- Email: [email protected]
Soybean trypsin inhibitors: Isolation, purification and physical properties
ARt’HITES
OF
RIOCHEMISTRY
Soybean
J. J. RACKIS,
From
AND
RIOPHYSICS
Trypsin
98, 471-478 (1962)
Inhibitors: Isolation, Physical Properties’-3
H. A. SASAME,4 R. K. MANN,5 A. K. SMITH the Northern
Regional
Research
Purification
and
R. L. ANDERSON
LaboratorvP
Peoria,
AND
lllixois
Received May 18, 1962 Two highly purified trypain inhibitors, designated SBTIA, and SBTI& , have been isolated directly from soybean whey solutions by chromatography on DESE-cellulose. The t\l-o inhibitors differ in physical properties, nitrogen content, and trypsin inhibitor activity. SBTIA, is identical to Kunitz’ crystalline soybean trypsin inhibitor, Molecular weight determinations and activity measurements indicate that SBTI4> and A? form trypsin-trypsin inhibitor complexes in the ratio of 1: 1. Commercial preparations of 5~ crystallized soybean trypsin inhibitor preparations frequently contain 550% impurities. A chromatographic method was developed on a preparative scale to purify these commercial inhibitors. INTRODUCTION
plants (1) have been att’ributed to inhibition of intestinal proteolysis by proteins having antitryptic activity. However, there appears to be no correlation between the effect of heat on the nutritive value of various legumes and the presence or absence of
Trypsin inhibitors are widely distributed in plants and animals (1, 2). Some inhibitars are globulins and others are polypep-
tides. Naturally occurring trypsin inhibitors were last reviewed in 1954 (3, 4). Growth-inhibiting properties of unheated :tlfalfa (5), raw soybeans, and many other
trypsin
‘Presented before the Division of Biological Chemistry, 140th Meeting, American Chemical Society, Chicago, Ill., Sept. 3-8, 1961. ?Abbreviations used: DEAE, diethylaminoethyl; SBTI, soybean trypsin inhibitor; SBTI(BX), commercial soybean trypsin inhibitor, five-times crystallized. 3DEAE-cellulose was purchased from Brown Company, Berlin, N. H. Mention of trade or company names does not imply endorsement by the U, S. Department of Agriculture over similar products or firms not mentioned. ’ Present address: Laboratory of Chemical Pharmacology, National Heart Institute, National Institutes of Health, Bethesda, Md. ’ Present address: Department of Biochemistry, University of Illinois, Urbana, 111. ‘This is a laboratory of the Northern Utilization Research and Development Division. .\gricultural Research Service. U. S. Department of +\griculture, Peoria, Ill.
inhibitor
(l).
Chicks (6) and rats (7) fed raw soybean meal develop hypertrophic pancreas. It has been suggested that pancreatic hypertrophy and excessive secretion of pancreatic juices are t,he result of a reaction to soybean trypsin inhibitor (7, 8). Crude SBTI preparations as well as SBTI (5x ) , isolated according to Kunitz (9)) produce these same physiological effects. The purity of these crystalline preparations, however, was not known. In a previous publication (loj , a chromatographic separation of the trypsin inhibitor activity of soybean whey proteins into two fractions was described. These protein components were designated as SBTIA, and A,, respectively. The present communication describes the chromatographic isolation, purification, and determination of the physical properties of SBTIAl , At,, and SBTI (5x).
471
47%
R$CKIS,
SASAME,
MANX,
PREI'ARATIOX OF WHEY PROTEINS AND CI-IR~MAT~GRA~H~ The whey proteins wer(’ prepared anct fmction:tt,ed actnording to previously described procedures (10) from Hawkeyc soybeans. 1958 crop. Two different 101s of D~AE-~~~l~~losc: Type 40 reagent grade, capacity 0.8 meq./g. and Srlrc~facel, l,eagent gratle, capacity 1.05 meq./g. were used. The prol.rin solution was clarified in a Scrlal tentrifngr: operating at a speed of 12,000 r.p.m. for 5 min. prior to addition to the column. The adsorbent was poured into the column, allowed to settle under gravity to a height of 43 cm., then compressed to a height of 39 cm. Protein content of the effluents was determined with Lowry’s phenol reagent (11) with phytnte-free acid-pre~ipitate(~ so\-bean protwin as a standard (12).
ANDERSON
AND SMITH
centrifugal nleas~ir~lnents. ~lectro~)~lorrsis was carried out at 2”. Mobility measurements were n&c on the :tsc,c?ntiingpnitcrns, with 1.06 as a m~gnifcation f:u*tor. Sedimcn tation ~~~I)er~~~~~n~s wcrc tlonc~ at room tcmprrnturc~ in n Spinc*o ultracentrifuge, model E. Sedimenl stion coneta,& were detcrminrtl nccordkg to Svedberg anti Pedersen (13). ~~oie~11~~11 weights were detcrrnincd from Trautman plots (14, 15). in :1 30-mm. double-sector ~811at room t (,n~wr:tture and three, rotor speeds. The rotor ~a< shaken between run,s. I’tart,ial specitic, volume: writ?; determined with a lo-ml. pycnometer at 25 Z? 0.1“ and two proiein concentrations in phosphate bllffcl with C~~arl~(~od’sequ:~tinn f 16).
1nhibitor.v xii&y against ttypsin was determined by Kunitz’ casein-digestion nlc,thotl (17). .-\ lot of commercial SBTI(5X) that was r*sentiall\ELECTBOPBORETICAND ~JLTRACENTRIF~JG~~ l~on~ogeneo~~~eicc~rophoreti~all~ and &tomatoANALYSIS graphically served as a standard. :\ctivity is c2;pressed in trppsin units (TIJ) numerically ~~(,u:11 Potassium phosphate buffer, pH 7.6, 0.1 ionic to micrograms of trypsin inhibited per microgram strength, was used for at1 e1e~trophoret.i~ and ultraof trypsin inhibitor. Sevfd prqtarationi: Of SBTI(5X) that were tested for purity wcrc (,t,tainrtl from various supply houses. R ESIJLTS
~~~R~MAT~~R,~P~I~ISOLATION AND PURIFKATION OF SRTIA,
L
2.4g2.0z ; 1.65
-
I
01 v’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ 0 4 8 12 16 20 24 20 32 3640 44 48 Effluent Tube Mumber Fro. 1. Stepwise elution diagram of whey proteins: 600-800 mg. dialyzed and lyophilized protein in 30 ml. of 0.01 M potassium phosphate buffer, pH 7.6; effluent fraction was 10 ml.; absorbancy is ~.heopticxl density at 750 rn@of a O.l-ml. aliquot of cfht~nt, with Fotin reagent (11). Vertieal arrows
indicate point of change of sodium chloride conIacLntmtion in buffer. Column dimensions of ad.wrImrt (Type 40) wcw 2.25 X 39 cm.
In Fig. 1 is shown the stepwisc elut,ion schedule used to isolate a crude ~reparatior~ of SBTIA, from lyophilized soybean whqprotein. Stepwiee increases of sodium chloride concen~sation in 0.01 M potassiur~l phosphate buffer pH 7.6, arc shown in tbcx legend. V’erbical arrows indicate the point of hange in the thltion buffer. Six fractions, designated according to tJleir order of elution, wwc obtained. Frncations I-IT? were discarded. Fraction V, corresponding to t.ube Nos. 33-40, was dialyzed against, cold dist’illed water and lyophilizctl. SBTIAl , in the combined tubes, was tq)proximately 855 pure. A yield of about 90 mg. prot,ein per run was obtained. There was a marked decrease in resolution betwrm Fractions IV? f’, and VI and a drcreasc in affinity of these proteins fol the adsorbent when about 1 g. of whey ~troteine was chroma~ogra~~hed under identical conditions as given in Fig. 1. For cxampl~. uo distinrt. peaks corresponding to tltPsc
fractions could he obt,ainctl. Trylkn inliibitor activity normally present in Fraction Y was now rlutcd with 0.15 JI instead of 0.17 M sodium chloritlc and contained only 50% SBTIA, There appeared to he little or no change in the concentration dr~wnclcncy of Fractions I-III. Tllc dccrc:~c in affinity and resolution of tllcw fractions is typical of substances liaring highly curvctl adsorlkion isotlrcrins; other factors, e.g., displacciucnt effects and c~hangw in hufl‘c~r capacity Ixcause of iuore protein, (aan 1~ implicating. Because of such Iwhr-ior, all t,he stepwire increases in salt concentration 1~1 to be iuatlc prior t,o elution of Fractions V and VI with 0.17 ant1 0.25 31 salt, rcspectiwlg. It was necessary to modify the clution wlicdule given in Fig. 1 if t,lic Stlcctawl DEXE-ccllulow was usctl in place of TTlw 40. The luodification consisted of chwnglng tile salt concentration in steps 2 and 3 to 0.14 ant1 0.18 111 sodium chloride, respectiwly. Othrwisc, the l)urit’y of PBTIA, in Flactiou ‘i’ \v:w dccrwsetl from 857; to 11cwly 7O’iZ. Tlio xwson for this hcliavior is probably not Iwcauw of tlic higher adsorptive calxwity of St~lcchwl, since Type 40 DEAEccllulosc, having tlw saint capacity ( 1.0 iucc~.,~g.I, (lid not require these changes in stqnviw c~lution. Clorrrspondence with tlw ulxnuf~\rtui~cl:’ indicates tliffcwnt lnwx~durcs wrv usccl to prqmrc tliesc cwliangtrs.
Two rc~clironintog~al,liic steps \vere ncccswry to purify HBTIX, from Fraction Y as shown in Fig. 2. Second rccllrolll:ttogr:ll~ll~ is indicated hy solid lines. Effluents fractions corresponding to tulw Kos. 36-41, incluwivcb. were wiul~inctl, dinlyzd ng:tinst csoltl tlistillctl water, ronwntratd to a.sinall wlui~~c, ant1 rc~~lirolllatogr:Il,llctl a sc~~~ntltiilw under iclcnticd conditions. Efflucnt~ fractions ( tub 3&i-41 inclusive 1 ncrc conil~inctl~ dialyzed against, tlist,illctl wat,csr for :tl~)ut 48 lw., and lyolhilizcd. Tlw extent of Imrification ant1 tlw yield at various stq)s i~w illustratrcl in Table I. SBTIX, , l)rq):krr(l iii this niannw~ is I~oi~iogcncous iii olcctroplwrcsis (Fig. 3-I) anal in ultl:l~enti.ifu~~~tion (Fig. 4;l ) ant1 is cllrol~~:~to~l~:~~~llie:lll~ purr as shown iu Fig. 5.1. T11c nlctllo(t of Ad1111 et ~1. I 18) ~vas usctl to calruhtc. t11cb s:ilt concc~ntlation of tllv elwnt in Fig. 5.
SHTTA- (Fraction VI, Fig. 1 I can 1): isolatcd cliroriiatogr:~~~l~i~~:ill~ lnirc’ ant1 sqwrated sinndt~ancousl~ from SBTTA, l)y eluting tlic m-hey protein with 0.25 J1 sotliuill chloride and collecting c~fflumts in tulw IVoa. 45-50, inclusive. It is ncccssary to cliw:n,tl the Iwotcins in tube Nos. 42-44, inclusive. Otjlicrwisc, in sword pmli will :iplm~~ in tllo clcrtropllorctic lxtttern of SBTIAZ . aftct l)ack-cotlil,cns:~tiori of 3 hr. or iuow. Ncxrl? 90% of the original SBTIA~ activity in tlltt
l.O2
-
Effluent
Tube Number
SBTI.4,
SBTIAI
Preparation -..~
.4ctivity”
Yield”
0.2
100
1.28
65
1.-l-1 1 .60
‘45 30
i2ctivity”
Y ieldh
0.18 1 .05
100
7;
Whole whey protein Fract,ion V First rechromatograph? Second rechromat’ograph?
“0
no
of trypsin inhibited t)y 1 pg protein in inhibit or prep:tr:ttion. :1mou111s in the whey :rnd t,hat SBTIA, b C:tlculated on the basis that SBTIA, and .4:, occur in FLI[II:L~ is 60% more active than SBTI.42 CLMicrograms
FIG. 3. Elcctrophoresis pattrrns of purified sopbc;m t rylwin irdrihi1ol.r; Pat i(,rn I. SBTIA, ; 11. SBTIA, ; III, SBTI(5x) ; pH 7.6, l)otassium plwphafe In!ffcr, 0.1 p ; proi& cwnwnt.ration Tisclills ~1~11; onl? l-1.2%, 120 min. for Patterns I and III, 150 min. for 1’31tcw~ II in n 2-1111. ascending patterns arc shown. Direction of migrnfion is io 11~~~ right.
SOYBEAN
TRYl’SIK
475
IP\THIBITORS
.’.--.,.-
_.,’
2.25
t
'.75-
2 g '.50h G E ‘-2s
.’
.’
%.oo-
; .’ .’
.’
.’
z 1 =.E
:j ,/‘i ,I ! .’ ; .’ ;
0.195
0.169;; 0.143
A\; 0.1'7
.’
i
0.09'2
'0
0.039
..;
0.2s;; .'
20
\1 0.065
j.’ .’
0.50-
s % E i
1
i’ .’
0.75-
0.247
1
0.22'
L.’
2.00-
0.273
,.c
40 I
60 ,.: 80
... '00
0.0'3
'20 I
140 I
160 I
180 Effluent lube Number Frc. 5, Gradient elution diagrams of purified soybean trypsin inhibitors; 4-5 mg. dialyzed and lyophilized protein in 5 ml. 0.01 M potassium phosphate buffer, pH 7.6; cfkcnt fraction was 1 ml. ; column dimensions of adsorbent were 1.5 X 17 cm. : flow rate 30 ml./hr.; gradient limit O-O.3 M sodium chloride in buffer; mixing chamber volunle was 80 ml.; absorbancy is the optical density at 750 rnp of 1 ml. of effluent with Folin reagent (11) ; Curve 8, SBTIA, ; 13. SBTI.1, : C, SBTI(5X): broken line and scale at right, represents salt concentration of cluant.
whey can be recovered in this mmner (Tablc I,. YBTIAa is homogeneous in electrophoresis !Fig. 3-11) and in ultracentrifugation (Fig. -1B) and is chromatographically pure as shown in Fig. 5R. PURIFICATIOS OF COMMERCIAL SBTI (5 x
j
The homogeneity of SBTI (5X ) purchased from three different supply houses differed widely as shown in the electrophoresis patterns of Fig. 6. The inhibitor preparation in Pat,tcrn IV (Lot I) is essentially homogeneous, and no additional peaks were formed during 3 hr. of back-compensation. Pattern V of Lot 2 shows the presence of a second sloxyer moving component (Peak A’). Peak CJ a faster moving component, is found in Pattern VI (IJot 3). SBTI(5x) preparations vary widely in purity and contnin protein components that, differ greatly
in electrophoretic mobility. Peak B in all three patterns has the same mobility value and represents the active trypsin inhibitor component in these preparations. The mobility and specific activity values of these trypsin inhibitor preparations are given in Table II. The gradient elution diagrams of these same preparations are shown in Fig. 7. These chromatographic experiments also indicate that commercial SBTI (5~ ) varies widely in purity. The elution diagram for Lot 1 indicates this preparation is chromatographically pure, but about 3% of protein was cluted with 0.5 M sodium chloride after gradient elution was stopped at tube No. 150. Lot 2 was fractionated into two chromatographic components. The component which is eluted in tube Nos. 80-110 is devoid of trypsin inhibitor activity. The protein of I,oh 3 elutcd in tube Nos. 150-180
476
RACKIS,
SASAME,
MANN,
ANDERSON
AND SMITH
FIG. 6. Electrophoresis patterns of different lots of commercial SBTI(5X); Pattern IV, Lot 1: Pattern V, Lot 2; Pattern VI, Lot 3; Pattern VI : 150 min. Oother condit,ions same as Fig. 3 TABLE
II
I':I.ECTR~P~~~RETI~~ MOBILITY ANI) SPECIFIC: ACTIVITY VALUES OF DIFFERENT hTS OF SBTI(5X) Lot”
Electrophoretic mobility X 10~~/sq. CIll./V./SCT.h
1 2 3
B A B B c
-
8.0 6, 6 8.0 8.0 9.-l
Protein concentration
“6 1.2
dicating aggregation to more rapidly sedimenting species. In each lot, the protein that elutes in tube Nos. 100-150 (peak at tube No. 126) when the salt concentration of eluant reaches 0.219 M sodium chloride,
Specific activityC
Lot l\r$ 0.95
1.0
0.79
1.1
0.54
; :
v-d
2.00
t
n Corresponds to t,he preparations used in Fig. 6. h Mobilities as measured on the ascending boundaries. cMicrograms of trypsin inhibited by 1 wg. of trypsin inhibitor.
represents the denatured form of SBTI (5 x ) . To elute the denatured inhibitor, the gradient elution was stopped at tube 150, and 69 ml. of 0.5 M sodium chloride in 0.01 M phosphate buffer, pH 7.6, was added to the column. If Lot 1 was 75% irreversibly denatured by heat and alkali (17), the undenatured form was eluted in tubes loo-150 and accounted for 25% of protein applied to the column. The remainder of the protein was cluted with 0.50 M sodium chloride in tubes 150-180, had no trypsin inhibitor activity, and represented the denatured form. There is little or no difference in the ultracentrifugal patterns of Lots 1 and 2. Lot 3 shows some evidcncc for a polydispersc system in-
-1.25 x $1.00 s
0 60
/' 80
,
I
I
100 120 140 160 Effluent Tube Number
1
FIG. 7. Gradient elution diagrams of different lots of commerical SBTI(5X); curve A, Lot 1; B, Lot 2; C, Lot 3; vertical arrow indicates point where gradient elution was stopped and 0.5 M sodium rhloride was added to colllmn: otherwise cvntlitions same as in Fig. ri.
SOYBEAN
TRYPSIN TABLE
PHYSICAL
PROPERTIES
AND
ANTITRYPSIN
Sedimentation Partial
specific
constnnt, S20,r volume,
nd./g.
Moleculltr weight, 9. mole-1 Hectrophoretic mobility value, sg.
III
ACTIVITY
Property
477
INHIBITORS
OF PURIFIED
SBTIA?
I SOS
2.29s
0.736
0.735
II ,300 -7.-t
x 10-5n
-8.0
TRYPUN INHIBITORS
SOYBEAIL
SBTIAl
__
21 ) Ii00 x 10 rw
---__
SBTI(5X)
2.30s 0.718 22) 700 -8.0 x lW”u
cm .lv./sec.
I~Lxt,inct~ion coefficient
(E), 1 mg./
ml. at 280 mp, pH 7.6 Specific activity, pg. trypsin
inhib-
ited/pg. inhibitorC1 Nitrogen content, yOb
0.912
0.904
0.900
1 Nl
1.05
1.0
14.9.i
15.68
16.50
tLAverage of five detjerminations. b On :t moisklre-free rend ash-free h:ks.
represents the active form of the soybean trypsin inhibitor and the active form elutes in the same position as SBTIAB (curve B, Fig. 5j. As a result, chromatographically pure trypsin inhibitor can be prepared from commercial SBTI (5~) by the stepwise elution schedule given for SBTIAZ (see Fraction VI, Fig. 1). PHYSICAL AND ENZYMIC PROPERTIES OF SBTIAl , A2 AND PURIFIED
SBTIA(5x) A comparison of some of the physical properties and trypsin inhibitor activity values of SBTIAI , A,, and purified SBTI(5x ) are given in Table III. Except for small differences in nitrogen content, SBTIAZ , SBTI (5~ ) , and Kunitz’ crystalline soybean trypsin inhibitor (17) have similar properties. The gradient eluCon diagram of a mixture containing equal amounts of SBTIA, and SBTI (5 x ) is identical to that of curve B, Fig. 5. Only one highly symmetrical peak having a mobility value of -8.0 x lo-” was formed during moving-boundary electrophoresis. No additional peaks were formed even after 3 hr. of back-compensation. SBTIA1 has different properties compared with either SBTIA- or SBTI (5x). Most of t,hc physical constants for SBTIA, are much lower, and SBTIA, is 607; more active on an equal-weight basis. DISCUSSIOX
Trautman plots were made to determine molecular weights with rotor speeds of
14,000, 24,000, and 48,000 r.p.m. The straight-line function of the Trautman plots for SBTIAl and AZ indicated that there were no concentration dependency effects and that no high polymers were present. Both the ultracentrifugal patterns (Figs. 4A and 4B) and Trautman plots indicated that the preparations were homogeneous. One microgram of SBTIA:! inhibits 1.05 pg. trypsin. If it is assumed that t,he formation of a trypsin-soybean trypsin inhibitor complex is stoichiometric (19) and the molecular weight of trypsin is 23,800 (20), then the molecular weight of SBTIA3 , calculated from inhibitor activity measurements, would be 22,600. This value closely agrees with that obtained by sedimentation analysis (21,600) but is lower than the reported molecular weight of 24,000 for Kunitz’ crystalline soybean inhibitor (17). The ult,racentrifugation patterns for SBTI (5 x ) at 11,000 r.p.m. showed the presence of a rapidly sedimenting component, which accounted for nearly 4% of the total area. However, this impurity did not interfere with the Traut’man plot measurements. One microgram of SBTIAl inhibit,s 1.60 pg. t,rypsin. If the formation of a trypsin-trypsin inhibitor complex of 1: 1 is assumed, the molecular weight of SBTIAl , calculated from activity mcasurement,s, is 14,400. A value of 14,300 was obtained by sctlimcntaCon analysis. The close agreement bet.wcen t’he molccular weight of SBTIA, obtained by ecdimcntation analysis and act’ivit’y measurements
478
RACKIS,
SASAME,
MANN,
and that reported by Kunitz (17) indicates that SBTIA, also contains only one active site on the molecule. A comparison of the physical and trypsin inhibitory properties of SBTIA, and SBTI (5~ ) show that these two proteins are identical and correspond to Kunitz’ inhibitor. Sedimentation analyses and activity measurements indicate that SBTIAl also has only one active site for reaction with trypsin. Birk (21) reported on the isolation of a trypsin inhibitor from soybeans using a procedure similar to that reported by Rackis et al. (10). On the basis of the information given, it appears that this inhibitor is different from t,hat of Kunitz’ inhibitor and may be the same as the inhibitor designated SBTIA, in this publication. Conflicting results have been reported regarding the growth-inhibitory properties of the soybean trypsin inhibitor. Commercial SBTI(5x) preparations are often utied in feeding experiments. It has been shown in this report t,hat, the preparations may contain ati much as 50% impurities. ACKiYOWLEDGMENTS The authors are indchted to Dr. S. &lander, Mr. G. E. Babcock. and Mr. R. L. Blumenshine for carrying out the llltr;c~c~ntl,ifugnl experiments.
I. Ii;.. i/I " Processed Plant Protein Foodstuffs” (A. M. Altschul, cd.), Chap. 5. Academic Press, New ‘L-ork, 1958. 2. HONAVAH,P. M., AND $OHONII(:, I(., .1. %i. Inrl. Kescarch (India) 1X, 202 (1959). 1. LIEXk:R,
ANDERSON
AND
SMITH
3. GREES, N. M., ASD ?;BuII:\T~, H., it “The Proteins” (H. Xeurath and K. C. B:Glcy, eds.), 1’01. II, Pt. B, p. 1057. :lmclwnic* I’wss, Krw York, 1954. M., AND ~,ASKOH.SKI, M.,
F., /'VW.
,%C. EXptl.
h%d.
l~fd.
104, 681 (1960). 8. HAINES, P. C., ANI) LYMAN, R. L., J. Nutrifiotz 74,445 (1961). 9. IZUNITZ, M., J. Gen. I’hysiol. 29, 149 (1946). 10. RACKIS. J. ,J., SAsAnq H. .I., :\NDRRSOR., R. I,., ATD SMITH, :2. K., J. Am, Chcm. Sot. 81, 6265 (1959). ROSEBROUGH, W. J., FAHR, A., AIXD 11. Lowau,O.H., RANDALL, R. d., J. Rid. C’hcnt. 193, 265 (1951). 12. SMITEI, A. K., .4ND Rac~irs, J. J., .I. A?,,. (:h<‘/,, Sot. 79,633 (1957). 13. SVEDBERG, T., AAT PEDFXSEK,I<. O., i,r “The Ultracentrifuge.” Oxford Univ. Press, Sew York, 1940. 14. TRAuTM.4N, It., J. I’h{/s. ~:hrtit. ho, 1211 (1956). 15. EHLAXDER, S. R., AND FOSTER, J. F’., .I. Pd/ntcr sci.37, 103 (1959). 16. CHARLWO~D, 1'. -t., J. II,,&. Clr,nw. Sov. 77, 776 (1957). 17. E;ITNITZ, R/I., J. Gn. Physiol. 30, 291 (1947). 18. ~\LM, R. S., WILLIAMS, J. P.. .~ND TJSELIIJS, .\., Acta Chem. Scnnd. 6,826 (1952). 19. &~NITZ, M., J. <:en. Physid. 30, 311 (1947). 20. CU~YWW~I.~M, L. ‘CV., JR., J. f&d. Chc~ru. 21 1, 13 (1954). 21.
OF
RIOCHEMISTRY
Soybean
J. J. RACKIS,
From
AND
RIOPHYSICS
Trypsin
98, 471-478 (1962)
Inhibitors: Isolation, Physical Properties’-3
H. A. SASAME,4 R. K. MANN,5 A. K. SMITH the Northern
Regional
Research
Purification
and
R. L. ANDERSON
LaboratorvP
Peoria,
AND
lllixois
Received May 18, 1962 Two highly purified trypain inhibitors, designated SBTIA, and SBTI& , have been isolated directly from soybean whey solutions by chromatography on DESE-cellulose. The t\l-o inhibitors differ in physical properties, nitrogen content, and trypsin inhibitor activity. SBTIA, is identical to Kunitz’ crystalline soybean trypsin inhibitor, Molecular weight determinations and activity measurements indicate that SBTI4> and A? form trypsin-trypsin inhibitor complexes in the ratio of 1: 1. Commercial preparations of 5~ crystallized soybean trypsin inhibitor preparations frequently contain 550% impurities. A chromatographic method was developed on a preparative scale to purify these commercial inhibitors. INTRODUCTION
plants (1) have been att’ributed to inhibition of intestinal proteolysis by proteins having antitryptic activity. However, there appears to be no correlation between the effect of heat on the nutritive value of various legumes and the presence or absence of
Trypsin inhibitors are widely distributed in plants and animals (1, 2). Some inhibitars are globulins and others are polypep-
tides. Naturally occurring trypsin inhibitors were last reviewed in 1954 (3, 4). Growth-inhibiting properties of unheated :tlfalfa (5), raw soybeans, and many other
trypsin
‘Presented before the Division of Biological Chemistry, 140th Meeting, American Chemical Society, Chicago, Ill., Sept. 3-8, 1961. ?Abbreviations used: DEAE, diethylaminoethyl; SBTI, soybean trypsin inhibitor; SBTI(BX), commercial soybean trypsin inhibitor, five-times crystallized. 3DEAE-cellulose was purchased from Brown Company, Berlin, N. H. Mention of trade or company names does not imply endorsement by the U, S. Department of Agriculture over similar products or firms not mentioned. ’ Present address: Laboratory of Chemical Pharmacology, National Heart Institute, National Institutes of Health, Bethesda, Md. ’ Present address: Department of Biochemistry, University of Illinois, Urbana, 111. ‘This is a laboratory of the Northern Utilization Research and Development Division. .\gricultural Research Service. U. S. Department of +\griculture, Peoria, Ill.
inhibitor
(l).
Chicks (6) and rats (7) fed raw soybean meal develop hypertrophic pancreas. It has been suggested that pancreatic hypertrophy and excessive secretion of pancreatic juices are t,he result of a reaction to soybean trypsin inhibitor (7, 8). Crude SBTI preparations as well as SBTI (5x ) , isolated according to Kunitz (9)) produce these same physiological effects. The purity of these crystalline preparations, however, was not known. In a previous publication (loj , a chromatographic separation of the trypsin inhibitor activity of soybean whey proteins into two fractions was described. These protein components were designated as SBTIA, and A,, respectively. The present communication describes the chromatographic isolation, purification, and determination of the physical properties of SBTIAl , At,, and SBTI (5x).
471
47%
R$CKIS,
SASAME,
MANX,
PREI'ARATIOX OF WHEY PROTEINS AND CI-IR~MAT~GRA~H~ The whey proteins wer(’ prepared anct fmction:tt,ed actnording to previously described procedures (10) from Hawkeyc soybeans. 1958 crop. Two different 101s of D~AE-~~~l~~losc: Type 40 reagent grade, capacity 0.8 meq./g. and Srlrc~facel, l,eagent gratle, capacity 1.05 meq./g. were used. The prol.rin solution was clarified in a Scrlal tentrifngr: operating at a speed of 12,000 r.p.m. for 5 min. prior to addition to the column. The adsorbent was poured into the column, allowed to settle under gravity to a height of 43 cm., then compressed to a height of 39 cm. Protein content of the effluents was determined with Lowry’s phenol reagent (11) with phytnte-free acid-pre~ipitate(~ so\-bean protwin as a standard (12).
ANDERSON
AND SMITH
centrifugal nleas~ir~lnents. ~lectro~)~lorrsis was carried out at 2”. Mobility measurements were n&c on the :tsc,c?ntiingpnitcrns, with 1.06 as a m~gnifcation f:u*tor. Sedimcn tation ~~~I)er~~~~~n~s wcrc tlonc~ at room tcmprrnturc~ in n Spinc*o ultracentrifuge, model E. Sedimenl stion coneta,& were detcrminrtl nccordkg to Svedberg anti Pedersen (13). ~~oie~11~~11 weights were detcrrnincd from Trautman plots (14, 15). in :1 30-mm. double-sector ~811at room t (,n~wr:tture and three, rotor speeds. The rotor ~a< shaken between run,s. I’tart,ial specitic, volume: writ?; determined with a lo-ml. pycnometer at 25 Z? 0.1“ and two proiein concentrations in phosphate bllffcl with C~~arl~(~od’sequ:~tinn f 16).
1nhibitor.v xii&y against ttypsin was determined by Kunitz’ casein-digestion nlc,thotl (17). .-\ lot of commercial SBTI(5X) that was r*sentiall\ELECTBOPBORETICAND ~JLTRACENTRIF~JG~~ l~on~ogeneo~~~eicc~rophoreti~all~ and &tomatoANALYSIS graphically served as a standard. :\ctivity is c2;pressed in trppsin units (TIJ) numerically ~~(,u:11 Potassium phosphate buffer, pH 7.6, 0.1 ionic to micrograms of trypsin inhibited per microgram strength, was used for at1 e1e~trophoret.i~ and ultraof trypsin inhibitor. Sevfd prqtarationi: Of SBTI(5X) that were tested for purity wcrc (,t,tainrtl from various supply houses. R ESIJLTS
~~~R~MAT~~R,~P~I~ISOLATION AND PURIFKATION OF SRTIA,
L
2.4g2.0z ; 1.65
-
I
01 v’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ 0 4 8 12 16 20 24 20 32 3640 44 48 Effluent Tube Mumber Fro. 1. Stepwise elution diagram of whey proteins: 600-800 mg. dialyzed and lyophilized protein in 30 ml. of 0.01 M potassium phosphate buffer, pH 7.6; effluent fraction was 10 ml.; absorbancy is ~.heopticxl density at 750 rn@of a O.l-ml. aliquot of cfht~nt, with Fotin reagent (11). Vertieal arrows
indicate point of change of sodium chloride conIacLntmtion in buffer. Column dimensions of ad.wrImrt (Type 40) wcw 2.25 X 39 cm.
In Fig. 1 is shown the stepwisc elut,ion schedule used to isolate a crude ~reparatior~ of SBTIA, from lyophilized soybean whqprotein. Stepwiee increases of sodium chloride concen~sation in 0.01 M potassiur~l phosphate buffer pH 7.6, arc shown in tbcx legend. V’erbical arrows indicate the point of hange in the thltion buffer. Six fractions, designated according to tJleir order of elution, wwc obtained. Frncations I-IT? were discarded. Fraction V, corresponding to t.ube Nos. 33-40, was dialyzed against, cold dist’illed water and lyophilizctl. SBTIAl , in the combined tubes, was tq)proximately 855 pure. A yield of about 90 mg. prot,ein per run was obtained. There was a marked decrease in resolution betwrm Fractions IV? f’, and VI and a drcreasc in affinity of these proteins fol the adsorbent when about 1 g. of whey ~troteine was chroma~ogra~~hed under identical conditions as given in Fig. 1. For cxampl~. uo distinrt. peaks corresponding to tltPsc
fractions could he obt,ainctl. Trylkn inliibitor activity normally present in Fraction Y was now rlutcd with 0.15 JI instead of 0.17 M sodium chloritlc and contained only 50% SBTIA, There appeared to he little or no change in the concentration dr~wnclcncy of Fractions I-III. Tllc dccrc:~c in affinity and resolution of tllcw fractions is typical of substances liaring highly curvctl adsorlkion isotlrcrins; other factors, e.g., displacciucnt effects and c~hangw in hufl‘c~r capacity Ixcause of iuore protein, (aan 1~ implicating. Because of such Iwhr-ior, all t,he stepwire increases in salt concentration 1~1 to be iuatlc prior t,o elution of Fractions V and VI with 0.17 ant1 0.25 31 salt, rcspectiwlg. It was necessary to modify the clution wlicdule given in Fig. 1 if t,lic Stlcctawl DEXE-ccllulow was usctl in place of TTlw 40. The luodification consisted of chwnglng tile salt concentration in steps 2 and 3 to 0.14 ant1 0.18 111 sodium chloride, respectiwly. Othrwisc, the l)urit’y of PBTIA, in Flactiou ‘i’ \v:w dccrwsetl from 857; to 11cwly 7O’iZ. Tlio xwson for this hcliavior is probably not Iwcauw of tlic higher adsorptive calxwity of St~lcchwl, since Type 40 DEAEccllulosc, having tlw saint capacity ( 1.0 iucc~.,~g.I, (lid not require these changes in stqnviw c~lution. Clorrrspondence with tlw ulxnuf~\rtui~cl:’ indicates tliffcwnt lnwx~durcs wrv usccl to prqmrc tliesc cwliangtrs.
Two rc~clironintog~al,liic steps \vere ncccswry to purify HBTIX, from Fraction Y as shown in Fig. 2. Second rccllrolll:ttogr:ll~ll~ is indicated hy solid lines. Effluents fractions corresponding to tulw Kos. 36-41, incluwivcb. were wiul~inctl, dinlyzd ng:tinst csoltl tlistillctl water, ronwntratd to a.sinall wlui~~c, ant1 rc~~lirolllatogr:Il,llctl a sc~~~ntltiilw under iclcnticd conditions. Efflucnt~ fractions ( tub 3&i-41 inclusive 1 ncrc conil~inctl~ dialyzed against, tlist,illctl wat,csr for :tl~)ut 48 lw., and lyolhilizcd. Tlw extent of Imrification ant1 tlw yield at various stq)s i~w illustratrcl in Table I. SBTIX, , l)rq):krr(l iii this niannw~ is I~oi~iogcncous iii olcctroplwrcsis (Fig. 3-I) anal in ultl:l~enti.ifu~~~tion (Fig. 4;l ) ant1 is cllrol~~:~to~l~:~~~llie:lll~ purr as shown iu Fig. 5.1. T11c nlctllo(t of Ad1111 et ~1. I 18) ~vas usctl to calruhtc. t11cb s:ilt concc~ntlation of tllv elwnt in Fig. 5.
SHTTA- (Fraction VI, Fig. 1 I can 1): isolatcd cliroriiatogr:~~~l~i~~:ill~ lnirc’ ant1 sqwrated sinndt~ancousl~ from SBTTA, l)y eluting tlic m-hey protein with 0.25 J1 sotliuill chloride and collecting c~fflumts in tulw IVoa. 45-50, inclusive. It is ncccssary to cliw:n,tl the Iwotcins in tube Nos. 42-44, inclusive. Otjlicrwisc, in sword pmli will :iplm~~ in tllo clcrtropllorctic lxtttern of SBTIAZ . aftct l)ack-cotlil,cns:~tiori of 3 hr. or iuow. Ncxrl? 90% of the original SBTIA~ activity in tlltt
l.O2
-
Effluent
Tube Number
SBTI.4,
SBTIAI
Preparation -..~
.4ctivity”
Yield”
0.2
100
1.28
65
1.-l-1 1 .60
‘45 30
i2ctivity”
Y ieldh
0.18 1 .05
100
7;
Whole whey protein Fract,ion V First rechromatograph? Second rechromat’ograph?
“0
no
of trypsin inhibited t)y 1 pg protein in inhibit or prep:tr:ttion. :1mou111s in the whey :rnd t,hat SBTIA, b C:tlculated on the basis that SBTIA, and .4:, occur in FLI[II:L~ is 60% more active than SBTI.42 CLMicrograms
FIG. 3. Elcctrophoresis pattrrns of purified sopbc;m t rylwin irdrihi1ol.r; Pat i(,rn I. SBTIA, ; 11. SBTIA, ; III, SBTI(5x) ; pH 7.6, l)otassium plwphafe In!ffcr, 0.1 p ; proi& cwnwnt.ration Tisclills ~1~11; onl? l-1.2%, 120 min. for Patterns I and III, 150 min. for 1’31tcw~ II in n 2-1111. ascending patterns arc shown. Direction of migrnfion is io 11~~~ right.
SOYBEAN
TRYl’SIK
475
IP\THIBITORS
.’.--.,.-
_.,’
2.25
t
'.75-
2 g '.50h G E ‘-2s
.’
.’
%.oo-
; .’ .’
.’
.’
z 1 =.E
:j ,/‘i ,I ! .’ ; .’ ;
0.195
0.169;; 0.143
A\; 0.1'7
.’
i
0.09'2
'0
0.039
..;
0.2s;; .'
20
\1 0.065
j.’ .’
0.50-
s % E i
1
i’ .’
0.75-
0.247
1
0.22'
L.’
2.00-
0.273
,.c
40 I
60 ,.: 80
... '00
0.0'3
'20 I
140 I
160 I
180 Effluent lube Number Frc. 5, Gradient elution diagrams of purified soybean trypsin inhibitors; 4-5 mg. dialyzed and lyophilized protein in 5 ml. 0.01 M potassium phosphate buffer, pH 7.6; cfkcnt fraction was 1 ml. ; column dimensions of adsorbent were 1.5 X 17 cm. : flow rate 30 ml./hr.; gradient limit O-O.3 M sodium chloride in buffer; mixing chamber volunle was 80 ml.; absorbancy is the optical density at 750 rnp of 1 ml. of effluent with Folin reagent (11) ; Curve 8, SBTIA, ; 13. SBTI.1, : C, SBTI(5X): broken line and scale at right, represents salt concentration of cluant.
whey can be recovered in this mmner (Tablc I,. YBTIAa is homogeneous in electrophoresis !Fig. 3-11) and in ultracentrifugation (Fig. -1B) and is chromatographically pure as shown in Fig. 5R. PURIFICATIOS OF COMMERCIAL SBTI (5 x
j
The homogeneity of SBTI (5X ) purchased from three different supply houses differed widely as shown in the electrophoresis patterns of Fig. 6. The inhibitor preparation in Pat,tcrn IV (Lot I) is essentially homogeneous, and no additional peaks were formed during 3 hr. of back-compensation. Pattern V of Lot 2 shows the presence of a second sloxyer moving component (Peak A’). Peak CJ a faster moving component, is found in Pattern VI (IJot 3). SBTI(5x) preparations vary widely in purity and contnin protein components that, differ greatly
in electrophoretic mobility. Peak B in all three patterns has the same mobility value and represents the active trypsin inhibitor component in these preparations. The mobility and specific activity values of these trypsin inhibitor preparations are given in Table II. The gradient elution diagrams of these same preparations are shown in Fig. 7. These chromatographic experiments also indicate that commercial SBTI (5~ ) varies widely in purity. The elution diagram for Lot 1 indicates this preparation is chromatographically pure, but about 3% of protein was cluted with 0.5 M sodium chloride after gradient elution was stopped at tube No. 150. Lot 2 was fractionated into two chromatographic components. The component which is eluted in tube Nos. 80-110 is devoid of trypsin inhibitor activity. The protein of I,oh 3 elutcd in tube Nos. 150-180
476
RACKIS,
SASAME,
MANN,
ANDERSON
AND SMITH
FIG. 6. Electrophoresis patterns of different lots of commercial SBTI(5X); Pattern IV, Lot 1: Pattern V, Lot 2; Pattern VI, Lot 3; Pattern VI : 150 min. Oother condit,ions same as Fig. 3 TABLE
II
I':I.ECTR~P~~~RETI~~ MOBILITY ANI) SPECIFIC: ACTIVITY VALUES OF DIFFERENT hTS OF SBTI(5X) Lot”
Electrophoretic mobility X 10~~/sq. CIll./V./SCT.h
1 2 3
B A B B c
-
8.0 6, 6 8.0 8.0 9.-l
Protein concentration
“6 1.2
dicating aggregation to more rapidly sedimenting species. In each lot, the protein that elutes in tube Nos. 100-150 (peak at tube No. 126) when the salt concentration of eluant reaches 0.219 M sodium chloride,
Specific activityC
Lot l\r$ 0.95
1.0
0.79
1.1
0.54
; :
v-d
2.00
t
n Corresponds to t,he preparations used in Fig. 6. h Mobilities as measured on the ascending boundaries. cMicrograms of trypsin inhibited by 1 wg. of trypsin inhibitor.
represents the denatured form of SBTI (5 x ) . To elute the denatured inhibitor, the gradient elution was stopped at tube 150, and 69 ml. of 0.5 M sodium chloride in 0.01 M phosphate buffer, pH 7.6, was added to the column. If Lot 1 was 75% irreversibly denatured by heat and alkali (17), the undenatured form was eluted in tubes loo-150 and accounted for 25% of protein applied to the column. The remainder of the protein was cluted with 0.50 M sodium chloride in tubes 150-180, had no trypsin inhibitor activity, and represented the denatured form. There is little or no difference in the ultracentrifugal patterns of Lots 1 and 2. Lot 3 shows some evidcncc for a polydispersc system in-
-1.25 x $1.00 s
0 60
/' 80
,
I
I
100 120 140 160 Effluent Tube Number
1
FIG. 7. Gradient elution diagrams of different lots of commerical SBTI(5X); curve A, Lot 1; B, Lot 2; C, Lot 3; vertical arrow indicates point where gradient elution was stopped and 0.5 M sodium rhloride was added to colllmn: otherwise cvntlitions same as in Fig. ri.
SOYBEAN
TRYPSIN TABLE
PHYSICAL
PROPERTIES
AND
ANTITRYPSIN
Sedimentation Partial
specific
constnnt, S20,r volume,
nd./g.
Moleculltr weight, 9. mole-1 Hectrophoretic mobility value, sg.
III
ACTIVITY
Property
477
INHIBITORS
OF PURIFIED
SBTIA?
I SOS
2.29s
0.736
0.735
II ,300 -7.-t
x 10-5n
-8.0
TRYPUN INHIBITORS
SOYBEAIL
SBTIAl
__
21 ) Ii00 x 10 rw
---__
SBTI(5X)
2.30s 0.718 22) 700 -8.0 x lW”u
cm .lv./sec.
I~Lxt,inct~ion coefficient
(E), 1 mg./
ml. at 280 mp, pH 7.6 Specific activity, pg. trypsin
inhib-
ited/pg. inhibitorC1 Nitrogen content, yOb
0.912
0.904
0.900
1 Nl
1.05
1.0
14.9.i
15.68
16.50
tLAverage of five detjerminations. b On :t moisklre-free rend ash-free h:ks.
represents the active form of the soybean trypsin inhibitor and the active form elutes in the same position as SBTIAB (curve B, Fig. 5j. As a result, chromatographically pure trypsin inhibitor can be prepared from commercial SBTI (5~) by the stepwise elution schedule given for SBTIAZ (see Fraction VI, Fig. 1). PHYSICAL AND ENZYMIC PROPERTIES OF SBTIAl , A2 AND PURIFIED
SBTIA(5x) A comparison of some of the physical properties and trypsin inhibitor activity values of SBTIAI , A,, and purified SBTI(5x ) are given in Table III. Except for small differences in nitrogen content, SBTIAZ , SBTI (5~ ) , and Kunitz’ crystalline soybean trypsin inhibitor (17) have similar properties. The gradient eluCon diagram of a mixture containing equal amounts of SBTIA, and SBTI (5 x ) is identical to that of curve B, Fig. 5. Only one highly symmetrical peak having a mobility value of -8.0 x lo-” was formed during moving-boundary electrophoresis. No additional peaks were formed even after 3 hr. of back-compensation. SBTIA1 has different properties compared with either SBTIA- or SBTI (5x). Most of t,hc physical constants for SBTIA, are much lower, and SBTIA, is 607; more active on an equal-weight basis. DISCUSSIOX
Trautman plots were made to determine molecular weights with rotor speeds of
14,000, 24,000, and 48,000 r.p.m. The straight-line function of the Trautman plots for SBTIAl and AZ indicated that there were no concentration dependency effects and that no high polymers were present. Both the ultracentrifugal patterns (Figs. 4A and 4B) and Trautman plots indicated that the preparations were homogeneous. One microgram of SBTIA:! inhibits 1.05 pg. trypsin. If it is assumed that t,he formation of a trypsin-soybean trypsin inhibitor complex is stoichiometric (19) and the molecular weight of trypsin is 23,800 (20), then the molecular weight of SBTIA3 , calculated from inhibitor activity measurements, would be 22,600. This value closely agrees with that obtained by sedimentation analysis (21,600) but is lower than the reported molecular weight of 24,000 for Kunitz’ crystalline soybean inhibitor (17). The ult,racentrifugation patterns for SBTI (5 x ) at 11,000 r.p.m. showed the presence of a rapidly sedimenting component, which accounted for nearly 4% of the total area. However, this impurity did not interfere with the Traut’man plot measurements. One microgram of SBTIAl inhibit,s 1.60 pg. t,rypsin. If the formation of a trypsin-trypsin inhibitor complex of 1: 1 is assumed, the molecular weight of SBTIAl , calculated from activity mcasurement,s, is 14,400. A value of 14,300 was obtained by sctlimcntaCon analysis. The close agreement bet.wcen t’he molccular weight of SBTIA, obtained by ecdimcntation analysis and act’ivit’y measurements
478
RACKIS,
SASAME,
MANN,
and that reported by Kunitz (17) indicates that SBTIA, also contains only one active site on the molecule. A comparison of the physical and trypsin inhibitory properties of SBTIA, and SBTI (5~ ) show that these two proteins are identical and correspond to Kunitz’ inhibitor. Sedimentation analyses and activity measurements indicate that SBTIAl also has only one active site for reaction with trypsin. Birk (21) reported on the isolation of a trypsin inhibitor from soybeans using a procedure similar to that reported by Rackis et al. (10). On the basis of the information given, it appears that this inhibitor is different from t,hat of Kunitz’ inhibitor and may be the same as the inhibitor designated SBTIA, in this publication. Conflicting results have been reported regarding the growth-inhibitory properties of the soybean trypsin inhibitor. Commercial SBTI(5x) preparations are often utied in feeding experiments. It has been shown in this report t,hat, the preparations may contain ati much as 50% impurities. ACKiYOWLEDGMENTS The authors are indchted to Dr. S. &lander, Mr. G. E. Babcock. and Mr. R. L. Blumenshine for carrying out the llltr;c~c~ntl,ifugnl experiments.
I. Ii;.. i/I " Processed Plant Protein Foodstuffs” (A. M. Altschul, cd.), Chap. 5. Academic Press, New ‘L-ork, 1958. 2. HONAVAH,P. M., AND $OHONII(:, I(., .1. %i. Inrl. Kescarch (India) 1X, 202 (1959). 1. LIEXk:R,
ANDERSON
AND
SMITH
3. GREES, N. M., ASD ?;BuII:\T~, H., it “The Proteins” (H. Xeurath and K. C. B:Glcy, eds.), 1’01. II, Pt. B, p. 1057. :lmclwnic* I’wss, Krw York, 1954. M., AND ~,ASKOH.SKI, M.,
F., /'VW.
,%C. EXptl.
h%d.
l~fd.
104, 681 (1960). 8. HAINES, P. C., ANI) LYMAN, R. L., J. Nutrifiotz 74,445 (1961). 9. IZUNITZ, M., J. Gen. I’hysiol. 29, 149 (1946). 10. RACKIS. J. ,J., SAsAnq H. .I., :\NDRRSOR., R. I,., ATD SMITH, :2. K., J. Am, Chcm. Sot. 81, 6265 (1959). ROSEBROUGH, W. J., FAHR, A., AIXD 11. Lowau,O.H., RANDALL, R. d., J. Rid. C’hcnt. 193, 265 (1951). 12. SMITEI, A. K., .4ND Rac~irs, J. J., .I. A?,,. (:h<‘/,, Sot. 79,633 (1957). 13. SVEDBERG, T., AAT PEDFXSEK,I<. O., i,r “The Ultracentrifuge.” Oxford Univ. Press, Sew York, 1940. 14. TRAuTM.4N, It., J. I’h{/s. ~:hrtit. ho, 1211 (1956). 15. EHLAXDER, S. R., AND FOSTER, J. F’., .I. Pd/ntcr sci.37, 103 (1959). 16. CHARLWO~D, 1'. -t., J. II,,&. Clr,nw. Sov. 77, 776 (1957). 17. E;ITNITZ, R/I., J. Gn. Physiol. 30, 291 (1947). 18. ~\LM, R. S., WILLIAMS, J. P.. .~ND TJSELIIJS, .\., Acta Chem. Scnnd. 6,826 (1952). 19. &~NITZ, M., J. <:en. Physid. 30, 311 (1947). 20. CU~YWW~I.~M, L. ‘CV., JR., J. f&d. Chc~ru. 21 1, 13 (1954). 21.