Further studies on thermosoluble proteins in serum and urine

671

CLINICA CHIMICA ACTA

FURTHER

STUDIES

ON THERMOSOLUBLE

PROTEINS

IN SERUM AND

URINE

JACQUES

R. POORTMANS

Laboratoire de l’Effort*, Universite’ Libre de Bruxelles, 28 avenue P. Higer, 1050 Bruxelles (Belgium) (Received

June 18, 1970)

SUMMARY

Thermosoluble proteins have been isolated from normal human serum and urine. The following proteins were identified in both media: tryptophan-rich prealbumin, ml-acid glycoprotein, a,-antitrypsin, a,HS-glycoprotein, Zn-a,-glycoprotein, transferrin, hemopexin, /&-glycoprotein I, &glycoprotein III, yG-fragments. Thermosoluble proteins in serum are exclusively related to the above components, while in urine 18% of heat-resistant proteins belong to other tissue proteins. Quantitative determination has shown that a,-acid glycoprotein and tryptophan-rich prealbumin constitute the major part of thermosoluble proteins in serum, while a,-acid glycoprotein and Zn-a2-glycoprotein are the major components in urine.

In 1931, Donaggiol described a reaction in urine, based on precipitation which appears upon mixing solutions of thionine and ammonium molybdate. Donaggio discovered that normal urine had no effect upon this reaction, while urine from physically tired or pathologic subjects inhibited this “obstacle phenomenon”. Donaggio’s reaction is thus said to be positive when most or all of the dye remains in solution. Other thiazine dyes, such as methylene blue or toluidine blue, may be used instead of thionine. Primarily utilized with urine, the reaction was applied to serum by Heremans who mentioned that the determination of Donaggio substances was intimately correlated (Y = 0.91) with that of thermosoluble proteins. In a previous study we came to the conclusion that not only one, but several components belong to this group of protein+. However, at the time, only a partial identification was attempted in serum. The present paper deals with a further identification of thermosoluble proteins in serum and urine, as well as their quantitative determination in both fluids.

* Director:

Prof. M. Segers. Clin. Chim. Ada, 30 (1970) 671-678

672 MATERIALS

POORTMANS

AND

METHODS

To one volume of fresh serum was added 1.5 volume of 0.1 M acetate buffer (pH 4.6). After 5 min boiling in a water bath, the mixture was centrifuged at 12000 g during IO min and the supernate was concentrated to approximately 7 g% of proteins by ultrafiltration under reduced pressure4 using cellulose dialysis membrane of 8/32 inch inflated diameter (Visking membrane). The fraction remaining in the bag was centrifuged and submitted to qualitative and quantitative determinations. Agar gel electrophoresis and immunoelectrophoresis were performed according to the techniques of Wieme6 and Scheideggere respectively. Double diffusion in agar was done using Ouchterlony’s technique’. Normal urines were collected from healthy male adults and pooled. After adjusting to pH 4.6 with acetic acid, the urine pool was submitted to the same conditions as described above for the serum. Pure yG-globulin was isolated by DEAEcellulose chromatography10 from a batch of fraction II of Cohn (Mann Res. Lab. No. C2445). The purity and homogeneity of isolated yG was tested with specific antisera against yG, y-chain, Faby-fragment, Fey-fragment, A-chain and x-chain. 150 mg of pure yG were dissolved in IO ml of 0.9 g% NaCl and then mixed with 15 ml of 0.1 M sodium acetate buffer, pH 4.6. The mixture was placed in a boiling water bath and treated as mentioned before. A final volume of 0.1 ml was achieved for the thermostable fraction. Pure yG-globulin was also submitted to papain digestion, according to the method of Porter4. Antisera

Antisera to albumin, u,HS-glycoprotein, Zn-a,-glycoprotein, haptoglobin, transferrin, 3 S y,-globulin, yG-globulin were obtained by footpad immunization of rabbits with pure antigen emulsified in complete Freund’s adjuvant. All antisera were tested against human serum and preparations of antigen by immunoelectrophorlesis and double diffusion in agar. Antisera to tryptophan-rich prealbumin, al-acidglycoprotein, cc,-antitrypsin, ceruloplasmin, a,Gc-globulin, cc,-macroglobulin, hemopexin, B,A-globulin, ,9,-glycoprotein, yA-globulin, yM-globulin and CRP-globulin were purchased from Behringwerke (Germany). Antisera to fragments of immunoglobulin-G (kappa, lambda and heavy chains, Fc-fragment, Fab-fragment) were obtained from Hyland Company (U.S.A.). Specific antisera to /&glycoprotein II, /?,-glycoprotein III, yD-globulin were a gift from Drs. Schwick and Storiko (Behringwerke) . Protein quantitation

Protein concentration was performed by the biuret method as described by O’Brien and Ibbotts. Quantitation of individual proteins was made using the antisera described above, by the single radial immunodiffusion method of Mancini et aL9. The amounts of antiserum incorporated into each agar plate were adjusted following preliminary experiments. Standards from Behringwerke (Hoechst) lot nr. 286 AX at different concentrations were included with each set of determinations. A pure preparation of the Zn-4-glycoprotein, generously provided by Dr. K. Schmid, Boston Univ., Boston, Mass., was used for estimation of this protein. Light chains were measured using their dimer form as standards. Clin. Chim. Acta, 30 (1970) 671-678

THERMOSOLUBLE

PROTEINS

673

Ultracentrigugation Analytical ultracentrifugation was carried out in a Spinco Model E ultracentrifuge equipped with Schlieren optics. The sedimentation velocity was determined at zoo with a 12 mm (4’) normal cell at 59780 rev./min. RESULTS

Agar gel electrophoresis emphasizes that thermosoluble proteins are widely distributed from the prealbumin position to the y position in both serum and urine (Fig. I). However, it must be stated that the electrophoretic pattern is entirely different in serum and in urine. The main fraction in serum is located in the cathodic area, while the separation in urine is spread over a broader zone. By immunoelectrophoresis, numerous plasma proteins can be identified as thermosoluble substances in serum and urine (Fig. 2). There was no analogy between urine and serum concerning the precipitin lines. Immunodiffusion pattern with specific antisera revealed that the following proteins are present in both serum and urine: tryptophan-rich prealbumin, albumin (trace), crl-acid glycoprotein, cc,-antitrypsin, a, HS-glycoprotein, Zn-a,-glycoprotein, transferrin, hemopexin, /3,-glycoprotein I, &glycoprotein III, yG fragments. The latter ones were found different in serum and urine. Only free kappa chain was identified in serum, while kappa and lambda chains, Fc-fragments were observed in urine. Monospecific antisera to yA, yM and yD fail to give a precipitin line against thermosoluble proteins. Fig. 3 shows immunoelectrophoretic analysis of the antigenic relations of serum thermosoluble proteins with yG-globulin and its subunits. When tested with antisera against y-chain and Fey-chain, no precipitin line is observed. This result, therefore, excludes the presence of heavy chain-related substances as components of serum thermosoluble protein. On the contrary, a precipitin line is obtained when using an anti-Faby and an antikappa chain. The immunoelectrophoretic mobility of this component however remains in the usual range of yG-globulin, which is slower than the Fey-fragment but faster than the Faby-fragment. It may be said that this component belongs to light chains of kappa-type. Quantitative radial immunodiffusion shows that their level amounts to 0.18 mg/Ioo ml of normal serum. In order to evaluate the possible origin of these serum free light chains from the original yG-globulin, as a consequence of the heat treatment, native yG was submitted to the same procedure as that described for the obtention of thermosoluble proteins. Fig. 4 reveals that, under heat treatment in acetate buffer at pH 4.6, no y component remains in solution. This result shows that the free light chains present in normal serum are not products due to the artificial degradation of yG. Analytical ultracentrifugation patterns of thermosoluble proteins are shown in Fig. 5. A discrepancy appears in these patterns between serum and urine. Three peaks are depicted in serum, i.e. 12.6 S (2%), 5.2 S (4%), 3.0 S (94%), while two boundaries are revealed in urine, i.e. 15 S (8%) and 2.0 S (92%). Table I gives quantitative data for individual thermosoluble proteins. It reveals that al-acid glycoprotein is the major component in both serum and urine. The distribution of the other proteins is variable in serum and in urine. The latter contains more cr,-glycoproteins than serum, particularly Zn-h-glycoprotein. Table I also emphasizes the importance of yG fragments and unknown proteins in urine, Clin. Chim. Acta, -jo (1970) 671-678

POORTMANS

Fig. I. Agar gel electrophoresis of thermosoluble proteins in serum and urine. The upper and lower patterns refer to normal serum (NS) and normal urine (NU). Thermosoluble proteins of serum (ThS) are mainly distributed in positions of fast migration, while those of urine (ThU) show a different pattern. Tryptophan-rich prealbumin (PA) and z,-acid glycoprotein (c(,AG) are predominant in serum.

Fig. 2. Immunoelectrophoresis of thermosoluble proteins in serum and urine. The upper pattern refers to normal serum (NS) Thermosoluble proteins of serum (ThS) and of urine (ThU) were visualized by use of antiserum against human serum. Using specific antisera, tryptophan-rich prealbumin (PA), albumin (Alb), cc-acid glycoprotein (qAG), cc,-antitrypsin (or,AT), a,HSglycoprotein (c(,HS), Zn-cr,-glycoprotein (Zna,), transfekrin (TF), hemopexin (HEM), &glycoprotein I (b,GP), light chain (L-chain) were identified. Clin. Chim.

Acta,

30 (1970)

671-678

THERMOSOLUBLE

PROTEINS

675

Fig. 3. Immunoelectrophoretic identification of the y related substances of thermosoluble proteins. In A and B, thermosoluble proteins have been allowed to diffuse against several antisera: antilambda chain (anti-A), anti-yG, anti-kappa chain (anti-x), anti-y heavy chain (anti-v), anti-y Fe fragment (anti-Fey). In C, thermosoluble proteins (2) and yG hydrolyzed with papain (1,3) were submitted to diffusion against an anti-Fey fragment (anti-Fey) and against an anti-Faby fragment (anti-Faby). The various antisera identify the yG-related substance to a kappa chain.

Fig. 4. Effect of heat treatment on yG. yG ( I ) and yG treated by heat (z) were submitted to immunoelectrophoresis using an anti-yG and an anti-kappa chain (anti-x). It is shown that heat treatment does not liberate light chain from the native globulin.

wherein their level amounts to nearly 20% of the whole protein content. The recovery of thermosoluble proteins in the concentrated fluids from the original medium prior to heat treatment is highly satisfactory. For example, in native serum we found the Cl&. Chim. Acta, 30 (1970)

671-678

POORTMANS

676

Fig. 5. Ultracentrifugation pa,tterns of thermosoluble proteins of serum and of urine. Photographs were taken after reaching full speed as indicated below each pattern. TABLE

I

THERMOSOLUBLE COMPONENTS

PROTEINS: EXPRESSED

QUANTITATIVE IN

PER

CENT

DETERMINATION

OF TOTAL

OF INDIVIDUAL

PROTEIN

Proteins

Serum

Urine

Tryptophan-rich prealbumin Albumin a,-Acid glycoprotein cr,-Antitrypsin Zn-a,-Glycoprotein a2 HS-glycoprotein Transferrin Hemopexin &Glycoprotein I Light chains @Fragments and unknown components

21.2

10.5

trace 62.0 1.1 0.5 0.2

0.6 0.1

12.5 0.3 1.5*

0.1

34.3 4.6 20.6 6.4 1.2

I.3 3.0 18.0

* This percentage is only related to unknown components.

level of thermosoluble proteins to be around 60 mg/roo ml of serum which is very close to the 57 mg/roo ml of protein (corrected by the concentration factor) found in our thermosoluble protein preparationzl. DISCUSSION

The present study puts forward some views concerning the thermosoluble proteins. Immunodiffusion and immunoelectrophoresis point out that these heatresistant proteins are widely distributed in serum and urine, but with a different pattern for both media. Ultracentrifugation studies reveal that these components are mostly of low molecular weight and have been identified mainly as &,-acid glycoC&z. Chim.

Acta, 30 (1970)

671-678

THERMOSOLUBLE

PROTEINS

677

protein, Zn-~~-glycoprotein, GUS-glycoprotein and ~~-glycoprotein I Several authors established, at least theoretically, a relationship between connective tissue and thermosoluble proteinP-14. The cycle of Jayle15, which represents a schematic view on the degradation of glycoproteins of connective tissue, suggests that thermosoluble proteins (Donaggio substances) and acid glycosaminoglycans form a soluble complex in the connective tissue. In response to stress (i.e., strenuous physical activity, infections) as well as in necrotizing diseases, these substances are dissociated and released into the blood stream. After filtration through the kidney, the thermosoluble proteins are excreted. The results obtained in the present investigation show that the Donaggio substances in serum are exclusively related to proteins found in normal serum. On the contrary, thermosoluble proteins of urine belong to plasma proteins and to other tissue proteins, presumably of kidney, urether, bladder and seminal glands origin. Several plasma proteins are heat-resistant in acetate buffer at pH 5.0, i.e. &,-acid glycoprotein, tryptophan-poor a,-glycoprotein, cr,-glycoprotein, cx$-neuramino-glycoprotein, hemopexin 16.According to this physico-chemical characteristic, the above glycoproteins would constitute the major part of the Donaggio substances. However, this assertion is not to be taken strictly, since in addition to the classical heat-resistant protein+, we have found that tryptophan-ricl~ prealbumin and & glycoprotein I were also thermosoluble proteins in the conditions used in the present study. Moreover, after heat treatment, tryptophan-rich prealbumin, &,-acid glycoprotein and ,$,-glycoprotein I were recovered at nearly 50% of their initial concentration in native serum. The recovery of the other thermosoluble proteins ranged from 0.1% (adds-glycoprotein) to zo/, (Zn-~~-glycoprotein). The concentration of light chains amounts to approximately I .8 tug/ml of normal serum. The physiological significance of excessive light chain production is not apparent, but it may explain its appearance in normal human urine. Since the kidney filters approximately 180 1 of plasma per day, our serum level of light chains found in the present investigation will give a maximum of 324 mg of light chains which might pass through the glomeruli. However, the renal pe~eability to plasma proteins is related to their molecular weight. Gregoire and Lambertl’ measured the sieving coefficient of light chains (dimer form) which they found to be 0.09. Therefore one may expect to have a glomerular filtration of ag mg of light chains per day. In addition, it has been shown that nearly 80% of the metabolism of this component was accounted for by kidney catabolism, the remainder being excreted as proteinurial*sls. When these data are applied to the present results, an approximate urinary excretion rate of 6 mg/z4h is obtained which is close to the values given by Vaughan et aLao (4 mg/z4 h). To conclude, one may state that the determination of total thermosoluble proteins in serum and urine remains questionable as a screening test for connective tissue damage. It would be more appropriate to determine specific acute phase reactants, i.e. cr,-acid glycoprotein or haptoglobin by radial immunodiffusion. REFERENCES

I A. DONAGGIO, Rev. Neural., 2 (1933) 155. 2 T. I?.HEREMANS, Rev. B&e Pathol. Mdd. ExDtl.. 26 (1958) 26~. ’ ’ _- ’ 3 j. POORTMANS, Clin.Chik Acta, 7 (1962) 334. 4 P. H. EVERALL AND G. H. WRIGHT, J. Med. Lab. Technol., 15 (1958) 209. 5 R. J. WIEME, Chin.Ckinz.Acta, 4 (1959) 317. C&s. Chim. Acta,

30 (I974

671-678

678

POORTMANS

6 J. Jo SCI~EIDEGGER, Ir&un. Arch. AtlavgyA#& ImmuaoE., 7 (1955) 103. 7 D. ULXSITERLONY, Acta R&W. Microbial. Salad., 20 (1949) 507. 8 D. O'BRIEN AND F. A. IBBOTT, Laboratory Manzcal of Pediatrics, hficvn- a& f/'btra-mi~v~ &achemical Techniques, Harper and Row, New York, rg62, p. 260. 9 G. MANCINI, A. D. CARBONARA AND J. F. HEREMANS, Immunochemistry, 2 (1965) 235. IO H. A. SOBER AND E. A. PETERSON, Federation Proc., 17 (1958) 1116. II R. R. PoRTER,B&~~~.J., 73 (1959) 119. 12 P. BUGARD, La Ff&gw, Masson, Paris, r960, p. 250. 13 J. F. HEREMANS, f&o. Be& P&ol. M&Z. E@&., 26 (1958) 127. rq F. TAYEhU, .&$tk.Ann. i9iociti;9ra. M&d., 16 (r954) 2Tj. 15 M. F. JAYLE AND G. BOUSSIER, ExptZ.Bvw. Biachiwa. h&d., 17 (rgyj) 157. 16 H. E. SCHULTZ~ AND J. F. HEREMANS, Molecular Biology of Hunzan Proteins,Vol. r, Elsevier Amsterdam, rg66. 17 F. GREGOIRE AND P. P. LAMBERT, C&n. Sci.,25 (x963) 243. r8 A.SOLOMON, T. AWALDMAN, J.L.FAHEYAND A.S.MACFARLANE, J.Clin. Invest., 43(rg64) 103. 19 R. D. WOCHNER, W. STROBER AND T. A. WALIBXAN~~,J. Exptf. Med., ~26 (1967) 207. 20 J. H. VAUGHAN, R. F. Jacox AND B. A. GRAY, 1. Clip. Innvest., 46 (1967) 266. zx J. R. Poortmans, J. App6. PhysioZG, in press. Clin. Chim. Acta, 30 (1970) 671-678