Studies on a hemagglutinin from Bauhinia purpurea alba seeds

ARCHIVES

OF

Studies

BIOCHEMISTRY

AND

BIOPHYSICS

on a Hemagglutinin TATSURO

161, 475-482 (1972)

from

IRIMURA

Bauhinia

AND

TOSHIAKI

purporea

alba

Seeds

OSAWA

Faculty of Pharmaceutical Sciences, University of Tokyo, Bunkyo-ku, Tokyo 113, Japan Received March 20, 1972; accepted May 4, 1972 A phytohemagglutinin has been purified from the seeds of Bauhinia pwpurea a&a. The homogeneity of the purified hemagglutinin was ascertained by ultracentrifugal analysis and electrophoresis on polyacrylamide gel. The purified hemagglutinin had a ~~20”value of 7.5 S and its molecular weight of 195,000 was estimated by sedimentatien equilibrium experiments. This hemagglutinin was found to contain 11.1% carbohydrate of which mannose (4.9%) and glucosamine (3.4yc) were the predominant sugars, with smaller amounts of xylose, glucose, fucose, and galactose. The purified hemagglutinin agglutinated erythrocytes regardless of their ABO and MN blood group types. In hapten inhibition assays with simple sugars, the so-called Makela’s group 2 sugars, particularly N-acetyl-n-galactosamine, were the most potent inhibitors for this purified hemagglutinin. From the titration assays with enzyme-treated erythrocytes and from the hemagglutination-inhibition assays using partially hydrolyzed trypsin fragments from erythrocytes as inhibitors, it was assumed that the receptor sites for this hemagglutinin were at least partly covered by sialic acid and mainly composed of alkali-labile carbohydrate chains on the cell surface.

Crude extracts of Bauhiniu purpurea seeds were first described as a satisfactory routine anti-N reagent by Boyd et al. (1). These investigators also found that, after dialysis, the extracts became nonspecific losing original anti-N specificity, and this fact was ascribed to the loss of certain diffusible materials during dialysis. Actually, it was shown that the anti-N specificity of the dialyzed extracts was recovered in the presence of a small amount of melibiose. On the other hand, Uhlenbruck and Dahr (2) found that certain Bauhinia seeds contained only nonspecific hemagglutinin. These extracts, regardless of anti-N specificity, have been found to be inhibited (3, 4) by what is called the Makela’s group 2 sugars (5), and it has generaly been assumed that Bauhinia receptor represents a fundamental structure of M,N blood group antigens (6, 7). The purpose of this investigation was to purify and characterize the hemagglutinin from Bauhinia purpurea alba seeds, and to clarify the detailed specificity of the purified

hemagglutinin in comparison with those of other phytohemagglutinins, being specific for the Miikela’s group 2 sugars. Based on these results, the nature of the receptor site for this hemagglutinin on human erythrocytes is discussed. MATERIALS

Purification of B. purpurea hemagglutinin. One hundred grams of finely powdered B. purpurea alba seeds (purchased from F. W. Schumacher, Sandwich, MA) was suspended in 1 liter of 0.15 M N&I0.15 mM sodium phosphate buffer (pH 7.0) and allowed to stand overnight at 4°C with continuous stirring. The clear supernatant fraction (crude extracts) obtained by centrifugation at 12,000gfor 20 min was directly subjected to (NHn)sSOd fraetionation. As shown in Table I, the precipitate which resulted between 0.4 and 0.7 saturation with (NH,) zSOI had the hemagglutinating activity, and it was dialyzed against distilled water until free of NH,+ and then lyophilized (crude hemagglutinin). Further purification of the active fraction was achieved by Sepharose 6B column chromatography as described in the legend of Fig. 1. The protein fractions eluted by 0.1 M lactose were combined, dialyzed, and lyophilized. The last traces of lac-

475 Copyright @ 1972 by Academic Press, Inc. All righta of reproduction in any form mserved.

AND METHODS

476

IRIMURA

AND TABLE

DETAILS

OF PURIFICATION

Dialyzed crude extracts (NH&S04 fractions 06.4 satn 0.4-0.7 satn (crude hemagglutinin) 0.7-1.0 satn Sepharose 6B fractions Peak A Peak B Biogel P-200 fraction (purified hemagglutinin)

I

OF Bauhinia

Yield from 100 g seeds bw)

Fraction

OSAWA

purpurea Minimum OMM cells

HEMAGGLUTININ hemagglutinating OMN cells

dose bg/ml)

for

ONN cells

3,556

5,600

5,006

5,000

1,000 750 50

10,000 625 >lO,OOO

10,000 625 >10,000

10,060 625 >lO,OoO

650 80 60

>10,000 80 . 40

>10,000 80 40

>10,000 80 40

Peak A

0.1 M Lactose

Peak 0

L 200

Fraction

number

FIG. 1. Sepharose 6B column chromatography of crude hemagglutinin. Three hundred and fifty milligrams of crude hemagglutinin were dissolved in 10 ml of 0.015 M phosphate buffer (pH 7.0) and dialyzed overnight against the same buffer, and then applied to a column (5 X 60 cm) equilibrated with the same buffer. After a large protein peak was eluted out, the column was further eluted with a buffer containing 0.1 M lactose. Fractions of 10 ml were collected, and the fractions corresponding to Peak B were pooled. tose were subsequently removed by gel filtration on a Biogel P-200 column. The active fractions were combined, dialyzed against distilled water, and lyophilized (purified hemagglutinin). Yields vere 60 mg/lOOg of dry seed flour. Enzymes. Trypsin (twice recrystallized) was purchased from Worthington, Vibrio cholerae neuraminidase from Calbiochem, Pronase from Kaken Co., Tokyo, Japan, bromelin from Nakarai Co., Kyoto, Japan. Highly purified samples of Clostridium perfringens neuraminidase and Streptomyces purpeofuscus neuraminidase, and subparticles of influenza virus strain Jap 305, possessing high neuraminidase activity, were kindly pro-

vided by Dr. Aoyagi, Institute of Microbial Chemistry, Kamiosaki, Tokyo. Specific activity of neuraminidases is defined as micromoles of Nacetylneuraminic acid formed per minute per milligram of protein under the condition described by Cassidy et al. (8). Phytohemagglutinins and antisera. Phaseolus vulgaris hemagglutinin was a product of Difco (PHA-M). Wisteria jloribunda hemagglutinin used in this study was a partially purified product obtained at 70% saturation of (NHI)&SO, from the crude extracts of the seeds according to the procedure previously described (9). Partially purified hemagglutinin from fberis amara seeds (purchased

HEMAGGLUTININ

FROM

from W. Atlee Burpee Co., Riverside, CA) was prepared by (NHJzSOa fractionation. The precipitate which formed between 0.25 and 0.5 saturation of (NHh)zSOd had hemagglutination activity and was dialyzed and lyophilized (Iberis amara hemagglutinin). Other phytohemagglutinins were extracted from the respective seeds by the method previously described (10). The absorbed rabbit anti-M and -N immune sere were prepared according to the method described by Race and Sanger (11) and used for typing of erythrocytes. Ultracentrifugation. Measurements of the sedimentation velocity of the hemagglutinin were performed in a Spinco Model E ultracentrifuge equipped with a Schlieren optical system at a speed of 59,617 rpm at 11.2”C. The concentrations of hemagglutinin tested were 0.25, 0.5, and 1% in 0.2 M NaCl. The ~“20~ was obtained by extrapolation to zero concentration according to the procedure described by Schachman (12). The sedimentation velocity experiments were also performed according to the band sedimentation method (13) in a Spinco Model E ultracentrifuge equipped with an uv optical syst’em at a speed of 52,640 rpm at 23°C in 1 M NaCl. Molecular weight determination. Molecular weight of purified B. purpurea hemagglutinin was measured by sedimentation equilibrium in 0.2 M NaCl-0.03 M sodium phosphate buffer (pH 7.0) at 15°C. This was performed with a Hitachi model UCA-1A ultracentrifuge according to the method of Yphantis (14). Disk electrophoresis. Disk electrophoresis in polyacrylamide gels wse carried out in 7.5yo gels in 0.04 M Tris-glycine buffer at pH 8.3 and also in 0.1 M B-alanine-acetate buffer at pH 4.3 according to the methods of Ornstein (15), Davis (16) and, Reisfeld et al. (17). Staining was performed with amido black in 7% acetic acid, and destaining in an electric field with 7% acetic acid. Amino acid analysis. The hemagglutinin was hydrolyzed with redistilled HCl in sealed tubes at 108°C for 24 and 48 hr. The amino acid content of the hydrolyzates was determined on a Hitachi KLA3D amino acid analyzer according to the method of Spackman et al. (18). Quantitative values for each amino acid were calculated as previously described (19). Tryptophan was determined on unhydrolyzed protein samples by the spectrophotometric method of Goodwin and Morton (20). Carbohydrate determinations. Neutral sugar was determined by the phenol method of Dubois et al. (21). Amino sugar was determined according to Garde11 (22). Hydrolysis for this assay was carried out with 4 N HCl for 8 hr at 100°C in a sealed tube. Sialic acid was determined by thiobarbituric acid

Bauhinia

purpurea

477

method of Aminoff (23). Hydrolysis of bound sialic acid for this assay was carried out with 0.05 N HzS04 for 1 hr at 80”. For the identification and the determination of the neutral sugars, gas-liquid chromatography was carried out after reduction to the respective alditol followed by trifluoroacetylation according to the method of Matsui et al. (24) as described earlier (25). The trifluoroacetylated alditols were separated at 140°C on a 1.8-m glass column packed with 27, XF-1105 on Gas Chrom 2 (SO-109 mesh). The heights of the peaks were determined, and the absolute amount present was determined from the ratio of each to that of trifluoroacetylated arabitol, derived from arabinose added as an internal standard. Hemagglutination assays. The titration and inhibition assays using human erythrocytes freshly obtained from a donor were carried out according to the methods previously described (25). The cells used for the inhibition assays on Glycine max and Iberis amara hemagglutinins were bromelin-treated and neuraminidase-treated cells, respectively. Treatments of hyman erythrocytes with various proteolytic enzymes. To 10% cell suspension in 0.05 M NaCl-0.05 M sodium phosphate buffer (pH 7.7) was added 0.25 mg of crystalline trypsin per 109 cells and the suspension was shaken gently for 3 hr at 37”C, and the cells were then collected, washed several times with chilled phosphate-buffered saline (pH 7.0) and used for the assay as a 3yo suspension. Pronase treatment was accomplished in the same fashion using 5.0 mg of the enzyme per lo9 cells, and bromelin treatment was similarly performed in 0.14 M NaC1-0.05 M acetate buffer (pH 5.5) containing 0.01 M CaC& by use of 10.0 mg of the enzyme per lo9 cells. Neuraminidase treatments of human erythrocytes. To 10% cell suspension in 0.05 M acetate buffer (pH 5.4) containing 0.14 M NaCl and 0.05 M CaCla wss added 7 X 10-l units (8 X 10-l units in the case of Clostridium perfringens neuraminidase) neuraminidase per lo9 cells and the suspension was gently shaken at 37°C for 1 hr. The cells were then collected, washed several times with phosphatebuffered saline, and used for the assay ss a 3% suspension. Treatment of cells with virus subparticles having 7 X 10-r units neuraminidase activity per 109 cells was performed on 10% cell suspension in 0.1 M NaC1-0.06 M sodium phosphate buffer (pH 6.0) at 37°C for 1 hr. Preparation of trypsin fragment from human erythrocytes. The preparation of trypsin fragment from human erythrocytes was mainly confined to the method of Winzler et al. (26). Packed washed cells (100 ml) were added to 100 ml of phosphatebuffered saline (pH 7.7) containing 25 mg of crystalline trypsin. The suspension was incubated for

478

IRIMURA

AND OSAWA

1 hr at 37°C with gentle agitation, then centrifuged at 2000 g in the cold and the supernatant fraction was removed by suction. To this supernatant fraction was added 50y0 trichloroacetic acid to a final concentration of 5y0 to precipitate the protein. The clear supernatant fraction was recovered by centrifugation, extracted with diethyl ether to remove trichloroacetic acid, neutralized with NaOH, desalted by means of ultrafiltration, and lyophilized to give white amorphous powder (yield 25 mg). Acid and alkaline treatments of trypsin fragment. Trypsin fragment (5 mg) was hydrolyzed in 2 ml of 0.05 N HtSO( under vacuum at 80°C for 1 hr. The hydrolyzate was neutralized with saturated solution of Ba(OH)2 and filtered (acid-treated trypsin fragment). To 1 ml of acid-treated trypsin fragment was added 1 ml of 0.2 M NaOH-0.8 M NaBH4 and the mixture was incubated for 33 hr at 20°C in a sealed tube, in the dark, under nitrogen. The excess borohydride wsa destroyed by the careful addition of 1 N acetic acid to pH 6.5 and an aliquot of the mixture was put on a small column of Sephadex G-10. The void volume fractions of the Sephadex G-10 eluate, containing carbohydrate linked to protein by alkali-resistant bond, were pooled and concentrated by ultrafiltration (alkalitreated trypsin fragment). RESULTS

PuriJcation of crude hmaggktinin. Table I summarizes data pertaining to the purification of crude hemagglutinin. Although the hemagglutinating activity was not observed in crude extracts of the seeds used in this study, the extracts developed distinct nonspecific hemagglutinating activity after dialysis as shown in Table I. Considerable enrichment of the hemagglutinating activity was achieved by (NH&SO4 fractionation of the crude extracts. Since the crude hemagglutinin thus obtained by (NH&SO* fractionation was effectively inhibited by Dgalactose, it was further purified by specific affinity chromatography on Sepharose 6B as shown in Fig. 1. Strong hemagglutinating activity was observed only in the fraction (Peak B) which was eluted by 0.1 M lactose. Purified hemagglutinin was obtained by gel filtration of Peak B on Biogel P-200. The minimum hemagglutinating dose of the purified hemagglutinin obtained was 40 pg/ml against human erythrocytes irrespective of ?tl, N blood groups. Further

+ FIG. 2. Polyacrylamide disc-gel electrophoresis of purified hemagglutinin. Electrophoresis was 1 hr at 5 ma/tube with 7.5% gels in 0.04 M Trisglycine buffer, pH 8.3.

tests for the homogeneity of the purified hemagglutinin were performed by ultracentrifugation and disc electrophoresis. Analytical results. Ultracentrifugation of purified hemagglutinin yielded a single symmetrical peak during the whole of the run. The sedimentation coefficient (~“20~) calculated from the sedimentation velocity data was 7.5 S. Molecular weight of 195,000 was estimated for the purified hemagglutinin by sedimentation equilibrium at a speed of 7640 rpm assuming a partial specific volume of 0.73 which was calculated from the analytical data (27, 28). The electrophoretic homogeneity of the purified hemagglutinin was confirmed by

HEMAGGLUTININ

FROM

disc electrophoresis on polyacrylamide gel. A single band was obtained at pH 4.3 as well as at pH 8.3 (Fig. 2.). The amino acid composition of the TABLE

II

CHEMICAL COMPOSITION OF PURIFIED B. purpurea HEMAGGLUTININ Amino acid ASP Thr Ser Glu Pro GUY Ala CYS Val Met He Leu Tyr Phe LYS His As Try Total

g/100 g

Carbohydrate

g/100 g

9.30 7.64 6.57 4.44 3.88 3.59 3.09 1.28 4.48 0.00 4.61 5.14 4.33 6.08 2.53 3.03 4.85 4.31 79.15

Mannose Xylose Glucose Fucose Galactose Glucosamine Total

4.9 1.1 0.9 0.5 0.3 3.4 11.1

INHIBITION

Bauhinia

purified hemagglutinin is presented in Table II. The most notable feature of the amino acid composition of the hemagglutinin is the absence of methionine and the high proportion of aspartic acid and threonine residues. About 79.2% of the dry weight of the purified hemagglutinin could be accounted for as amino acid residues. If one adds the weight of carbohydrate moiety (ll.l%), the recovery is about 90%. Gas chromatographic determination of the carbohydrate moiety revealed that the major neutral sugar constituent was mannose (4.9 %) and the remainder of the neutral sugar was made up of smaller amounts of xylose, glucose, fucose, and galactose. Further, only glucosamine was detected as a component amino sugar by the amino acid analysis of the purified hemagglutinin. Quantitative data from the foregoing carbohydrate analyses are included in Table II. Inhibition assays on purified hemagglutinin. The results of inhibition tests of the purified hemagglutinin with simple sugars are given in Table III, and, for comparison, those tested on enzyme-treated cells are also listed in Table III. In all cases, the most active inhibitor is N-acetyl-D-galactosamine and,

TABLE III ASSAY OF PURIFIED HEMAGGLUTININ Minimum

479

purpurea

WITH SUGARS

concentration (maa) completely inhibiting hemagglutinating doses for

four

Sugars Intact cells D-Glucose D-Galactose D-Mannose n-Fucose L-Rhamnose D-Xylose L-Arabinose N-Acetyl-n-glucosamine N-Acetyl-n-galactosamine Lactose Melibiose Raffinose Maltose Cellobiose Trehalose N-Acetyllactosamine Di-N-acetylchitobiose

>lOO 1.57 >loO >lOO 50 >loo 25 >lOO 0.78 1.57 1.57 6.25 >lOO 100 >lOO 1.57 >lOO

Trypsin-treated >lOO 0.78 >lOO >lOO 25 100 25 >lOO 0.78 0.39 0.78 3.13 >lOO 50 >lOO 0.78 >lOO

cells

Neuraminidase-treated cells >lOO 25 >lOO >lOO 25 50 25 >100 3.13 12.5 12.5 50 >lOO 25 >lQO 3.13 >lOO

480

IRIMURA

AND OSAWA

in general, the so-called M&kela’s group 2 sugars are potent inhibitors. It is also apparent from Table III that the sugars are more inhibitory against trypsin-treated cells, but less inhibitory against neuraminidase-treated cells, than against untreated cells. Effects of enzyme treatments of erythrocytes on the hemagglutination titers of the purified hemagglutinin. Table IV shows the effects of

various enzyme treatments of human erythrocytes on the hemagglutination titer of the purified hemagglutinin. The hemagglutination titer was increased appreciably by the treatment with any of the proteolytic enzymes and neuraminidases regardless of the blood group of the cells. Further, as shown in Table IV, successive treatments of cells with trypsin (3 hr) and neuraminidase (1 hr) or vice versa resulted in greater in-

TABLE

IV

EFFECT OF ENZYMES ON AGGLUTINABILITY OF HUMAN ERYTHROCYTES WITH B. purpurea AND I. amara HEMAGGLUTININS

Titer Enzyme treatment

Purified B. purfufea hemagglutinina OMM

Untreated Trypsind Bromelind Pronased C. perfringens neuraminidaaee S. purpeofuscus neuraminidase” Influenza virus Jap 3O56 Trypsin after V. cholerae neuraminidase V. cholerae neuraminidase after trypsin

ONN

l/l

l/l

V3

W3 l/8

l/f3 l/8

l/16 l/64 l/64 l/64 l/64 l/128

l/16 l/64 l/64 l/64 l/64 l/128

l/16 l/64 l/64 l/64 l/64 l/128

l/256

l/256

l/256

l/l l/8

V. cholerae neuraminidaae”

OMN

I. antara hemagglutininb OMM

OMN

ONN nc n

l/l l/l

l/l l/l

l/f3

l/8

l/8

V3

l/8

l/8

l/32

l/32

l/32

a Initial concentration: 40 &ml. b Initial concentration : 80 mg/ml. c No agglutination. d Three-hour treatment.. 8 One-hour treatment. TABLE INHIBITION

V

ASSAYS OF VARIOUS PHYTOHEMAGGLUTININS WITH TRYPSIN FRAGMENT OBTAINED FROM HUMAN ONN ERYTHROCYTES

Hemagglutinins

Purified B. purpurea Iberis amara Ricinus communis Phaseolus vulgaris Glycine max Wistaris jloribunda @Protein concentrations albumin m the standard.

Minimum concentration (pg protein/ml)a completely inhibiting four hemagglutinating doses Intact trypsin fragment

Acid-treated trypsin fragment

Alkali-treated trypsin fragment

> 1720 > 1720 1720 860 >172O > 1720

5 150 1500 150 300

40 >64O 10 640 300 300

were determined by the method of Lowry et al. (32), using bovine serum

HEMAGGLUTININ

FROM

crease of the agglutinability of the cells with the purified hemagglutinin or I. amara hemagglutinin than in the case of the treatment with either enzyme alone. It is of interest to note that the increase in the agglutinability of the cells is larger if the trypsin treatment precedes the neuraminidase treatment. Inhibition assays of various hemagglutinins with trypsin fragment from human erythrocytes. Table V shows the inhibitory activity of the trypsin fragment obtained from human ONN cells as well as those of its products after acid and subsequent alkaline hydrolysis against various phytohemagglutinins. In general, the inhibitory activity of the trypsin fragment against these phytohemagglutinins was remarkably increased after mild acid hydrolysis in which about 94% of neuraminic acid was found to be released, whereas notable difference in specificity between the phytohemagglutinins relevant to M , N blood groups, the purified B. purpurea hemagglutinin and I. amara hemagglutinin, and other phytohemagglutinins tested was observed in the experiments using alkali-treated trypsin fragment as inhibitors. DISCUSSION

The hemagglutinins from Bauhinia purpurea, Iberis amara, and Vicia graminae seeds have generally been assumed to bind to the fundamental structures of M,N blood group antigens (6, 7). However, most of the work using these hemagglutinins has been done with crude extracts of the seeds or with partially purified products. In view of the recent observations from this laboratory (9, 19) that the crude extracts of certain plant seeds contain two kinds of hemagglutinins differing in specificity from each other, highly purified preparations are required for the elucidation of the structural requirements of the receptor sites with which these hemagglutinins can bind on erythrocyte membrane. In the present work, the hemagglutinin from Bauhinia purpurea alba seeds was purified by specific adsorption on Sepharose 6B column and subsequent displacement with lactose. Specific affinity chromatography on Sephadex G-50 column had been

Bauhinia

purpurea

481

successfully applied for the purification of concanavalin A by Agrawal and Goldstein (29). The purified hemagglutinin was found to be hemogeneous by ultracentrifugal analysis and electrophoresis on polyacrylamide gel. The sedimentation constant, s”2oW, of this preparation was 7.5 S and a M, of 195,000 was estimated from the sedimentation equilibrium experiments. Further, the glycoprotein nature of this purified hemagglutinin, containing 11.1% carbohydrates, was established by chemical analyses. In titration assays, the purified hemagglutinin was found to be nonspecific regarding M , N blood groups. Since Boyd et al. (1) reported that the addition of a small amount of melibiose made the nonspecific Bauhinia extracts N-specific, the oligosaccharides having n-galactose residue as a nonreducing terminal, lactose, melibiose, and raffinose, were added in various concentrations to the purified hemagglutmin, but the development of anti-N specificity could not be observed. In the hemagglutination-inhibition assays using simple sugars as inhibitors, it was reveaIed that the purified hemagglutinin was inhibited by the so-called Makela’s group 2 sugars, particularly by N-acetyl-ngalactosamine. These results are in good coincidence with those obtained on the crude extracts of the seeds by other investigators. (2-4). Although all of proteolytic enzymes and neuraminidases tested in this study enhance the agglutinability of cells with the purified hemagglutinin, the fact that the greater amounts of inhibitory sugar are required for the inhibition assays on the neuraminidase-treated cells than for those on trypsin-treated cells seems to imply that the neuraminidase renders much stronger affinity with the purified hemagglutinin to the receptor sites on the cell surface. This fact also suggests that the receptor sites of the purified hemagglutinin are, at least partly, covered by sialic acid and it is likely that N-acetyl-n-galactosamine or n-galactose residue next to sialic acid is most important for the interaction with the hemagglutinin. However, accurate interpretation of the results must be done aft.er the binding

482

IRIMURA

studies of labeled hemagglutinins with the enzyme-treated cells. The different nature of receptor sites even among the hemagglutinins which are specific for the Makela’s group 2 sugars was demonstrated by the inhibition assays using trypsin fragment obtained from human ONN cells and its products after acid and alkaline hydrolysis as inhibitors. Thus, the alkaline reductive cleavage decreased the inhibitory activity of the trypsin fragment against both Bauhinia and Iberis hemagglutinins, whereas the inhibitory activity against other phytohemagglutinins tested was rather increased by this alkaline cleavage. These facts suggest that the receptor sites for Bauhinia and Iberis hemagglutinins, both of which have generally been regarded to bind with the fundamental structure of M,N blood groups, are mostly composed of the alkaline-labile carbohydrate chain such as the one reported by Thomas and Winzler (30). Although final conclusion must await the inhibition assays using highly purified hemagglutinins, these facts also suggest that the other phytohemagglutinins tested can more effectively bind to the alkalineresistant carbohydrate chain, possibly similar to the one isolated by Kornfeld and Kornfeld (31) from human erythrocytes. Detailed structural feature of the receptor site with which the Bauhinia hemagglutinin bind on cell surface and its possible relation to M, N blood group antigenic structure remain to be elucidated. Along these lines further work is now in progress in this laboratory. ACKNOWLEDGMENT

We are grateful to Dr. T. Aoyagi, Institute of Microbial Chemistry, Tokyo, for generous gifts of purified neuraminidases. REFERENCES 1. BOYD, W. C., EVERHART, D. L. AND MCMASTER, M. H. (1958) J. Immunol. 81,

414. 2. UHLENBRUCK, G., AND DAHR, W. (1971) Voz Sang 21, 338. 3. BOYD, W. C., AND WASZCZENICO- ZACHARCZENKO, E. (1961) Transfusion 1,223. 4. PARDOE, G. I., UHLENBRUCK, G., AND REIFENBERG, U. (1971) J. Med. Lab. Technol. 28,255. 5. M&CELA, 0. (1957) Ann. Med. Exp. Biol. Fenn.

36, Suppl. 11.

AND

OSAWA

6. ROMANOWSKA, E. (1964) Vor Sang 9,578. 7. UHLENBRUCK, G. (1969) VOX Sang. 16,200. 8. C~SSIDY, J. T., JOURDIAN, G. W., AND ROSEMAN, S. (1965) J. Biol. Chem. 240.3501. 9. TOYOSHIMA, S., AKIYAMA, Y., NAKANO, K., TONOMURA, A., AND OSAKA, T. (1971) Biochemistry 10, 4457. 10. OSAWA, T. (1966) Biochim. Biophys. Acta 116, 507. 11. RACE, R. R., AND SANGER, R. (1954) Blood Groups in Man, p. 78. Blackwell, Oxford. 12. SCHACHMAN, H. K. (1959) Ultracentrifugation in Biochemistry, Academic Press, New York. 13. VINOGRAD, J., BRUNNER, R., KENT, R., AND WEIGLE, J. (1963) Proc. Xat. Acad. Sci. U.S.A.

49, 902.

14. YPHANTIS, D. A. (1960) Ann. N.Y. Acad. Sci. 88, 586. 15. ORNSTEIN, L. (1964) Ann. N.Y. Acad. Sci. 121, 321. 16. DAVIS, B. J. (1964) Ann. N.Y.

Acad. Sci. 121,

404. 17. REISFELD, R. A., LEWIS, U. J., AND WILLIAMS, D. E. (1962) Nature London 196,281. 18. SPACKMAN, D. H., STEIN, W. H., AND MOORE, S. (1958) Anal. Chem. 30,1185. 19. MATSUMOTO, I., AND OSAWA, T. (1969) Biochim. Biophys. Acta 194,180. 20. GOODWIN, T. W., AND MORTON, R. A. (1946) Biochem.

J. 40, 628.

21. DUBOIS, M., GILLES, K. A., HAMILTON, J. K., REBERS, P. A., AND SMITH, F. (1956) Anal. Chem. 28,350. 22. GARDF,LL, S. (1953) Aeta Chem. Stand. 7, 207. 23. AMINOFF, D. (1961) Biochem. J. 81,384. 24. MATSUI, M., OKADA, M., IMANARI, T., AND TANIURA, Z. (1968) Chem. Pharm. Bull. 16, 1383. 25. MATSUMOTO, I., AND OSAWA, T. (1970). Arch. Biochem. Biophys. 140, 484. 26. WINZLER, R. J., HARRIS, E. D., PEKAS, D. J., JOHNSON, C. A., AND WEBER, P. (1967) Biochemistry 6, 2195. 27. GIBBONS, R. A. (1966) in Glycoproteins (Gott-

schalk, A., ed.), p. 29. Elsevier, Amsterdam. 28. SCHACHMAN, H. K. (1957) Methods Enzymol. 4. 32. 29. AORAWAL, B. B. L., AND GOLDSTEIN, I. J. (1967) Biochem. Biophys. Acta 147.262. 30. THOMAS, D. B., AND WINZLER, R. J. (1969). J. Biol.

Chem. 244,5943.

31. KORNFELD, R., AND KORNFELD,

S. (1970) J. Biol. Chem. 246, 2536. 32. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (1951) J. Biol. Chem. 193, 265.