of the individual bands, dialysis, and weighing, did not’ exceed 8% of the total protein weight. Xoreover, after the elution of the main al-glycoprotein band from the gel-electrophoretic column, the analysis of the carbohydrate components of this band gave results which

RESULTS CHARACTERIZATION OF THE al-GLYCOPROTEIN

ISOLATED

Physical properties. The protein precipitated from t#he aqueous methanolic extract with acetic buffer at pH 4.5 in the cold consisted entirely of a whitish-grey gelatinous sediment which became colorless and transparent on drying by inversion, and drainage

FIG. 1. Disc-gel electrophoresis of the isolated orI-glycoprotein from plasma of various individuals (samples 1-5) compared with that of crystalline “orosomucoid” prepared by the method of Schmid (sample 6) (see footnote 7 in text).

ISOLATION

FIG. 2. Electrophoretic

FROM

mobility

PLASMA

283

OF AN LU&LYCOPROTEIN

of the pooled

sample

of the isolated

ml-glycoprot,ein

(sample 1) compared with that of Schmid’s orosomucoid (sample 2) at pH values ranging from 1.9 to 8.6. Both preparations exhibited identical electrophoret.ic mobility at all pH values; sample 3: normal human serum run at pH 8.6 in 0.1 I* barbital buffer; a, application lines. -

were virtually identical with those obtained with the protein not subjected to the geldisc electrophoretic separation. No lipid components were detected by either osmium or Sudan III staining of the electrophoretic strips. The isoelectric point of the isolated (Yeglycoprotein was e&mated by electrophoretie technique. Runs were performed at ten different pH values under the conditions outlined in Experimental Procedure. The isoelectric point was at pH 3.0 in a pyridineformic acid-acetic acid buffer. Figure 2 shows the mobilities of the al-glycoprotein at different pH values. C’ltracentrifugal analysis. Sedimentation analysis of several preparat’ions from individual subjects, and of a sample of pooled preparations from several subjects, revealed only a singIe symmetrical boundary (Fig. 3) Concentrat’ions over t,he range of l-6 mgf’ml were examined. The sediment’ation constant, extrapolated to zero concentration (s~~~,~), as determined 011 the pooled sample and run in isotonic saline, was 3.4s. The sediment’ation coefficient demonskated a relatively small concentration dependence. At 6 mg/ml the s20,W value was 3.1s. Chemical analysis. The isolated al-gIycoprotein was analyzed for the carbohydrate and protein content (Table I). The carbohydrate composition of our protein is very similar to that reported for “orosomucoid.” Of interest are variations in the percentrage of various carbohydrate components in sam-

FIG. 3. Ultracentrifugal analysis of the 01~. glycoprotein isolated from plasma of two different subjects (upper and lower pattern). The analysis was carried out in barbital buffer, pH 8.6, O.lr, at, 59,780 rpm. Left, after 64 minutes; center, after 112 minutes; right, after 144 minutes; all three photos taken at a phase plate angle of 45”.

pies from different individuals, this, howa conspicuous ever, apparently without change in the electrophoretic, ultracentrifugal and immunological behavior of this protein (see below).5 It should be pointed out that values reported by other investigators for the concentrations of various carbohydrate components of a-glycoproteins (2, 14-17) were obtained as a rule on pooled specimens. Following an extensive dialysis of the isolated al-glycoprotein, the results 5 Corresponding molar ratios of various carbohydrate components in samples from different individuals were as follows (mean =I=SD) : hexose/ hexosamine, 1.25 f 0.17; sialic acid/hexosamine, 0.76 + 0.05; fucose/hexosamine, 0.10 f 0.05.

284

KORNEL,

SCHROHENLOHER, TABLE

CHEMICAL

COMPOSITION

OF THE

-

ISOLATED

AND

CALDWELL

I

CWGLYCOPROTEIN 7” of total

COMPARED

WITH

THAT

OF OROSOMUCOID

glycoprotein __

Preparation Hemsamine

HeXOSe

Sialic

,2;!

acid

hydrate

~ai-Glycoprotein (our method, 13 subjects) Range Mean Pooled ai-glycoproteina (our method) al-Acid glycoprotein (Schmid)r’ Orosomucoid

Concentration in plasma (w/100 ml)

13.4-17.8 15.1 f 1.3 14.3

10.1-12.3 11.0 f 0.7 12.0

13.2-15.9 14.4 f 0.7 13.9

0.5-2.4 1.1 f 0.6 0.9

41.7 41.0

13.6

11.2

14.1

0.7

39.6

15.0

12.0

12.0

1.0

41.0

-

-

57.6-95.7 78.2

75.0

0 This sample was obtained by pooling oci-glycoprotein isolated from 20-ml plasma specimens from 27 subjects. The pooled preparation was purified by precipitating possible contaminants at pH 3.7 with ammonium sulfate added to 70% of saturation, followed by an extensive dialysis of the supernatant fluid: the ai-glycoprotein was then re-precipitated with ethanol (357$ concentration) in the presence of barium ions, at -15”, and again dialyzed. b Values given here are those obtained in our laboratory for Dr. Schmid’s preparation. c After Winzler (2), composite data of several published figures.

of chemical analyses were virtually identical with those performed on the crude material. Immunological studies. The antigenic characteristics of the isolated ocl-glycoprotein were investigated by immunoelectrophoresis and Ouchterlony agar diffusion. Immunoelectrophoresis of several preparations from various plasma samples, in which antisera to pooled human serum were used, consistently demonstrated a fairly well-defined precipitin line in the al-globulin region (Fig. 4). In addition, in some instances, two or three fainter precipitin lines were noted, all of them of a-globulin mobility. Samples of normal human serum were run simultaneously as controls. A precipitin line corresponding to albumin was also frequently noted, This line was not seen when antisera were used which were first absorbed with a slight excess of human serum albumin (the absorbed antisera did not further precipitate human serum albumin). The absorption of anti-albumin antibodies did not affect formation of precipitin lines by the absorbed antisera

in the 01~region.

The Ouchterlony agar diffusion studies demonstrated that the cul-glycoprotein preparations

from various

individuals

contained

components which were very similar

anti-

FIG. 4. Immunoelectrophoresis of the oci-glycoprotein preparation; upper part, actual photograph of the plate; lower part, diagrammatic sketch. Normal human serum was applied to the upper sample well, the cri-glycoprotein preparation to the lower well. The antisera to pooled human serum were placed in the upper and lowest troughs; the antiserum in the latter was first absorbed with an excess of human albumin. The middle trough was left empty.

genitally. The principal precipitin lines given by preparations of albumin-absorbed antisera from different patients gave reactions of identity with each other and with prepara-

ISOLATION FROM PLASMA OF AN o(l-GLYCOPROTEIN

FIG. 5. Ouchterlony agar diffusion of various oil-glycoprotein samples. Antiserum to pooled human serum, absorbed with an excess of albumin, was placed in the center well; aI-glycoprotein samples from different individuals were placed in the peripheral wells. The principal precipitin lines of all preparations gave react,ions of ident,ity. tions from pooled normal sera (lpig. 5). As was observed on immunoelectrophoresis, certain preparations gave several precipitin lines (Fig. 5, samples l-3, and 6). The additional precipitin lines were usually much weaker and were located between the principal line and the antiserum well. Ko precipitin lines were obtained with al-glycoprotein preparations with commercial antisera to orosomucoid.6 The goat antiorosomucoid antiserum gave a single line with pooled normal human serum. However, the monkey anti-orosomucoid antiserum failed to precipitat,e with the same pooled human serum. COMPARISON OF PROPERTIES OF al-GLY~OPROTEIN ISOLATED BY OUR R!~ETHODWITH THOSE OF “OROSOMUCOID”’ Electrophoretic mobility and estimation of isoelectric point. Both preparations, the LYE6 Antiserum to human orosomucoid prepared in the goat’ was obtained from Hyland Laboratories,

285

FIG. 6. Ouchterlony agar diffusion of cur-glycoproteins prepared by our method (wells 1 and 3) and by the method of Schmid (well 2) ; the lack of immunological identity between the two preparations is demonstrated. Antiserum to pooled human serum (prepared in rabbit), absorbed with an excess of human albumin, was placed in well 4. Note also the stronger antigenicity of our preparation (same protein concentrations were used for both preparations). glycoprotein isolated by our method and Dr. Schmid’s al-acid glycoprotein (“orosomucoid”), exhibited identical electrophoretie mobility at ten different pH values ranging from 1.9 to 8.6 (Fig. 2). The isoelectric point determined by the electrophoretic method was at pH 3.0 for either preparation. A slight difference between this and the one of 2.5: reported by Schmid for “orosomucoid” is probably due to different buffers used in his and our studies.8 Ultracentrifugal analysis. Both preparations yielded identical results with respect to their homogeneity (a single symmetrical Los Angeles, California, and that prepared in the monkey was obtained from Mann Research Laboratories. 7 We are indebted to Dr. Karl Schmid of the Boston University, who kindly supplied crystalline “orosomucoid” (preparation AW-2) prepared by his method (14). * The influence of the nat,ure of buffer upon the isoelectric point of orl-glycoprotein was investigated by Schmid, who compared properties of his al-preparation with those of Winaler’s (14); Winzler reported an isoelectric point of 1.8 for “orosomucoid” isolated by his method (15).

2%

KORNEL,

SCHROHENLOHER,

AND

CALDWELL

(a) determining the amount of the OLDboundary, cf. Fig. 3) and sedimentation properties. The .sO*~, zuvalue was found to be glycoprotein co-precipitating with all other plasma proteins during their removal with 3.4s for both our cul-glycoprotein and Dr. methanol, and (b) estimating the amount of Schmid’s “orosomucoid.” Chemical analysis. Table I shows that our the oil-glycoprotein not precipitated by our method from the aqueous methanolic superpooled preparation closely resembled Dr. Schmid’s crystalline al-acid glycoprotein as natant fluid: (a) The methanol-precipitated proteins to its composition and concentrations of the various carbohydrate components. The mean were resuspended in isotonic saline, and the al-glycoprotein isolation procedure was twice values of the estimations performed on individual specimens from 14 subjects also close- repeated on this suspension. The additional ly agreed with those of the pooled specimens, amount of the al-glycoprotein recovered in both Dr. Schmid’s and ours. The total car- this manner was less than 6% of the total bohydrate content of both preparations was Lul-glycoprotein initially isolated. (b) The aqueous methanolic supernatant also very similar. fluid, from which the oil-glycoprotein was Immunological studies. The oll-glycoproprecipitated by our method, was combined tein isolated by our method and Dr. with 0.66 volume of absolute et’hanol and Schmid’s “orosomucoid” did not give identity precipitin lines on an Ouchterlony agar 0.01 volume of aqueous 40% (w/v) barium diffusion plate with antisera to pooled hu- acetate, to precipitate any proteins which man serum, obtained from various sources would not be completely removed by our (cf. Experimental Procedure). Moreover, at procedure. No additional al-glycoproteins were present in this precipitate. similar protein concentrations, Dr. Schmid’s Recoveries of added cY1-glycoprotein to preparation gave a much fainter precipitin plasma specimens prior to the isolation proline than our al-glycoprotein (Fig. 6). The cedure ranged between 90 and 97 YG. same was true on immunoelectrophoresis, where both preparations exhibited an crlDISCUSSION globulin mobility. Lack of immunological The al-glycoprotein isolated by the methidentity was also demonstrated between our od described in this paper closely resembles preparation and “orosomucoid” prepared “orosomucoid” of Winzler (16) and “acid by the mesod of Weimer et al. (15).g glycoprotein” of Schmid (14) [the latter two are considered to be identical (2)]. The comPROPERTIES OF THE GQ-GLYCOPROTEIN parison of the properties of our preparation ISOLATED FROM TISSUES with those of the one prepared by Schmid’s Rat kidneys and livers were homogenized method revealed identical electrophoretic in isotonic saline, and the cY1-glycoprotein and ultracentrifugal behavior and very similar was extracted and separated as described in this paper. The physical properties (solu- chemical composition of both. However, the results of the immunological studies rebility and precipitability) of the isolated tisvealed differences: (a) lack of immunological sue protein, and its ultracentrifugal and electrophoretic behavior, were the same as identity between the two preparations; and (b) stronger antigenic properties of our prepthose of the protein isolated from plasma. aration when tested against human antisera. Similar immunological differences were also METHOD EVALUATION seen between our preparation and that of The reproducibility of the method deWinzler. From the data obtained, it seems scribed, expressed as standard error calcureasonable to assume that the protein isolated from a series of 10 quadruplicate and lated by our method is actually “oroso20 duplicate estimations, was 4 3 %. mucoid” (or, using Dr. Schmid’s term, “CQThe yield of the method was examined by acid glycoprotein”), and that the method described yields a virtually intact protein, 9 We are indebted to Dr. Richard J. Win&r, University of Buffalo, for supplying a sample of whereas other methods (14, 15) possibly re“orosomucoid” prepared by his method. sult in its partial denaturation.

ISOLATION

FROM

PLASMA

Work is in progress aimed at a final elucidation of this important point. If our assumption is right, that one of the proteins is the denatured form of the other, it should be possible to t,ransform one into the ot’her by simply applying to one of t,he proteins the procedure used for t,he isolation of the other. Further evidence, even more convincing, as to which of the two proteins is more is expected to be derived from “natural,” immunizat~ion experiment,s which are presently being undertaken. At t,his time, t’he possibility should also be considered that the two preparations may represent two different, glycoprotein species; however, this seems to be less likely. The importance of the measurement’ of a-glycoproteins in plasma has been repeatedly emphasized (16, 17), and increased blood and urinary levels of these proteins have been found in patients with rheumatoid arthritis (16-H), as well as in malignant’ neoplasia (16-20). The function of Lyl-glycoproteins is still obscure. It has been shown, however, that their concent,ration corresponds t,o the degree of activity of the inflammatory process (21--.23). It’ has been suggested that these proteins constitute an important metabolic product of connective tissues (24-26). Furthermore, a possible relation of glycoproteins to tissue proliferation and neoplastic growth has been considered (27,28). More recent,ly, crl-glycoprotein contents of leukocytes have been shown to be markedly increased in chronic myeloid leukemia (29). Large scale clinical investigations, however, have been handicapped by lack of suitable routine methods for a separate estimation of individual glycoprotein species. It is likely that’ different glycoproteins have different sites of origin and that their levels reflect different pathological processes (16, 30). nletjhods available and used for the estimation of plasma glycoproteins in clinical studies measure not one single protein component but a group of several glycoproteins. One of the most widely used of such methods is that of Winzler et al. (31), for the est’imation of the “seromucoid fraction” of blood. This met’hod, as well as its various modifications, is based on the solubility of these proteins in 0.6 N perchloric acid (while other

OF AN LU,-GLYCOPROTEIN

287

proteins precipitate) and their subsequent precipitation with phosphot’ungstic acid. Other routine procedures for t’he estimat)ion of glycoproteins, based 011 the electrophoretie separation of a-globulins (32, 33), also lack precision and specificity. As pointed out by Winzler (a), the progress in our understanding of the origin and significance of glycoproteins will depend 011 the availability of specific methods for the estimation of individual protein species. The techniques used by Weimer et al. (15) for the separation of “orosomucoid,” and by Schmid (14) for the preparation of “al-acid glycoprotein,” rendered invaluable service in the study of these proteins; however, they are too complex for use in clinical investigation. The method described in this paper presents the advantages of being simple enough for routine use, and specific. It permits isolation and quantitation’O of cyl-glycoprotein from 24 plasma samples by one technician within one working day. Furthermore, t,he method can easily be applied to urine, thus permitting renal clearance studies of t#his protein. l\lIeasurement of its concentration in the synovial fluid and in various tissues would be also of considerable int’erest. ACKNOWL

EI)GRIENT

We are grateftd to l)r. Richard J. Winzler for reviewing the manuscript, and for his helpful suggestions, and to I>r. Karl Schmid for his interest in this stttdy and for supplying crystalline orosomucoid. The skillful technical assist,ance of Mrs. Frances W. Wells and Miss Mildred Heard and the secretarial assistance of Miss Carolyn Clark and Mrs. Linda Ford are acknowledged with thanks. REFERENCES 1. KORNEL, L., J. Lab. Clin Med. 54, 659 (1959). 2. WINZLER, R. H., in “The Plasma Proteins” (F. W. Putnam, ed.), Vol. 1, p. 317. Academic Press, New York (1960). L., J. Clin. Endowinol. Metab. 24, 3. KORNEL, 956 (1964). 4. DAVIS, B. J., Ann. h-.E’. itcad. Sci. 121, 404 (1964). H. K., “Ultracentrifugation in 5. SCHACHMAN, Biochemistry.” Academic Press, New York (1959). 1oThe quantitation is readily performed by any of the color reactions measuring either the “proteo” or “glyco” moiet,y, or both.

288

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SCHROHENLOHER,

6. CALDWELL, R. C., AND PIGMAN, W., Biochem. Biophys. Acta 101, 157 (1965). Quantitative Or7. CLARK, E. P., “Semimicro p. 37. Academic Press, ganic Analysis,” New York (1943). 8. DISCHE, Z., Methods Biochem. Anal. 2, 327 (1955). 9. DISCHE, Z., AND SHETTLES, L. B., .J. Biol. Chem. 176,595 (1948). 10. BOAS, N. F., J. Biol. Chem. 204,553 (1953). 11. PIGMAN, W., HAWKINS, W. L., BLAIR, M. G., AND HOLLEY, H. L., Arthirtis & Rheum.at. 1, 151 (1958). 12. GRABAR, P., Methods Biochem. Anal. 7,l (1959). 13. OUCHTERLONY, O., Acta Pathol. Microhiol. Scan& 32, 231, (1953). 14. SCHMID, K., J. Am. Chem. Sot. 76, 60 (1953). 15. WEIMER, H. E., MEHL, J. W., AND WINZLER, R. J., J. Biol. Chem. 186, 561 (1950). 16. WINZLER, R. J., Methods Biochem. Anal. 2, 279 (1955). 17. GHEENSPAN, E. M., Advan. Intern. Med. 7, 101 (1955). 18. NETTLELBLADT, E., AND SUNDBLAD, L., Arthritis & Rheumat. 4, 161 (1961). 19. WINZLER, R. J., AND SMYTH, I. M., J. Clin. Invest. 27, 617 (1948). 20. SHETLAR, M. R., SHETLAR, C. L., RICHMOND, V., AND EVERETT, M. R., Cancer Res. 10, 681, 1950. 21. DE LA HUERGA, J., DUBIN, A., KUSHNER, D.

AND

22. 23. 24. 25. 26. 27.

28.

29. 30.

31.

32.

33.

CALDWELL

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