Physico-chemical studies on four macroglobulins

CLINIC.4 CHIMICA ACT.4

420

PHYSICO-CHEMICAL

STUI)IES

ON FOUR

VOL. 4 (1959)

MACROGLOBULINS

The formation of a precipitate of protein when certain sera are diluted with water was described by BR~~H~IACHAHIAND SES 1 and by s1.4~. WALDENSTR~RI~ diluted serum I in 16 and observed the bulk and appearance of the precipitate. Many other workers have used the “cater-euglobulill test” as a screening test for macroglobulinaemia. MACKAY cf al.” state that false positives are obtained notably in multiple myeloma while BICHEL, BINC ASD HARB~E 5, LLLJESTKAND AND OLHAGEN~, SANDKtiHLER7, I;ERRIM4N ASD ,/4NDERSON8 and MACKAY et ~1.~ repOrt negative results with sera containing macroglobulins. The stability of some proteins has been reported, STEIN AND ~VERYIXEIMEX~~ state that their “dilution fraction“ is remarkably labile while LERXER ASD GREENIGXG 11 reported a cryoglobulin as being stable over a long period. Electrophoresis of m~~croglob~llins has been recorded. Many reports show migration with the y-globulins while MACKAY et ad.3, FERRIMAN AND ANDEIISON~ and BICHIX, BISG AND HARIIOE 5 report P-globulin mobility and SKrros et al.‘2 record /!and u,-mobility. JIM AND STlillu’KA~~~~ and SEHOS lsreport macroglobulins showing a positive periodic acid-Schiff staining reaction for protein-bound polysaccharide. t’ltraviolet absorption spectra have been examined bv LEHNEII xw GRIXNHENG 11, IXRNER, BARNUM 4ND ‘1I;ATSON14, BaKL<, R13313~ AND \VK~ZLEL<~~and M~XWEXA, V.1.v r)Kri SCHAAF AND Ht%MAN I6 giving data for cryoglobulins and macroglobulins indicating a similarity

to the y-globulins.

GRWKR,

GRU.NX AND HijL
the spcc-

tra of three macroglobulins wit-h y-globulin and indicated a reduced tryptophan and tvrosinc content. The isoelectric precipitation of euglobulins was reported by GREEN 18 and by SANDOK AND CHEDDAHA14. SANDOR 2oand V~r~ccrss ASL) I.A~ROSSE 21 describe a euglobulin having an isoelectric point between PH 7.0 and 7.6 while JIM Asr) STEINK.~RIP’~ recorcl the maximum insolubility of a macroglobuIin paper describes some properties of four macroglobulins.

at pH 6.4. The present

;\IA’I’EIII_ALS AND METHODS Sera from a number of patients with rheumatic diseases have been encountered which on dilution with water yield a precipitate of protein (euglobulin). Case I (AR) was first observed in liovembcr 1953 when the water precipitable cuglobulin accounted for 1.75 g per IOO ml of serum. Eight months later a concentration of 2.80 g/loo ml was recorded and in August 1957 the concentration was 3.90 g/100 ml. The serum albumin had ranged from x.1 to 1.4 g/100 ml and the total y-globulins from 2.4 to 6.5 g/100 ml of serum during this period. Over a period of three years a survey of a large number of sera from patients attending a Kheumatic Clinic revealed nine further cases giving a positive “water” test in which serum euglobulin assays ranged from 0.41 to 1.48 g/100 ml. Three of these cases showing increased serum y-globulins (case 2 HC 2.1 g/100 ml; case 3 NG 3.4 gjroo ml; case 4 AMP 2.0 g/Ioo ml) together with case I lZ&erlc~~S p. .$“3

VOL. 4 (r959)

PHYSICO-CHEMICAL

STUDIES

ON M:\CKOGLOBULINS

421

(AK) were examined in the ultracentrifuge. The sera were found to contain macroglobulins. The euglobulins from these four cases were examined by a variety of techniques.

Precipitation of euglobulin from the sera examined reached a maximum after 15 min in the dilution range I in 20 to I in 50 where a nearly linear relation was obtained between dilution and optical density at 590 rnp. Flocculation usually occurred within 30 min. Quantitative assays were carried out by adding 0.2 ml of serum to 4.8 ml of water and measuring the optica density after r5 min. 0.05 ml of a saturated solution of sodium chloride was added to dissolve the globulin and the resulting optical density subtracted from the first reading. *Assayed protein solutions precipitated with sulphosalicylic acid were used as standards. Assays performed by centrifuging off the precipitate from 0.5 ml of serum after dilution with 15 ml of water and determination of the protein by a micro-Kjeldahl method showed close agreement with the turbidimetric

analysis.

Judged

by the amount

of precipitate

produced

on dilution

with water,

sera rc-

main stable when stored for at least one year at -25". They are stable at room temperature for several days but are unstable at ~7~. Sera repeatedly thawed at 37’ from the frozen state showed a progressive reduction in the amount of “euglob~~li~~”precipitated. Freeze dried sera stored at room temperature for up to one year and initially containing 1.20 and 0.86 g euglobulin/roo ml gave no precipitation after reconstitution and dilution

Isolated

with water.

macroglobulin

was dissolved

in veronal

buffer (pH 8.6,

0.1

X)

and ex-

amined by paper electrophoresis. Papers were dried at IIOO and stained with bromophenol blue, Sudan black and with periodic acid-Schiff stain using the technique of KI~IW AND

C~R~SWALI,

22.Results

PHYSICO-CHEYICAL

are given in Table

PROPERTiES

MACROGLOBULINS

OF THE

FROM

THE

I. Starch

SERUM FOUR

AND

gel elcctrophoresis

was

ISOLATED

CASES

Case Yt$

I (AK) 2 (HC) 3 WG) 4 (AMP)

F

F F 1;

29 55 55 58

45.8 30.2 ‘9.3 28.5

L.80 1.22 1 .qH 0.48

y-globulin y-globulin y-globulin y-globdin

+ -t + +

278 ----

:;; 278

‘4.3 13.4 ‘3.5 14.0

carried out using hydrolysed potato starch at a concentration of zz :.i, w/v in 0.03 M borate buffer at pa 8.6. Intense staining at the point of insertion of the sample serum or protein was seen consistent with the observations of SILBERMAN 23.The patterns for whole serum showed no otherwise abnormal characteristics.

A. E. STEEL

422

VOL. 4 kk59)

~‘ltm-violet absorption Clear solutions of the macroglobulins were prepared in I 9{,v/v hydrochloric acid solution at a final protein concentration of 50 mg/roo ml and optical densities measured in the range 240 to 300 rnp using silica cells in a “Gnicam” S.P. 500 spectrophotoand for y-globulin are shown in Fig: I. meter. Curves for the four macroglobulins

0.8 0.6

240

250

260

300 270 280 290 Wavelength rn/.&

Fig. 1. Cltra-violet absorption curves. Curve G: Human y-globulin, Curve1 :iVacroglobt~lins from cases AK and AMP, Curve 2 : Macroglobulks from cases NG and HC.

0

6

5

7

8 P”

l:ig. 2. Optical density produced by mixing serum (1 vol.) from cases of macroglol,ulinaenria with buffer (40 voIs., 0.0~ M) over 5 rsngeof pi values (curves AK, SG, and AMP). Curve f-‘is given by a saline solution of the protein isolated from case _kK, curve N with a normal serum and curve SM from a serum containing a 7Seuglobulin.

p H p Y~CZ ? $2z a t zon cwves Measured volumes of serum were added to buffer solutions range of 5 to 9 to give a final dilution

(:I1 = 0.02) in the pH

of I in 40. The optical density

was measured after 15 min at 590 rnp. Fig. z illustrates containing macroglobulin, an isolated macroglobulin, human serum.

of the mixtures

the results obtained with sera a 7Seuglobulin and normal

DISCUSSION A positive water dilution test combined with a very high serum viscosity indicates the possible presence of high molecular weight proteins. The four sera examined in this paper had these properties and contained macroglobulins when examined in the ultracentrifuge (see STEEL **, for data). The macroglobulins appear to be stable when stored at low temperatures but are unstable at 37@. It was frequently observed that solutions of macroglobulins yield precipitates of protein which are difftcult to redissolve on storing at z0 or at room temperature. One stored sample after redissolving gave only a single slow peak in the ultracentrifuge. Some samples of macroglobulin h’efeveaces 9, x/r,j

vo=. 4 (1959)

PHYSICO-CHEMICAL

STUDIES

ON MACROGLOBULINS

423

were found to dissolve on heating, an observation also noted by SANDK~HLER’. Analysis by electrophoresis at pH 8.6 showed these globulins to possess low mobility. Tests at other pH values (pH 4.5 to IO) indicated slower migration that other serum proteins. A stippled effect at the origin on the paper strips was noted in some experiments and may be due to precipitation of the macroglobulin at this point. All four macroglobulins showed a strong PAS staining rea,*tion for glycoprotein. From the apparent failure to penetrate starch gel blocks on electrophoresis it would appear that these y-macroglobulins combine size and asymmetry in their molecules since the normal S-19 a,-glycoprotein of serum migrates into the gel. It will be demonstrated in a subsequent report that these macroglobulins show very high viscosities in solution. All the sera gave marked precipitation in the region of pH 6.3 to 6.5, a saline solution of one of the globulins gave a peak precipitation at pH 6.4. Ultra-violet absorption analysis indicated a similarity to the y-globulins although extinctions were slightly lower.

Euglobulins containing macroglobulins have been isolated from four sera by dilution with water and examined for stability, electrophoretic mobility and staining reaction, ultra-violet absorption and isoelectric precipitation.

REFERENCES

1 1:. hr. BRAHMACHARI .4ND P. B. SEN, Indian Med. Gaz.. 58 (1923) 295. 2 R. H. I?. %A, China Med. J., 38 (1924) 35. 3 J. WALDEMTRBM, .4&a Med. Sand.. 117 (1944) ~16. a I. R. MACKAY,L. I. TAFT AND E. F. \VooDs,~rit. Med.J., I(I9j7) 561. SJ. BICHEL, J. BIPI'GAND N.HARBOE, Acta Med. Sand., 138 (1950) I. 6 A. LILJESTRAXD AND B. OLHAGEN, .4&z Med. &and., 151 (1955) 42.j. 7 St. SANDK~~HLER, Klirz.Wochschr., 33 (1955) 536. 8 D. G. I;ERRIMAN AND A. B. ANDERSON, BYit. Med. J., 2 (1956) 402. 9 I.R. ~IACKAY,N.ERIKSEN,.-\.G.~~OTULSKYANDW.VOLWILER,A~.J.M~~.,~~(I~~~)~~~. lo 1.. STEIN AND E. WERTHEIMER, .4xn. Trap. Med. Parasitol.. 36 (1942) 17. 11 A. B. LERNER AND G. Ii.GREENBERG, J. Biol. Chem., 162 (1946) 429. I* ;\.H. SEHON, L. GYENES, J. GORDON, M. RICHTER AND B. ROSE, J.CZin. Iwest., 36(1957) 13 R. T. S. JIM AND R. C. STEINKAMP, J. Lab. Clin. Med., 47 (1956) 540. I4 A. B. LERNER, C.1'. BARNUM AND C. J. \VA~sox <4m. J. Med..%., 214(1947) 416. I5 D. F’. BARR, G. G. READER AND C. H. \VHEELER, AWZ. Internal Med., 32 (1950) 6. I6 E. MANDEMA, P. C.VAN DER SCHAAFAND T. H. J. HUISMAN, J.Lab. C&n.Med.,45 (1955) Ii H. D. GROOMER, E. GRUNZE .~ND H. HGRNER, lilix.Wochschr., 33 (1955) 588. I* h. 2%.GREEN, J. .lm. Chem. .%c., 60 (1938) 1108. I8 G. SANDOR A?iD L. CHEDDAHA, Bull. sot. chim. biol.. 31 (‘949) ‘5’5. 2o G. SANDOR, Bull. sot. chim. biol., 33 (1951) 1483. 21 R. VARCUES AND S. LABROSSE, Pvesse we’d., 62 (1954) 1740. 22 E. K61w AND 4. GR~~XWALL, Scaxd. J. Clin. &Lab. znvest., 4 (1952) 244. 23 H. J. SILBERMAN, Lancet, 2 (1957) 26. 24 A. E. STEEL, Clipt. Chim. .-1&a, (1959) 1x1the press. Received

November

456.

261.

7th, 1958