Physical, physicochemical and chemical characteristics of macroglobulinic serum mucoprotein

PHYSICAL,

PHYSICOCHEMICAL OF MACROGLOBULINIC

AND CHEMICAL SERUM

CHARACTERISTICS

MUCOPROTEIN

:n recent years the carbohydrate-contailting components of serum have been studied vigorously; these studies have followed three main iines: (I) the nature of these compounds and their chemical and physicochemical properties, (2) the elucidation of their physiological significance, and (3) their role in some pathological states. The first two points have been dealt with by many authors (see the reviews by WINZI.F:K* and by JAYLE AND BoussrEn~), but as regards the third point it is only known that some ‘relation exists between the percentage of these caubohydrateprotein components and various diseases. Of the different pathological states, macroglobulinemin is the case where the most constant and significant changes have been observed. Furthermore, it has been shown that macroglob~~li~~ itself contains a large amount of carbohydrate”. It seemed of interest, therefore, to investigate whether changes in the structure of mucoprotein could be observed in ~~acroglot?uli~~emia. The present work deals with the physicocl~e~~lical and chemical ~haract~si%ation of mucoprotein isolated from a serum containing typical ~~acroglobl~lirls.

Nucoprotein was prepared from freshly drawn blood serum of a patient with typical macroglobulinemia. The method employed for the major mucoprotein component was essentially that of %‘EIMER, nilm~ AND WIXZLER”. The mucoprotein was further purified by dialysis against saturated ammoniuln sulfate solution. Xucoprotein was also isolated and purified from pooled serum of normal adult subjects, Electrophorctic experiments were done with the moving-bound~~r~~ PerkinElmer apparatus; mobility data are given in 1.’ x IO-~ cm%ecm-1V-1. All the runs were made with buffer systems according to MILLER AND GOLDER~. ~edi.m~nta~ion studies were made in the Spinco electrical centrifuge at 59,780 rev./min. The values of the sedimentation constant, corrected for viscosity and density of water to 20~ are given in Svedberg units (S”pO,, x 10-13). Diffusion measurements were made at -+-I’ in the electrophoresis separation cell from photographs taken at different times. The results were calculated by the heightarea method. The values of the diffusion constants corrected for viscosity and. density of water to zoo, are given in D x IO-? cm*sec-‘. The average molecular weight was determined by light-s~atterillg measurements with the Dual xooo Brice-Phoenix photometer at 546 nq. The solutions mere clarified by ultracentrifugatjon 0s by filtration through Celia filters ~~~~rnbr:~n Filter

-4. CAPUTO, Ill. SANTORO

560 Gesellschaft).

The

molecular

weight

was

calculated

by

the

DEBYE

formula6

H c/t = ,‘I + L B c, where c is the protein concentration, T the turbidity, H the Debye factor and B a constant for the solvent. The protein content of the preparation was determined by the Biuret reaction’. The amino

acids content

of the mucoprotein

hydrolysate

was determined

by high

voltage paper electrophoresis according to DOSE -IND CAPUTO~. Supplementary analyses were made to detect spcctrophotometricallys the content of tyrosine and tryptophane, without loss during the hydrolysis. The amount of sugars was estimated by the orcinol reaction as galactose-mannose”‘. The hexosamine

content

was determined

by the method

Fig. 2. Light scattering data of normal (0) and macroglobulinic (0) serum mncoprotein.

Fig. I. Mectrophoretic mobility as a function of PH for normal (0) and mncro~loln~linic Serum mucoprotein (0) determined in MILLER ~ixD GoLnrcR" Imfiers, ionic stren@h 0. I. Uranic

acid was detected

of ELSON AND MORGAN II.

by the carbazole

method

of DIsCHE’~.

The isolation

and

estimation of the different types of sugars were accomplished by paper chromatography with the system, butyl alcohol-acetic acid-water, 4 : I : 5. The sugar spots were revealed

by the phloroglucinol

method

of UOREFREUND .~ND DISCHE’~.

RESULTS

The electrophoretic

studies of the mucoprotein

were carried out over a pH range

from T to IO. Between PH I and 7 with acetate and phosphate buffers of the same ionic strength, the compound behaved as a monodispersed system and migrated as a single homogeneous symmetrical component. Above PH 7 the boundary became somewhat broader, and at high alkalinities a second small slower component appeared in the rlectrophoretic pattern. Alma: t similar results were observed for normal mucoprotein. Fig. I shows the curve obtained by plotting the electropho;ctic mobilities against the PH values. The isoelectric point of the macroglobulinic mucoproteins appeared at pH 2.9 while that of the noTma mucoprotein was at pH 3.2. In the ultraccntrifuge the mucoprotein behaved as a molecularly monodispersed system and in the diagram a single homogeneous symmetrical component was present. The value of the sedimen-

MACROGLOBULINIC

SERUM

MUCOPROTEIN

561

tation constant extrapolated to zero protein concentration, was S020,w = x.10 for the macroglobulinic mucoprotein and S”,~,, = 3.04 for the normal mucoprotein. In order to ascertain the stability of the mucoprotein a series of ultracentrifugations were made at different pH values. A stability region between pH 3 and 7 was found for both the macroglobulinic and normal mucoproteins. In this PH range the sedimentation constant did not change, whereas below pH 3 and above pn 7 it gra-

Proteitt cam. Preparation ___.____~ Normal serum 0.5 mucoprotein 0.7 0.s mean value

D”,O,w x IO-~

Pre@ration

5.49 5.22 5.61

Macroglobulinic mucoprotein

5.44

Proteits

cont.

DJzO,u, x IO-?

0.4 0.5 0.0

5.50 .%I* 5.34

mean xxlue

5.31

dually decreased from 3.1 to 2.95. It must be emphasized that the sedimentation diagram was always characterized by the presence of a single component. In Table I the values of the diffusion constants for various concentrations of mucoproteins are reported; clearly, the protein concentration had no detectable effect on the diffusion constant. Partial specific volume measurements were made in pyknometers by plotting the weights of the contents of the pyknometer for different mucoprotein concentrations against the concentration expressed as a weight fraction. The average value found was 7 = 0.6s both for macroglobulinic and normal mucoprotein. The molecular weight calculated from the formula rll = RTs/ll(~ - PQ) was about 43,000 for the macroglobulinic mucoprotcin and about 41,ooo for the normal one. The frictional coefficients f/jO, calculated from sedimentation and diffusion constants, were respectively 1.42 and 1.41. The average molecular weight was calculated by light-scattering measurements. In Fig. z the values of H +c/t are plotted against the protein concentrations. Extrapolation to zero protein concentration gave a value of I/&' = 21 for the macroglobulinic mucoprotein and I/J~ = 21.7 for the normal one. The alwage molecular weights were therefore 47,500 and 46,000. Chemical

atzalysis

In Table II are given the data on the chemical composition of the macro~Iobuli~lic and normal mucoproteins. To ascertain the type of hexoses, aminated sugars and uranic acids, paper partiTABLE

II

._ .._ ._____..~~~ Sitrogen Phosphorus Sulphur Protein Hexose Hesosamine Uranic acid

rr.4

II.0

0.01

0.02

r.00 74 15 6 0.3

I .oo 71 I7 10 0.Z

The figures are given as g per loo g of mucoprotein. Cliw.Chiitl. A&,

j (1960)559-563

562

A. CAPUTO.

I

M. SANTORO

2

Aspartic Giutamic Histidine

11.3 14.7 0.9

37 47 3

Arginine Lysine Valine

3‘3 4.9 2.I

Serine Threonine Glycine

I.1

5

5.7 0.7

23 4

Leucine Isoleucine Cystine/l

5.4 4.’ 0.7

20

I4lethionine I’rolinc Tryptophane Tyrosine I’llen~lalani~e

9 16

8

’ .4 j.N

0,s

5

‘5 3

4.3 3.9 0.X

17 ‘4 3

0.9 I.9 0.9

3 x 2

1.L 2.2 0.7

3 9 2

2.3 3.8

0 1T

‘.5 3,s

6 rr

Column I : g of amino acid per IOO g of protein. Column 2 : residues ular weights assumed: 47,500 for macroglobulinic mucoprotein protein)

tion

chromatography

indicated

the presence

was

6 71

used. The spots relealed

of fucose, mannose,

of amino acid per moie (molecand 46,000 for normal muco-

by the phloroglucinol

glucosamine

and glucuronic

reaction

acid.

To obtain further information, the amino acid composition of the mucoprotein hydrolysate was estimated. The values obtained are reported in Table III. Clearly, the main difference in the amino acid constitution consists of an increased amount monoaminodicarboxylic acids and a decreased amount of diaminomonocarbosylic

of

acids. UISCUSSION

All these results indicate

that the difference

between the mucoprotein

component

isolated from normal and that from macroglobulinic serum is not very great. Most of the physical and physicochemical properties of the macroglobulinic nlllcopl-otein are similar to those formd for the analogous normal component, both by us and by other workers. However, the isoelectric point of the normal component is lower than that calculated for the macroglobulinic mucoprotcin. It should be noted that the isoelectric point of the normal component is somewhat higher than the T.alue of 1.S previously obtained by ‘VC’EIMER et ul.” This discrepancy may be due to the different buffer systems employed for the pH range r-3; the graph given by WEIMER & al. is no different from ours from pH 9-3, ix. the range where the buffers used were similar. One of the most significant points of this investigation concerns the size of the mucoprotein molecule. The molecular weight of 41,000 calculated from sedimentation and diffusion for the normal component is very near the value given by \vEIMER et d. and is only slightly lower than the value of 46,000 found by the light-scattering

JlACIWGLOBULINIC

SERI:M

MI~COI’ROTEIN

50.3

method. This second figure should bc considertad as much more litwty, bvcnuse it rc’precnts an avtxragc wx+$1t drtermination. From the data reported it can hrcn conctudcd that the mucoprotein motccuk i> not spherical (j/p I ._I]) but somcMhat obtatc. \I’hitc no apprcciabtc diffcruncrs \vcrc obs;rr\wl in thv phykxt and phy5icochcniicat prop~rtic5, ttw nincroglot)ulinic mucoprotcin diffvrcd from thv norm;lt oncb in ww-at chcmicat aspwts. The protein compontwt of the macrogtol~ulin mucoprotA~ \vas slightly tiigtlt~r than that of the normal onv, \vhilc thv contat of sugprs and ailinated sugar> UYIY considcrabty lowt’r. I)cyitc~ this ctu;tntit;lti\.r diffvrc*ncv, t htl MIIN t\ys of sugars mtt aminatvd suprs wcrc i only Cy. TII(~ ttiff(wnw is dutl to thus tligt1(7- contcat of both qxutic and gtutamic ncidr;.