Chromatographic and electrophoretic studies on pas-positive components of serum macroglobulins

CLINICA

CHIMICA

I75

ACTA

CHROMATOGRAPHIC VE COMPONENTS

AND ELECTROPHORETIC OF SERUM

S. ISRAEL-BUDNICK,

Department Univevsity

ofChile, Santiago

and Pathological (Chile)

(Received

June

~rntl,

ON PAS-POSITI-

MACROGLOBULINS’k

J. A. MORXLES-MALVr, MAGASICH

o_f Physiological

STUDIES

M. SAPAG-HAGhR

Chemists?,

Faculty

AI\‘D A. V.4LLEG.4.

of Chemistry

and

Pharmacy,

1967)

SUMMARY

An ascending chromatographic technique for the detection of serum proteins is described using Whatman No. I paper developed with citrate buffer pH 5.1. Differences in the protein distribution permit one to distinguish between normal and certain

pathological sera. 2-Thiobarbituric acid was used as location reagent for sugar-containing

proteins

in the chromatographic technique. It has the advantage of being more rapid than the PAS technique and, moreover, only the complexes formed with some pathological sera are fluorescent when submitted to ultraviolet light. Paper electrophoretic studies of the precipitates obtained after treating different serum samples with a 2% boric acid solution, demonstrated that they are made up of a mixture of globulins. When analysed by means of paper chromatography, an elongated pattern typical of the proteins contained in the serum of rheumatic and hepatic

patients can be clearly observed. After electrophoresis of the boric acid precipitates and staining with Schiff reagent, important differences appeared in the serum samples obtained from different diseases. In the serum of rheumatic patients a single zone corresponding to the ccZ fraction was obtained. In the serum of cirrhotic patients, this single fraction was located between the /? and y fractions.

ISTRODUCTIOS

The separation of serum macroglobulins such as ultracentrifugation, taking advantage

can be achieved by physical methods, of their high molecular weight. Chemi-

cal methods that permit their identification using common laboratory procedures have been usedlIZ, as that reported by Badin3. This last technique is based on the selective precipitation of serum macroglobulins and is used for the diagnosis of rheumatic diseases. The present report deals with the application of Badin’s method to other * Work

supportccl

hy the University

of Chile,

Research

Law No. Clin.

I I 575.

Chim.

Acta,

18 (1967)

175-181

176

MORALES-MALVA

et a/.

diseases in which macroglobul~ns, especially the ~~-lnacroglobulin fraction (10 S yglobulin), are slightly increased&. Paper chromatography and electrophoresis were used in order to study the fluctuations and localization of the periodic acid-Schiff (PAS)-positive substances of macroglohulins in different pathological sera. As the colour obtained with Schiff’s reagent is nonspecific, because several serum components, e.g. glycoproteins, mucoproteins give positive reactions, the studies were performed mainly with the precipitates. Using this method, a partial purification of macroglobulins by precipitation is obtained, and they can be separated from the supernatant where the substances capable of masking the reaction remain. The PAS method was used because the ~~-macroglob~llin, as well as the a,-matroglobulin5, contain carbohydrates”. The purpose of this work was to know, using a simple method, whether the increase of macroglobulins in different diseases is due exclusively to the quantitative variation of a single fraction or whether, in addition, qualitative differences existed between MATERIAL

the fractions. AND

METHOIX

Normal sera, as well as sera from patients

with cirrhosis,

hepatitis,

nephropa-

thies and rheumatic diseases were studied. As described by Badins, a 2% boric acid solution was used to precipitate some serum samples. These precipitates were centrifuged at 500 g (3000 rev./min.) for IO min at room temperature and washed twice with the same boric acid solution. Paper chromatography and electrophoresis of the boric acid precipitates and the original serum samples

were carried out simultaneously.

I. Chromatogva$hy Ascending chromatography

was done on Whatman

No. I paper and on What-

man No. I silica-treated paper? using citrate buffer* pK 5.1 or IOS. IO to I5 ,ul of the different serum samples were applied. The pellets from their boric acid precipitate were applied directly on the paper sheets with a small spatula. Total proteins were proteins by the PAS reaction* detected with Bromophenol blues, sugar-containing or by the z-thiobarbituric acid method’*, both modified as follows: PAS stainiq. The chromatograms were first immersed for IO min in a solution of 19," mercuric chloride in absolute ethanol. The excess of this coagulating solution was eliminated by allowing the sheets to drain at room temperature until the superficial gloss disappeared. Afterwards they were left in the dark for 75 min, in a I'$!, solution of potassium periodate in I.5 iS sulfuric acid. Then the papers were washed for IO min, in a bath containing distilled water, drained at room temperature and when the gloss disappeared, they were immersed for 5 min in a bath with Schiff’s reagent. Two formulas of Schiff’s reagent have been used with equally good results: (a) Basic fuchsin Distilled water Sulfur dioxide (b) Basic fuchsin Roiled distilled

2.5 g I000 ml enough for decoloration. 2.0 g

water

Cli?7. C&W. Acta, I8 (1967) 175-181

400 ml.

PAS-POSITIVE

COMPONENTSOF MACROGLOBULIKS

I77

Heat to 50” and filter. Add to the filtrate: z iV Hydrochloric Potassium Activated

acid

metabisulfite charcoal

Allow to stand, stoppered,

I0 ml 4 g rg

at o-4” for 12-18

h. Filter

and add at once, slowly,

and

under stirring 2 N

Hydrochloric

acid

Lo ml.

After 5 min in Schiff’s reagent, the sheets were washed for z min in distilled water and placed for 5-10 min in a sulfurous bath made of I?/, potassium metabisulfite in 0.1 N HCl. Finally,

the papers, still wet, are quickly

washed with 90% ethanol

and allowed to dry at room temperature. 2-Thiobarbitwic acid technique: The damp chromatograms IO min in a coagulation sulfate in 300,& ethanol.

were immersed

for

reagent made up of a saturated solution of anhydrous zinc The papers were then left for 3-5 min at room temperature,

and thereafter placed for 30 min in an oxidant solution (1% potassium periodate in 2.5 N sulfuric acid. This solution must be kept and used in the dark). Next, the papers were washed for IO min in a bath containing 50 parts ethyleneglycol, 50 parts acetone and 0.3 parts of sulfuric acid d = 1.84, and immersed, finally, for I min in a bath containing a 6% solution of dithiobarbituric acid in I N sodium hydroxide solution. The colour was developed by heating the papers at 100’ for IO min. The brownreddish colour obtained can be studied under ultraviolet light. 2. Electro$horesis Electrophoresis

was carried out at room temperature on Whatman No. I paper ionic strength 0.075, pH 8.2, in the electrophoretic strips using Michaelis bufferI chamber, and a 75% dilution of the same buffer as impregnating so1ution12. 15 ,ul of whole serum were used and the boric acid precipitates corresponding to 0.2 ml of the same serum samples were applied directly to the papers. At IIO V, separation time was 16 h. Total proteins12 and PAS-positive substances as described for chromatography, were stained on the papers. RESULTS Chromatography on Whatman No. I paper sheets and citrate buffer pH 5.1 were found to be the best working conditions. The chromatograms of serum samples obtained from patients with hepatitis, cirrhosis or active rheumatic disease, stained for total proteins, showed elongated zones usually extending through the entire chromatogram, from the application point to the solvent front. Normal sera gave a compact spot on the upper part of the chromatogram, shaped like a flame or an arrow’s point (Fig. I). This general distribution pattern was also observed when the chromatograms were stained for sugar-containing proteins with Schiff’s reagent or by the z-thiobarbituric acid method (Fig. I). Chromatography of the boric acid precipitates showed a different distribution pattern for normal and pathological sera (Fig. 2). Electrophoresis of the boric acid precipitates demonstrated that they were not a single fraction. Indeed, when stained for total proteins, clZ-,p- and y-globulins could Clin.

Chim.

.4cfa,

18 (1967)

175-181

178

MOKAIXS-MALVA

et al.

Fig. 1. Comparative chromatography of serum samples. Ccntre: Left , Bromophenol blue sta.ining of a normal serum; right, and of serum from a rheumatic patient. Ix ,ft : The same serum sa1nples stained with z-thiobarbituric acid. The marked area shows the fluores ;ccnt zone. Right: The same scram samples stained with Schiff’s reagent. In all three chromate grams the rheumatic scrum spot is more elongated than that of normal serum.

Pig. I. Chromatography of boric acid precipitates obtainccl from pat hological sera. Upper: I’AS staining. Lower : Bromophenol blue staining. Centre: hepatitis. Right : both samples, cirrhosis. Left: active rhcumat .ic disease and ncphrop; Ithy. This last sample presents a distribution very close to normal serum.

PAS-POSITIVE

‘79

COMPONENTS OF MACROGLOBULINS

be discerned. In some cases, cc,-globulin fraction invariably absent (Fig. 3). When sugar-containing

was also detectable. Albumin was proteins were stained with Schiff’s

reagent, a single zone was obtained corresponding to cr,-globulins in the precipitates obtained from serum of rheumatic patients, and to /&globulin in the precipitates obtained from serum of cirrhotic patients (Fig. 4).

The z-thiobarbituric acid method reported by WarrenlO is based on the measurement of the colour compounds synthesized by the coupling of z-thiobarbituric acid with the products of periodic oxidation (deoxy sugars, z-keto-3-deoxy sugars and sialic acid). As the oxidant solution proposed by Warren was not suitable for chromatography, the oxidant employed for the PAS staining in electrophoresis was used. The chromatograms of serum samples from cirrhotic patients, usually containing smaller amounts of proteins than normal sera, generally showed increased quantities of PAS-positive substances. Similar results were obtained with PAS staining by electrophoresis of the same samples. Electrophoresis of the boric acid precipitates demonstrated that albumin was absent and some globulinic material was lost. The diagrams of these precipitates were similar to those obtained

with the original serum with respect

to the globulin

frac-

Fig. 3. Electrophoresis of two serum samples and their corresponding boric acid precipitates. Right: hepatitis. Left: active rheumatic disease. Staining: Bromophenol blue. _\lbumin and czlglobulin arc absent in the strips corresponding to the boric acid precipitates. Clin. Chim.

Acta,

18 (1967) 175-181

MORALES-MAL’i’rZ et 621.

Fig. 4. Electrophoresis of: (from left to right) a. Boric acid precipitate of a serum obtained from an active rheumatic patient. Staining: Bromophenol blue. b. Same as above. Staining: Schiff’s reagent. c. Boric acid precipitate of a serum of a cirrhotic patient. Staining: Bromophenoi blue. (1. Same as above. Staining: Schiff’s reagent. The Schiff staining of the precipitates shows a single zone corresponding to r,-globulin in the rheumatic sera and between the /?and y fractions in the cirrhotic sera.

tions (Fig. 3). Rut, when PAS-positive

substances

were determined,

the zones corre-

sponding to muco- or glycoproteins (a,, clg, /3or r) were not stained as in normal sera. In this way it can be demonstrated that the precipitation technique partially purifies macroglobulins. Schiff staining of the chromatograms of sera from nephrotic or rheumatic patients demonstrated that these sera have more PAS-positive substances than normal sera. When stained with 3-thiobarbituric acid, lighter spots were obtained, but the method has the advantage of being more rapid and, moreover, only the above mentioned pathological sera produced fluorescent spots under ultraviolet light. Chromatograms of boric acid precipitates obtained from sera of active rheumatic patients and patients with hepatitic diseases, showed elongated spots when stained for total proteins, while PAS staining produced shorter zones with precipitate from rheumaticsera than from hepatitic sera (Fig. 2). The typical electrophoretic diagram of cirrhosis presents, in the zone corresponding to ,#- and y-globulins, a single stained fraction. The differentiation of the two globulins was difficult to perform and the densitometric curve of this fraction resembled a “camel hump”. The authors believe that the merging of these two fractions was due to an increase in ¯oglobulins (also called yr- or y 19 S-globulins), migrating in the space between /3- and y-globulin (Fig. 4). Deutsch and Morton’3 have demonstrated that even though /&-macroglobulin has a sedimentation constant of 19 to 20 S, and a molecular weight of approximately one million, the sulfhydryl groups of cysteine can further break down the molecule. These fragments, considered as monomers, have a sedimentation constant of 6.5 S, a molecular weight of 160000, and an electrophoretic migration corresponding to a,globulin.

PAS-POSITIVE

COJlPONENTS

Examining

OI; MACROGLOBULINS

the results

with active rheumatic

diseases,

obtained,

181

it was concluded

with a shorter

that

distribution

the sera of patients

pattern

in the chromato-

grams and PAS-reactive only in the cc,-globulin zone, may carry the monomeric molecules. On the other hand, the sera of patients with cirrhosis, with their longer chromatographic pattern and PAS reaction in the /I-fraction, could contain increased amounts

of /7,-macroglobulins

(Fig.

4).

ACKNOWLEDGEMEST

We are indebted

I 2 3 4

to Miss Carmen Urrutia

for technical

assistance.

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Acta,

18 (1967)

175-181