Physical studies on three lectins from the seeds of Abrus precatorius

43

Biochimica et Biophysica Acta, 621 (1980) 43--52 © Elsevier/North-Holland Biomedical Press

BBA 38341

PHYSICAL STUDIES ON T H R E E LECTINS FROM THE SEEDS O F A B R US PRECA TORIUS

MARY S. HERRMANN and W. DAVID BEHNKE

The Department of Biological Chemistry, University of Cincinnati Medical Center, Cincinnati, OH 45267 (U.S.A.) (Received June 7th, 1979)

Key words: Lectin; A brin; Agglutinin; Tryptophan accessibility; (Abrus precatorius)

Summary The physical properties of three lectins from the seeds of the Abrus precatorius plant, abrin C, abrin A and the Abrus agglutinin, were studied. All three exhibited similar circular dichroic (CD) spectra in the near-ultraviolet having negative maxima at 286 and 293 nm. In addition, D-galactose induced similar conformational alterations in the three proteins as observed through changes in the near-ultraviolet CD from 280 to 295 nm. The near-ultraviolet CD spectrum o f the toxic subunit of abrin C was very different from that of the parent molecule. The fluorescence emission spectra of the three proteins were also studied. All exhibited fluorescence near 335 nm which is quenched 9% b y galactose. Iodide quenching of fluorescence using the Stern-Volmer analysis indicated different tryptophan accessibilities in the presence and absence of D-galactose for the Abrus agglutinin. The results suggest that there is a saccharide-induced conformational change which buries several partially exposed tryptophan residues. A comparable analysis of the closely related Ricinus agglutinin revealed that its t r y p t o p h a n residues are more buried than those of the Abrus agglutinin and, unlike the Abrus agglutinin, there was no saccharide-induced change in tryptophan accessibility.

Introduction

Lectins is a term originally proposed b y Boyd [ 1] to describe a class of proteins which causes agglutination of erythrocytes. Much interest has arisen concerning lectins since it was discovered that t u m o r ceils are more easily agglutinated b y lectins than the corresponding normal cells [2]. The seeds of the A brus precatorius plant contain the lectin abrin which is unlike many other

44 lectins in that it is toxic to animal cells [3,4]. Abrin has been shown to be more toxic towards t u m o r cells than for normal cells. Abrin was found to suppress the growth of solid Ehrlich ascites t u m o r and fibrosarcoma in mice and Yoshida sarcoma in rats [5,6] and, in addition, demonstrated a strong inhibitory effect in nude mice bearing solid human cancers [7]. In another publication, we described the purification of three proteins from the seeds of A. precatorius, abrin C, abrin A, and Abrus agglutinin [8]. Both abrin C and abrin A were found to be toxic lectins, however, abrin C was more like the abrin described b y others. The Abrus agglutinin has been shown to be non-toxic to animal cells and a p o t e n t agglutinator of erythrocytes [9,10] and as such is a more ,typical lectin. Abrin C has a molecular weight of 65 600 and is composed of t w o subunits, a sugar-binding subunit (B-chain) and a toxic subunit (A-chain) [10]. Abrin A is 62 500 daltons also having t w o unlike subunits and the Abrus agglutinin is a tetramer of 134 900 daltons [8]. The three proteins from A. precatorius are closely related both chemically and physically. All three are giycoproteins b u t not metalloproteins [8]. Circular dichroic studies demonstrated that all three have almost identical secondary structures, being dominated by fl-pleated sheet [8]. Furthermore, all three lectins specifically bind D-galactose; abrin C and abrin A are monovalent while the agglutinin is bivalent [ 8,9]. Another toxic lectin, ricin, has been obtained from the seeds o f Ricinus communis. Ricin is like abrin in many respects including the mechanisms of its toxic action, sugar specificity, sugar content, molecular weight (64 000) and number of subunits [4,9,12]. In addition to ricin, these seeds also contain a bivalent agglutinin, the Ricinus agglutinin which is almost identical to the Abrus agglutinin in b o t h its physical and biological properties [9]. In this communication, we further compare the three Abrus proteins by examining some physical properties including circular dichroism and fluorescence. The comparison also includes the closely related Ricinus agglutinin. Materials and Methods Materials. Mature seeds of the A. precatorius plant were obtained from Dr. Julia Morton of the Morton Collectanea, University of Miami in Coral Gables, FL. Monosaccharides were purchased from Sigma Chemical Company. Pierce Chemical Company was the source of guanidine hydrochloride. Sodium iodide and sodium thiosulfate were purchased from Fisher Scientific Company. Other chemicals used were reagent grade. Methods. The proteins from the A. precatorius seeds were purified as per Ref. 8. Protein was routinely quantitated by measuring its absorption at 280 nm and for abrin C [13], 14.6 using the following extinction coefficients (E280:12.9 1~ for the agglutinin [14] and 11.8 for abrin A [13]. The extinction coefficient used for abrin C A-chain was 7.87 as earlier described [14]. Absorption spectra were recorded on a Cary Model 15 double-beam spectrophotometer using a 0--1.0 slide wire. Measurements were made at ambient temperature (22QC) using quartz cells of 1 cm pathlength.

45

Circular dichroic spectra were obtained at ambient temperature (22 ° C) with a Cary 61 spectropolarimeter. Standardization of the spectropolarimeter was accomplished using a 1 mg/ml aqueous solution of (+)-10-camphorsulfonic acid as specified by Varian Associates. All CD spectra were recorded in strain-free quartz cylindrical cells of 1 cm pathlength. Ellipticity is expressed as mean residue ellipticity, [0 ]22 = (O/lO)(M/lc)with units of degree/cm -2 per drool, where 0 is the observed ellipticity in degrees, M is the mean residue weight, l is the pathlength in centimeters, and c is the concentration in g/ml. Molecular eUipticities are not corrected for the refractive index of the solvent. Protein concentrations used in this region of the spectrum were 1--2 mg/ml. Fluorescence emission spectra were recorded on a Perkin-Elmer MPF 44A spectrofluorometer used in the ratio mode. The sodium quenching experiments were performed using a Baird Atomic Fluorospec Model SF-100 spectrofluorometer with an Engelhard Hanovia 15-W power supply. For both instruments a Bausch and L o m b omnigraph 2000 recorder was used. The temperature was maintained at 25°C b y means of a circulating temperature bath. Fluorescence spectra were measured using quartz cuvettes of 1 cm pathlength at right angles to the incident light. Fluorescence quantum yields, Q, were obtained by comparison of the emission spectra of a standard, tryptophan, and the u n k n o w n sample excited at 290 nm. The u n k n o w n Q was calculated from the following equation: Qunknown

=

Qstandard X

Funknown

,./

Astandaxd

Fstandard /x Aunknown

where F is the relativefluorescence determined by integratingthe area beneath the fluorescent spectrum and A is the absorbance at 290 nm. The Q of the standard tryptophan was taken to be 0.13 [14]. Sodium iodide quenching experiments were performed by the addition of small increments of an aqueous NaI solution (1 M) which contained a small amount of sodium thiosulfate (10 4 M) to suppress triiodide formation. The emission spectra were adjusted for the dilution effect mathematically. Control experiments consisted of adding NaCl instead of NaI to rule out ionic strength effects. Results

Circular dichroism (CD) in the near-ultraviolet The near-ultraviolet CD spectra of abrin C and the A brus agglutinin are presented in Fig. 1. Negative maxima occur at 293 and 286 nm for b o t h lectins, the agglutinin having slightly greater ellipticity. Most likely, the peak at 293 nm and possibly the peak at 286 nm arise from tryptophan residues. There is a small a m o u n t o f negative ellipticity above 300 nm indicating some contribution from the disulfide bonds. The CD spectrum of abrin A in the near-ultraviolet is shown in Fig. 2. It is very much like the other t w o Abrus proteins having negative maxima at 285 nm and 293 nm. However, abrin A has greater ellipticity in this region especially at 293 nm. Also, unlike the other two proteins, the ellipticity above 300 nm

46

I0" 0

:"

:

:

:

°° t

:

'!

Te -IO-

E -20" N ~ -30-

40

x -40-

". ~

~-50"

~ -60-

70"

-80 T I

270

I 280

~,

I 290 hrfl

I 300

I 310

I

27O

I

I 290

)%rim

I

I 310

Fig. 1. N e a x - u l t r a v i o l e t C D s p e c t r a o f a b r i n C a n d a g g l u t i n i n . N e a r - u l t x a v i o l e t C D s p e c t r a o f A b r u s agglutinin ( ), a b r i n C ( . . . . . . ) and Abrus agglutinin after the addition of 5 ' 10-4 M galactose (...... ) ( 8 0 % s a t u r a t i o n ) . P r o t e i n c o n c e n t r a t i o n w a s 1 m g / m l a n d t h e b u f f e r u s e d w a s 3 0 m M NaCI, 1 0 m M Tris, p H 7 . 7 . A P a t h l e n g t h o f 1 c m w a s u s e d w i t h a full s c a l e d e f l e c t i o n o f 0 . 0 5 °. Fig. 2. N e a r - u l t r a v i o l e t C D s p e c t r u m o f a b r i n A. N e a r - u l t r a v i o l e t CD s p e c t r a o f a b r i n A w i t h o u t ( ) ) 5 • 1 0 -4 M g a i a c t o s e a d d e d . ( 8 0 % s a t u r a t i o n ) . C o n d i t i o n s w e r e t h e s a m e as u s e d i n Fig. 1.

a n d w i t h (. . . . . .

forms a peak centered at 306 nm. All three proteins have a broad positive band from 250 to 270 nm which undoubtedly contain contributions from tyrosine and phenylalanine residues as well as disulfide bonds. The response of the three Abrus proteins to the binding of sugar is also similar as all exhibited an increase in amplitude. The saccharide-induced change in the agglutinin (Fig. 1) and abrin C (data not shown) are almost identical; both underwent an increase in amplitude primarily from 280 to 297 nm. The change in the abrin A spectrum (Fig. 2) is primarily an increase in the 285 nm peak. The near-ultraviolet CD spectrum of the abrin C A-chain was also determined and appears in Fig. 3. Quite surprisingly, its ellipticity is entirely positive while

50, 40. 30.

[e]

20.

IO O' -I0" -20' 250

ZTO

290

310

)~lnm Fig. 3. N e a r - u l t r a v i o l e t C D s p e c t r u m o f a b r l n C A - c h a i n . N e a ~ u l t r a v i o l e t C D s p e c t r u m o f a b r i n C A - c h a i n a t a c o n c e n t r a t i o n o f 0 . 6 4 m g / m l u s i n g a p a t h l e n g t h o f 1 e m a n d a full s c a l e d e f l e c t i o n o f 0 . 0 2 ° . B u f f e r c o n d i t i o n s w e r e t h e s a m e as i n Fig. 1.

47 that of the intact abrin C molecule is entirely negative. This could be due to conformational changes incurred when the subunit is separated from the B-chain. The spectrum is characterized by maxima at 294 nm and 287 nm which are probably caused by the two tryptophan residues in the A-chain (perhaps the 1La and 1Lb indole transitions). There is a maximum at 279 nm and a broad peak with a maximum at 265 nm in the region where tyrosine and phenylalanine chromophores display Cotton effects. Fluorescence studies The results of CD studies in the near-ultraviolet suggest that one or more tryptophan residues change environment when the A brus proteins go from the unbound to the bound state. Since fluorescence emission is usually caused almost entirely by tryptophan residues, the fluorescence properties of the A brus proteins were studied. (Abrin C and abrin A each contain ten while the agglutinin contains 27 +- I tryptophan residues.) Fig. 4 shows the fluorescence emission spectrum of the agglutinin at an excitation wavelength of 280 nm. The emission maximum occurs at 334 nm with a quantum yield of 0.13. It was found for the agglutinin, as well as the other proteins studied, that the fluorescence emission results entirely from tryptophan residues with no tyrosine components. This was accomplished by comparing spectra excited at 270 and 290 nm as previously described [17]. The fluorescence spectrum of the agglutinin in the presence of D-galactose is also shown in Fig. 4. D-Galactose induces a reduction in the quantum yield along with a 2 nm shift in the emission maximum. The difference spectrum (see Fig. 4) peaks at 350 nm revealing that the tryptophan residues which are quenched by D-galactose are exposed to the solvent. The fluorescence spectrum of abrin C was similar to that of the agglutinin except that its maximum occurred at 335 nm, with a quantum yield of 0.17. The higher wavelength of its maximum indicates that its tryptophan residues are more exposed to the solvent. The fluorescence spectrum of abrin A indicates

w

~n

I0

_o mm

8

-nr" mc ~0 m~

o

•.J u.

6

~z

~4

m

O.

2

...°" I 300

I

°'°O,o..., I 340

I

I 300

!

I 420

)~inm

Fig. 4. F l u o r e s c e n c e e m i u i o n s p e c t r u m o f Abru8 agglutinin. F l u o r e s c e n c e e m i s s i o n s p e c t r u m o f A b r u s agglutinin at a concentration of 0.13 rag/m1 with an excitation wavelength of 280 nm( ). - . . . . . , the e m i s s i o n s p e c t r u m after the a d d i t i o n o f 5 • 1 0 - 4 M g a l a c t o s e ( 8 0 % s a t u r a t i o n ) , a n d . . . . . . . an enlargem e n t o f the difference b e t w e e n these t w o spectra (see scale o n the r i g h t ) . B u f f e r u s e d w a s 3 0 r n M N a C I , 10 mM Tris, pH 7.7.

48 TABLE

I

FLUORESCENCE

D A T A F O R A B R US P R O T E I N S

AND RICINUS AGGLUTININ

Agglutinin A brus E m i s s i o n m a x i m u m (A = 2 8 0 n m ) Emission maximum (A = 290 nm) Quantum yield Emission maximum with sugar bound Emission maximum of the difference spectrum Fluorescence quench due to sugar Shift in emission maximum due to I(7.5 • 10 -2 M) Emission maximum of the difference spectrum

334 nm 338 nm 0.13 332 nm 355 nm

*

9 ± 0.5% --5 nm

Abrm C

AbrinA

335 nm 339 nm 0.17 333 nm 355 nm

331 nm 335 nm 0.12 329 nm 354 nm

Ricinu8 329 nm 330 nm 0.11 328 nm +318 nm/--350

nm **

+5%

350 nm

9%

7%

0

0

--1 nm

340 nm

335 nm

335 nm

* Sugar used was D-galactose. ** The difference spectrum of the Ricinus agglutinin exhibits two maxima, i n c r e a s e i n f l u o r e s c e n c e a n d o n e a t 3 5 0 n m d u e t o a d e c r e a s e ( s e e F i g . 5).

one at 318 nm due to an

that its tryptophan residues are more buried than those of abrin C, since its m a x i m u m occurs at a lower wavelength (331 nm). Both abrin C and abrin A have their fluorescence spectra quenched by D-galactose, the same as occurred with the agglutinin. Table I is a summary of all the fluorescence data. The fluorescence of the A brus agglutinin was compared to that of the Ricinus agglutinin. The circular dichroism of the Ricinus agglutinin in both the near and far-ultraviolet are close to that of the A brus agglutinin indicating similar secondary and tertiary structures [18,19]. However, the fluorescence of the t w o agglutinins proved to be different. The emission spectrum (see Fig. 5) of the Ricinus agglutinin has a m a x i m u m at 329 nm which suggests that its tryptophan residues tend to be more buried than those o f the A brus agglutinin.

I0

o w

e

uJ .J u.

' 0 . 0 ~', ,-0.5":"

I 300

I

I 340

!

I 380

I

i 420

)~ nm

F i g . 5. F l u o r e s c e n c e

emission spectrum

of Ricinus agglutinin. Fluorescence

emis~flon s p e c t r u m

of Rlcinus

agglutinin at a c o n c e n t r a t i o n o f 0 . 1 4 m g / m i w i t h a n e x c i t a t i o n w a v e l e n g t h o f 2 8 0 n m . - . . . . . , t h e e m i s s i o n s p e c t r u m after the a d d i t i o n o f 5 • 1 0 - 4 M g a l a e t o s e , a n d . . . . . . . an enlargement of the difference spectrum

between

these two spectra (see scale on right). Buffer conditions

w e r e t h e s a m e as i n F i g . 4.

49

ABRUS

I0'

AGGLUTININ

RICINUS

AGGLUT ININ

z

~8 o~ t~ bJ >

r~

2' °

I 300

.

"

•

I

I 340 )~,

I Ilm

I 380

..*

°

I

"l 300

"

"**

I

I 340 X,

I

.

I .... 380

!"

nm

F i g . 6. F l u o r e s c e n c e q u e n c h i n g d u e t o I - . Q u e n c h i n g o f t h e f l u o r e s c e n c e e m i s s i o n s p e c t r a o f A b r u s and R i c i n u s a g g l u t i n i n s d u e t o I - . A n e x c i t a t i o n wavelength o f 2 9 0 n m w a s u s e d a n d t h e i n i t i a l protein concentration w a s 0 . 1 3 m g / m l . - - , without, and ..... , with 7.5 • 10 -2 M sodium iodide ........ the difference s p e c t r u m . D i l u t i o n o f the protein s o l u t i o n d u e t o a d d e d N a I w a s c o r r e c t e d m a t h e m a t i c a l l y . B u f f e r c o n d i t i o n s w e r e t h e s a m e as i n F i g . 4.

When sugar is b o u n d to the Ricinus agglutinin the fluorescence increases, the maximum of the difference spectrum occurring at 320 nm. This is n o t due to tyrosine fluorescence because the effect is greatest at an excitation wavelength of 295 nm, and least at an excitation of 270 nm. The difference in the tryptophan residues between the t w o agglutinins was examined further b y observing the quenching of the protein fluorescence by I-. The fluorescence spectrum of both agglutinins with 7.5 • 10-2 M NaI added is shown in Fig. 5 along with the initial fluorescence. There is a 5 nm shift in the emission spectrum of the Abrus agglutinin to a shorter wavelength while that of the Ricinus agglutinin remains unchanged. The difference spectrum in each case is also shown in Fig. 6. For the Abrus agglutinin, the maximum of the difference spectrum occurs at 350 nm which indicates that more exposed tryptophan residues are being preferentially quenched by NaI. The maximum of the difference spectrum of the Ricinus agglutinin occurs at 340 nm so that the tryptophan residues being quenched are partially exposed. Quenching due to I - occurs through a collisional process and has been shown to adhere to the Stern-Volmer relation (see Fig. 7 [16]). According to the relation, the slope, KQ, of a plot of initial fluorescence (F0) divided by fluorescence (F) in the presence of NaI versus the NaI concentration is an indication of tryptophan availability. Fig. 7 shows the results of such a plot for the Abrus agglutinin. The fact that the points are linear indicates that the tryptophan residues being quenched are homogeneous in their degree of exposure. KQ was found to be 3.5 M -1 which is much smaller than that determined for free tryptophan of 10.5 M -1. To demonstrate that the quenching of agglutinin fluorescence is a consequence of collision by I- rather than a nonspecific ionic strength effect, it was shown that comparable concentrations of NaC1 had no effect on the fluorescence. The same experiment was performed in the presence of saturating amounts of D-galactose. At low concentrations of I-, the line runs parallel to the line

50

1.3-

Fo/F = I + Ko[NoT~

Fo/F 1.2-

I.I-

I

I

o.ot

I

I

I

I

I

I

o.o2 0.05 o.o4 0.05 0.06 o.o7 0.08 ['NAT], M

Fig, 7. S t e r n - V o l m e r p l o t of A b r u s agglutinin. S t e r n - V o l m e r p l o t o f the A b r u s agglutinin in the absence (e) and presence (o) o f 1 0 -3 M galactose. The data were c o l l e c t e d as described in Materials and Methods and p l o t t e d a c c o r d i n g to the e q u a t i o n s h o w n . The slope o f the line w i t h o u t galactose was f o u n d to be 3 . 5 . The c o n d i t i o n s used to generate the data were identical t o t h o s e described in Fig. 6.

without sugar, indicating that some tryptophan residues with the same degree of exposure are being quenched. However, at higher iodide concentrations, the slope (KQ) is reduced indicatingthat some residues with less exposure are being quenched. Apparently, the accessible tryptophan residues change from homogeneity to heterogeneity of exposure when sugar is bound. The experiment was repeated using D-glucose instead of D-galactose as D-glucose fails to protect erythrocytes from agglutination by the A brus agglutinin [9]. As expected, the changes in Fig. 7 did not occur. However, when D-fucose was used, results comparable to those with V-galactose occurred. Apparently, the Abrus agglutinin binds fucose in a similar manner to the binding of galactose. The NaT quenching experiment was then performed with the Ricinus agglutinin (Fig. 8). In this case, the slope (KQ) decreases with increasing iodide concentrations, and there is essentially no change when D-galactose is added. Stern-Volmer plots of both agglutinins (Figs. 7 and 8) intercept the ordinate above the expected value of Fo/F = 1. While there does not appear to be an

1,2

% 1.1"

I O.Oi

I

0.02

I

0.03

I

0.04

I

O.OS

I

I

O.Oe O.oT

[NaZ],M

Fig. 8. S t e r n - V o l m e r p l o t o f R i c i n u 8 agglutinin. S t e r n - V o l m e r p l o t o f the R i c i n u s aggluttnin in the absence ( e ) and presence (o) o f 1 0 -3 M galactose. All c o n d i t i o n s w e r e the s a m e as in Fig. 7.

51

explanation for this, it is not unprecedented as a similar plot of gene 32-protein also intercepted the ordinate above 1 [17]. It has been suggested to us that a small amount of I- may produce significant quenching probably due to binding rather than do dynamic quenching. The NaI quenching experiment was also performed on abrin A and abrin C (data not shown) in order to compare the three Abrus proteins. The findings were somewhat different than those obtained for the agglutinin. Both abrins had very little shift in the emission maximum as a result of NaI quenching, and consequently, the maximum of the difference spectrum is close to the initial emission maximum (Table I). Abrin C by Stern-Volmer analysis gave a straight line with a much higher KQ than that of the agglutinin indicating that its tryptophan residues are much more exposed. This agrees with its higher quantum yield. The Stern-Volmer plot for abrin A resulted in a KQ of 4.7 which is less than abrin C and more than the agglutinin. Perhaps part of the reason the aggiutinin has a lower KQ is that the two additional subunits cause some tryptophan residues to be protected from the solvent. Abrin A undergoes a small change in its Stern-Volmer plot when galactose is included (data not shown). The change is similar to that of the agglutinin (Fig. 7), but the net change is smaller. Discussion The near-ultraviolet CD spectra of the three A brus proteins proved to be very similar to one another indicating comparable tertiary structures. The similarity went further as all three spectra exhibited an increase in ellipticity when galactose was added. In the area of the spectrum where the change is greatest (285--295 nm), the most likely groups involved are tryptophan side chains [20]. There is undoubtedly some contribution from disulfide bonds as well in this region because of the tapering off of the curves to the baseline up to 320 nm [21]. However, since the absorhance of disulfide chromophores tend to be much weaker than aromatic chromophores [22], the observed change was tentative assigned to tryptophan chromophores. The three A brus proteins also had very similar fluorescence properties and the fluorescence of each was quenched slightly by the addition of D-galactose. The fact that similar saccharide-induced changes were observed for all three proteins using both CD and fluorescence suggest that these three lectins undergo comparable changes in conformation upon the binding of D-galactose and that this change involves several tryptophan residues. The cumulative results indicate that some of the ten exposed tryptophan residues (probably four; data from a modified Stern-Volmer [16] not shown) are completely exposed on the surface of the A brus aggiutinin and remain such when D-galactose binds. The remainder of the ten residues (probably six) interact with solvent and the I-, but are not available to the releatively large modifying agents. It is these latter residues which move to a more protected environment upon sugar binding. It was previously demonstrated that the Abrus and Ricinus agglutinins have closely related far-ultraviolet CD spectra and that this type of spectrum is unique among lectins and that this type of spectrum is unique among lectins [18]. A comparison of the near-ultraviolet CD spectrum of the Abrus aggluti-

52

nin to that previously found for the Ricinus agglutinin [19] reveals that the two are similar in this area of the spectrum as well. Also like the A brus agglutinin, the Ricinus agglutinin exhibited a saccharide-induced increase in ellipticity in the near-ultraviolet. However, the major change occurred at a lower wavelength, from 270 to 290 nm. In this area of the spectrum, the change is most likely due to tyrosine side chains rather than tryptophan as was suggested by these authors [19]. The fluorescence emission data of the Ricinus agglutinin revealed its tryptophan residues to be more buried than those of the A brus agglutinin. It also responded quite differently to the addition of sugar than the A brus proteins. Stern-Volmer analyses indicated that the tryptophan residues of the Ricinus agglutinin do not change availability when galactose is bound unlike the A brus agglutinin. Furthermore, its pattern of tryptophan availability is comparable to that of the Abrus agglutinin in the sugar-bound conformation. This difference may be the reason for the Ricinus agglutinin having a larger binding constant than the Abrus agglutinin (15 000 versus 8000 M -1 [9]). These results are consistent with the near-ultraviolet CD results which also indicates a saccharideinduced change to tryptophan residues only in the case of the A brus agglutinin. These fluorescence studies show, therefore, that the Ricinus agglutinin and possibly ricin as well, bind carbohydrate in a different manner than the A brus agglutinin. The finding of a major structural difference between these two agglutinins was unexpected as they have been notable for their similarities. References

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