430s
Vol. 77
11.HEIDELRERCER
[ C O S T R I R U T I O N FROM T H E DEPAR’I’MEST O F h I E D I C I X E , COLUMBIA VXIVERSI’TY, C*OI.LECE O F P H Y S I C I A N S A N D ASI) THE PRESBYTERIAN I-IOSPITALI
Immunological Specificities Involving Multiple Units of Galactose.
SURGEONS,
111
BY MICHAELHEIDELBERGER RECEIVED MARCH 21, 1955
Quantitative data are given on the precipitation of antibody in Type S I V antipneumococcal horse sera by a number of gums containing galactose such as gum arabic aut1 its partial degradation product, the gum of .4cucin pycnnntha, the arabogalactans of Jeffrey pine and larch, the galactan of the seeds of Strychnos nux vomica, karaya gum and carob mucilage. The results are compared with earlier data on the cross reactivity in Type XIV serum of blood group substntices, lung galactau, arid tamarind seed polysaccharide. Multiple recurrences of non-reducing galactose end groups suffice for reactivity in Type XIV antipneumococcal sera, but gums with very few such end groups and a number of 1,3-, and/or 1,6-, and/or 1,3,filinked galactose residues precipitate the antiserum as readily. The above galactose linkages are generally assumed to be 8-.
I t was recently shown that the galactan isolated from the residues of beef lungs used for the production of heparin precipitated more than one-quarter of the antibody in a Type XIV antipneumococcus serum.* This demonstrated a similar linkage or arrangement of some, a t least, of the galactose units in the lung galactan to the as yet unknown linkages and arrangement of the galactose in the type specific capsular polysaccharide of Type XITpneumococcus (S XIV). Alt that time S XIV was thought to be composed of three residues of galactose to one of N-acetylglu~osamine~ b u t it is now knowii to contain glucose in a d d i t i ~ r i . ~ The precipitation of Type XIV antiserum by tamarind seed polysaccharide (je1lose)j was of assistance in the interpretation of the results with lung galactan, since all of the galactose in jellose, which also contains xylose and glucose, is in the form of nonreducing end groups. I t appeared evident, therefore, t h a t multiple galactose end groups surSccd to permit cross reactivity with the Type X I V system. I n view of the implications of these instances of cross reactivity for a parallel study of the fine structure of the specific polysaccharide of Type XIV pneumococcus it was decided to examine the reactivity toward Type XIV antipneumococcal serum of other natural galactosecontaining polysaccharides of more or less known structure. The results are given below, since they supply an alternative structural feature requisite for this type of cross reactivity and add to the presently meager knowledge of the relation between the chemical constitution of the natural nntiqeris and their imniunological specificity. Experimental Materials and Methods .--The ar,ibogalactan of the Jeffrey pine and its product of rnlld acidlc hydrolysise were kindly supplied b> Prof \Y Z . H i?\id, the gum of ilcacin (1) A preliminary account of this work appeared in Abstracts, 126th Meeting. American Chemical Society. S r p t . 1!154, GD. Carried o u t under t h e Harkness Research Fund and a grant from t h e Rnckefeller Foundation. Technical assistance by Check hl. SooHoo and John Adams is gratefully acknowledged. ( 2 ) M . Heidelberger, 31. L. Wolfrom, \V, Brock N-eely and Z . Dische, THISJ O U R N A L , 77, 3511 ( 1 9 5 ) . Xow designated Paper I of this series. (3) W. F . Goebel, P. B. Beeson and C . I,. Hoagland, J . Bioi. Chenl., 129, 455 (1939). (4) M . Heidelberger, S. A. Barker and M. Stacep, Scicrzce, 120, 781 (1934). T h e glutamic acid reported in this ahitract appears due t o a dialyzable impurity, b u t glucose also occurs in a sample of one of the original preparations kindly supplied by Dr. Goehel. (5) E. T’. White and P. S. Rao, THISJOURKAL, 75, 2617 (1953). (6) W. H. Wadman, A. R . Anderson a n d W. Z. Elassid, ibid., 76, 4097 (1934).
pycnantha (golden wattle),’ karaya gum from Cochlospermum gossypium,* larch arabogalactan and its partial hydrolytic product, and carob mucilage (gum gatto)$ by Prof. E. L. Hirst, and the galactan of the seeds of Strychnos nux z ~ o m i cby~ ~Prof. ~ J. K . N. Jones. A commercial sample of gum arabic was in part converted to a degraded arabic acid.” The specific polysaccharide of Type XIV piieumococcus (S XIV) was donated by E. R . Squibb and Sons, kindness of Mr. T. D . Gerlough, and the Type XI\’ antipneumococcal (anti-Pn) horse serum by the Division of Laboratories of the Xew York Stcite Department of Health, Dr. G. Dalldorf, Director. Quantitative estimations of antibody nitrogen precipitated in the homologous arid cross reactions were carried and are given in the Tables. All mixtures for analysis were allowed t o stand in a bath a t 0 ” for six days to two weeks, depending upon the extent and rapidity of interaction of the system under study. All washings were carried out with chilled saline with the tubes immersed in ice-water xiid centrifugations were run at 0’ also.
Results and Discussion 1.
Gum Arabic and Related Products. a.
Gum Arabic.--Because of the reactivity in Type XIV anti-Pn serum of lung galactan, with its nonreducing end groups and 1,3- and 1,6-, presumably @-, galactose linkages, i t could be predicted that gum arabic, in which the galactose is similarly bound,16 would also react. This was readily verified, as noted in Table I, but the amount of antibody nitrogen was only one-fifth as large, 43 pg., a t the nlaximuni, as t h a t precipitated by lung galactan, about 250 pg. Possibly this is due to the presence in gum arabic of very much more glucuronic acid, which is known to be a potent determinant of immunological specificity. This will be discussed in greater detail in a forthcoming comniunication on the specificity of Type I1 pneumococcus. Few of the arabofuranose residues in gum arabic are attached t o galactose end groups and presumably none to the internal 1,3- and 1,6bound galactose units. One would therefore expect that mild hydrolytic removal of part of the araboI-, Hirst a n d A . S. Perlin. J. C h e m . Soc., 2622 (1954).
. I,. Ilirst and S. Dunstan. ibid.. 2332 (1953). . L. Hirst and J . K . ri.Jones, i b i d . , 1278 (1948); F . Smith, THISJ O I ~ R K A I . , 7 0 , 3249 (1928). (101 P. Andrews, I,, Hough and J. K. N Jones, J . Chem. S a . , 806 (19%). (11) 0. T. -4very. hl. Ileidelberger ani1 W. F. Goebel, J . E x p e r . .\fed., 42, 709 (192.5). (12) hl. Meidelberger and F. E . Kendall, . I . E x p e r . d i e d . , 6 5 , 55.5 (19321; 61, 559 (1935). (13) &I.Heidelherger, C . hI. h l a c l e o d , S . C Kaiser and B. Robinson, ibid., 83, 303 (1946); R . Markham, Biochcm. J . , 36, 790 (1942). (14) E. A . Kabat and hf. AI. hfayer, “Experimental Immtinochemistry,” C . C. Thomas, Springfield, Ill.. 1948. ( 1 5 ) For prohable structure, 6.F. Smith, 1. C h e w S a c . , IORT, (1940).
IMMUNOLOGICAL SPECIFICITIES INVOLVING MULTIPLEUNITS O F GALACTOSE
Aug. 20, 1955
furanose would have little effect on the reactivity of the gum in Type XIV anti-Pn serum and this is borne out by the values shown in Table I. The greater inhibitory effect of the hydrolyzed gum a t higher concentrations is perhaps due to the splitting of other linkages and the consequent smaller size of the molecules of hydrolyzed gum. TABLE I
4309
for a slight concentration of galactose with respect to rhamnose and glucuronic acid, the ratios of the sugars for which analyses were made remain much the same as in the original gum. TABLE I1 PRECIPITATION O F 3.5 ML. C-ABSORBED TYPE)LIV ANTIPNEUMOCOCCUS HORSESERUMWITH 1.75MG. GUMARABIC Antb, -In
precipitate, fig.~ 1
~-Ratios-
Antb. PRECIPITATION OF 1.0 ML. TYPEXIV ASTIPNEUMOCOCCUS Gal. GluGum N curGUMS pptd,," pptd., gum Rham- Galac- onic -Gal_ Glu- CUT. HORSESERUM635 B Y ARABOGALACTANS A N D RELATED pg. pg, pptd. nose tose acid Rham. cur. R h a m . AT 0" 138 104 0.8 16b 77* 17 5 Ob 4.6 1.1 The antiserum contained 47 pg. anti-C nitrogen and 920 Original gum 4 4 1.1 fig. a n t i 8 XIV nitrogen per ml. Volumes actually used ranged from 1.0 ml. 1 -+ 2 dilution to 2.0 ml. undiluted a 10/8 X sum of three sugars for which analyses are given. serum, but all results are calculated to 1.0 ml. "C-ab* Mean of three analyses. sorbed" serum refers to serum deprived of its antibody to C-substance, the somatic polysaccharide of pneumococcus. Amount of polysaccharide used, mg.
-Antibody G u m arabic Partially Inhydrotact lyzed
0.05 30 .1 .2 .3 .5 45 .6 1 43 2 2.5 37" 3 5 6 10
32b
nitrogen (clg.) precipitated hyAraboArabogalactan, galactan, Jeffrey pine larch G u m , Acacia ParParPycitantha tially tially InhydroI n - hydroWhole Cser. absd. t a c t lyzed t a c t lyzede
108 35
46
80
21
64 126
30 28
85
173 35 30 9
218
171 181 191
245
72 83
91 92
173
9Od 2 20@ 187 2 138 a Supernatants of all tubes combined: with pine arabogalactan, 202 pg. N; the supernatant from this, with S XIV, gave 805 pg. K , total 1046. Gum arabic precipitated 41 pg. N from C-absorbed serum. bC-absorbed serum gave 29 pg. N, the maximum, a t this level. The supernatants with 2 mg. of larch galactan, gave 73 pg. N ; with 0.6 mg. of partly hydrolyzed galactan, 67 pg. N, with 1 mg., 74 pg, N. c Combined supernatants from these and preceding tubes, mean ?; already pptd., 176 pg.; with S XIV, 774 fig. N; with lung galactan, 26 pg. N, followed by S XIV, 713 fig., total 915. dCombined supernatants with pine arabogalactan, 113 pg. S ; supernatant from total 975. a Analyses with Cthis with S XIV, 779 pg. 9, absorbed serum.
b. Gum of Acacia pycnantha.-In this gum7 arabinose and galactose are distributed in much the same fashion as in gum arabic, but there is only one-third as much glucuronic acid. One would therefore not expect i t to precipitate less antibody than gum arabic, but the figures shown in Table I are comparable to the somewhat lower values characteristic of the degraded gum arabic. Possibly factors of molecular size and shape enter in this instance, as well. 2. Arabogalactan of Jeffrey Pine.-Relatively little of the galactose in this substance is in the form of non-reducing end groups. Evidently a favorable distribution of the 1,6- and 1,3,6-galactose TABLE I11 OF 1.0 ML. TYPEXIV ANTIPNEUMOCOCCUS PRECIPITATION HORSESERUM635 BY 1-ARIOUS GALACTOSE-CONTAINING POLYSACCHARIDES AT 0" Amount of Polysaccharide used, mg.
--Antibody nitrogen (fig.) precipitated byHuman blood Galacgroup A tan, Strychsubst., nos data from Lung Karaya Carob nux ref. 19b galactan Jelloae gum mucilage uomtca
0,015 .04 .1
.3 .4 .5
2 66 134
103 179
86
17 2Od 18
8 94
178
126 154 153#
14
.6
12
159
