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IV. Amino acid composition of the globulins from Lupinus angustifolius and Lupinus luteus
VOL. 22 (1956)
BIOCHIMICAET BIOPHYSICA.\CTA
269
LUPIN SEED PROTEINS IV. AMINO ACID COMPOSITION OF T H E G L O B U L I N S FROM
L U P I N U S A N G U S T I F O L I U S AND L U P I N U S L U T E U S by TH. GERRITSEN
National Chemical Research Laboratory, South A ]rican Council/or Scientific and Industrial Research, Pretoria (South A ]riea)
In Part I and I I of this series, J O U B E R T 1,2 reported on the isolation and the physicochemical data of the seed globulins of the blue lupin (Lupinus angusti/olius) and the yellow lupin (Lupinus luteus). In the protein fraction of the seed of L. angusti[olius he found two globulins with ultracentrifugal sedimentation constants of 11.6 and 7.8 S.U. (Svedberg Units), and an albumin with a sedimentation constant of 1.6 S.U. 1 These are referred to hereafter as components L.a.-slL 6, L.a.-sT. * and L.a.-sl. e respectively. Afterwards we found t h a t the protein fraction of the seed of L. luteus differed from t h a t of L. angusti[olius in t h a t three components instead of two were precipitated with 85 % saturated a m m o n i u m sulphate. These three globulins had sedimentation constants of 11.6, 7.4 and 2.0 S.U. * and are designated as components L.1.-Sn.s, L.l.-sT. 4 and L.1.-s2. o respectively. The present paper reports the amino acid composition of the five globulins mentioned above, as determined b y the chromatographic method of MOORE AND STEIN3. The albumin fraction L.a.-sL6 was not analysed, as it was impossible to obtain it free from the yellow coloration which washed out during the alcohol and water washings of the blue lupin seed meal. PETRI AND STAVERMAN 4 have determined the amino acids of the total protein of sweet yellow lupin seed b y microbiological assay, while HOLMES5 has done the same for L. pilosis. The amino acids of the individual proteins of lupin seeds have not, however, been determined previously. EXPERIMENTAL
Preparation and purification T h e five globulins were isolated as described in P a r t s I a n d II of this series. E a c h globulin w a s purified b y r e p e a t e d f r a c t i o n a t i o n s u n t i l t h e criteria of p u r i t y , discussed in t h e following p a r a g r a p h , were satisfied. T h e purified p r o t e i n s were finally dialysed free f r o m salt. freeze-dried, a n d stored as d r y w h i t e powders.
Criteria o/ purity T h e u l t r a c e n t r i f u g a l a n d electrophoretic h o m o g e n e i t i e s were u s e d as criteria of p u r i t y . T h e i m p u r ities in t h e final p r o d u c t s were n o t m o r e t h a n 2 - 4 %, e s t i m a t e d f r o m t h e u l t r a c e n t r i f u g e d i a g r a m s (Fig. I). W h e n it was discovered t h a t t r y p t o p h a n was a b s e n t f r o m proteins L.a.-s~. 8, L.l.-s~. 4 a n d
Re/erences p. 273.
27o
VOL. 22 (1956)
T. GERRITSEN
TABLE AMINO ACID ANALYSES NO. of residues = N u m b e r of residues nearest Fraction: L.l.-s,.o* Mol. weight: 27,600 Total N: 16.62% N recovery: 98.20o/0
Amino acids
Fraction: L.l.-sT.4* Mot. weight: z73,ooo Total N: 17.36 o/~ N recovery: 99.o8%
0/0 mean
No. ol residues
lO.O8 I. 19 4.93 39.25 2.53 2.2o 1.3o 1.57 o.45 3.76 11.35 0.82 3.87 0.66 l.O 7 9-44 7.61
21 3 13 74 6 8 4 4 l 8 24 I 7 1 2 15 17
in duplo
Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Histidine Lysine Arginine Cysteine + cystine/2 Tryptophan Amide-NH2-groups
Total * See text.
lO.21 ; 9-94 i. 20 ; i. 18 4-94; 4.92 38.98; 39-53 2.59; 2.47 2.21 ; 2.18 1.29; 1.3o 1.6o; 1.53 0.45 ; 0.45 3.75; 3.77 11.38; 11.32 o.8o; 0.85 3.96; 3.77 o.65 ; o.67 i. I i ; 1.o2 9.26; 9.61 7.4o; 7.82 . . 3.75 : 3.74
. 3.75
.
. (65)
105.83
209**
% in duplo
14.58; 15.25 3.o6 ; 3.03 5.91 ; 5.76 22.97; 23-58 4.30; 4.26 3.2o; 3.27 3.13; 2.98 3.95; 4.27 . . . 5-75; 6.1o 9.33; 9.55 5.58; 5 .I2 5.48; 5.35 i .92 ; 1.64 4.17; 3.62 12.2o; 12.27 1.64; 1.68 . . 2.50; 2.56
mean
14.92 3-o5 5.84 23.28 4.28 3.24 3.06 4 -11 . 5.92 9.44 5.35 5.42 i .78 3.9o 12.24 1.66
198 44 96 274 64 75 60 61
2.53
(274)
78 125 51 57 2o 46 121 24
.
1Io.o2
** A m i d e - N H 2 - g r o u p s not included.
L.l.-s,.o, this was used as an additional criterion of p u r i t y . The purification of these proteins was repeated until t h e y were free from t r y p t o p h a n as estimated colorimetrically according to SHAW AND MACFARLANE 6. Since the accuracy of the d e t e r m i n a t i o n of a m i n o acids b y the m e t h o d of MOORE AND STEIN is only of t h e order of 3 %, the presence of 2 - 4 % of i m p u r i t y in t h e individual proteins should not affect the final results significantly. Fig. I. Sedimentation diagrams of t h e final purification stages of lupin seed proteins. (t = ca. 2o min; r = ca. 60,000 r.p.m.). F r o m 1 --+ r, L u p i n u s l u t e u s fractions s2.0, sT.4 and Sll.6 , L u p i n u s a n g u s t i / o l i u s fractions ST.8 and Sll.6. H y d r o l y s i s o] the p r o t e i n s
Acid hydrolysis was carried o u t in dilute solution, according to SCHRAM et a L L A b o u t 60 m g of protein w a s dissolved in 2oo ml 5 N redistilled hydrochloric acid, and refluxed for 24 hours. The excess hydrochloric.acid waS r e m o v e d i n v a c u o a n d the h y d r o l y s a t e dissolved in distilled w a t e r and m a d e up to io ml. The p H of the solution w a s b e t w e e n i.o and 2.o. T h e solution was used directly or k e p t in the dark at - - 15 o C until required. According to SCHRA~t et al. 7 this m e t h o d of hydrolysis causes no destruction of a m i n o acids. This was b o r n e o u t in t h e p r e s e n t w o r k b y t h e fact t h a t the recovery of a m i n o acids in the h y d r o l y s a t e was n o t less t h a n 97.5 % (Table I). Consequently we did not t h i n k it necessary to follow the elaborate m e t h o d of VAN DER SCHAAF AND HUISMANs for the d e t e r m i n a t i o n of losses during hydrolysis, for these proteins. R e f e r e n c e s p. 273.
No. o#. residues
1,394"*
VOL. 22 (I956)
LUPIN SEED PROTEINS IV
271
*F LUP1N SEED PROTEINS O the calculated n u m b e r per molecule. Fr.~ction.L.l.-sla.8* Mol. weight." 336,00o T~dd N: I7.49% ,V recovery." 99.7 o % %
Fraction: L.a.-sT., ~ Mol. weight." I8Gooo Total N: 27.zo°/o N recovery: 99.47%
in duplo
mean
No. o! residues
13.32; 13.33 3.83; 3.55 4.75; 4.32 26.45; 26.27 5.oo; 4.48 4-65; 4.45 3.43; 3-46 4-77; 4.48 o.48; o.39 5.70; 5.74 8.78; ~.62 3.85; 3.68 5.32; 5.16 2.93; 2.81 4-39; 4 .ol 11.43; ii.46 1.7o; 1.74 1.59; :.61 2.28; 2.32
13.33 3.69 4.54 26.36 4-74 4.55 3.45 4.63 0.44 5.72 8.70 3-77 5.24 2.87 4 .20 11.4. 5 1.72 1.6o 2.30
336 lO 4 146 602 138 204 13o 134 IO 164 224 7° lO6 62 96 222 48 26 (484)
113.3o
Methods
2,822**
% in duplo
mean
13.34; 13.53 2.36; 2.21 5.28; 5.56 25.32; 26.20 3.89; 3.74 3.34; 3.48 2.53; 2-50 3.59; 3.4 °
~3.4, 2.29 5.42
6.00 7.60 4.74 4 .82 2.23 4.43 13.5o 1.68 --2.45
5.95
5.90; 7.51 ;
4-83; 4-84; 2.36; 4 .21; 13.31; 1.66; 2.20;
25.75
3 .82 3.41 2-52 3.50
7.59 4.79 4.83 2.30 4.32 13.41 1.67 2.33
lO7.35
Fraction.. Mol. weight: Total N : N recovery."
L.a.-sta., ~ 336,oo0
I8.39% 97.51 °/o
No. o/ residues
in duplo
mean
183 35 93 316 60 82 51 ,54 -82 IO5 48 53 27 54 I4O 25 -(264)
t3.o2; :2.74 3.65; 3.7 ° 4-85; 4.58 25.39; 25.39 4.76; 4.77 4.24; 4.20 3.38; 3-34 4.32; 4.21 o.56: 0.44 5.6I: ,5.57 8.38 ; 8.49 3.72; 3.72 5.35; 5.32 3.15; 2.9r 3.8o: 3.96 14.37; 14.31 1.74; 1.86 1.6o; 1.63 2.11: 2.14
12.88 3.68 4.72 25.39 4-77 4.25 3.36 4-29 0.50 5.59 8.44 3.72 .5.34 3.03 3.88 14.34 1.8o :.62 2.i 3
1,4o8"*
%
113.73
No. o~ residues
324 lO 4 :52 588 14o 19o 126 124 :2 144 216 7° io8 66 90 278 5° 28 (,48 )
2.8~o**
o/estimation
The q u a n t i t a t i v e c h r o m a t o g r a p h y was performed on columns of Dowex 5 ° according to the technique described b y MOORE AND STEIN s . T r y p t o p h a n was determined on a solution of the protein in lO% sodium hydroxide b y BLOCK AND BOLLING'S modification of the m e t h o d of SHAW AND MACFARLANEe. This fast m e t h o d gives reliable results for p u r e proteins. Cysteine plus cystine was e s t i m a t e d in a 4-hour acid hydrolysate, after reduction with bisulphite b y a slight modification of t h e m e t h o d of PARK AND SPEAKMAN9. Amide nitrogen was estimated b y determining the a m m o n i a released on hydrolysing 3o0 m g protein w i t h 15 ml 2 N HC1 for 2 h o u r s at 12o °. RESULTS
The amino acid compositions of the five proteins are listed in Table I. Duplicate analyses were obtained on two different hydrolysates of each protein. The duplicate values for tryptophan, cysteine plus cystine, and amide nitrogen were obtained on separate portions of protein. The molecular weights of fractions L.a.-s~. 8 and L.a.-s:l.6 are I8i,ooo and 335,ooo:. Those of fractions L.l.-s~. 4 and L.l.-s:Ls were estimated b y Dr. F. J. JOUBERT to be I73,ooo and 336,000. From the sedimentation rate, the molecular weight of fraction L.l.-s~. o must be about 25,ooo. The calculated molecular weight from the R e f e r e n c e s p . 27.3.
272
"r. GERRVrSEN
VOL. 22 (1956)
contents of all amino acids with less than ten residues per molecule gives an average value of 27,600. DISCUSSION
The globulin fractions L.a.-SlL 6 and L.l.-Stl.S show several points of similarity. Their sedimentation constants, molecular weights and behaviour in the electrophoretic field are very much the same. Each can be reversibly dissociated, into an s7.16 component in the case of the blue lupin seed 1, or an sT.,0 component in the yellow*. Thus from their physico-chemical properties, the two proteins would appear to be identical. However, the nitrogen contents of the two fractions are different (17.5% in L.l.-slL 6 and 18.4% in L.a.-sll.~ ), and the two proteins show a significant difference (p < o.oi after five analyses) in the number of arginine residues per molecule (222 in L.l.-SlL 6 and 278 in L.a.-s11.~ ). The other differences in amino acid composition are not significant. Fraction L.a.-s~.,~ has 912 acid, and 882 basic groups while fraction L.l.-Sll.~ contains 938 acid and 864 basic groups, per molecule. These differences are also not significant, so that the similarity in the electrophoretic mobility of the two proteins is explainable. The amino acid analyses confirm JOUBERT'S 1 conclusion that the dissociation product of L.a.-s11.6 is not identical with L.a.-s~. s. The same holds for L.l.-slL ~ and L.l.-s~. 4. The most striking difference in amino acid compositions is that the S~l.6 fractions contain tryptophan, which is completely absent from the lighter fractions. Fractions L.l.-sT. 4 and L.a.-sT. s show certain broad similarities in composition, for example in the absence of tryptophan and methionine. The difference between the amounts of most amino acids present is, however, greater than the experimental error of the determination. This supports the physical evidence that these proteins are not identical. It is interesting to note that in both lupin seeds, tryptophan is present only in the stx.s fractions. The very small amount of methionine in the proteins investigated and in particular its complete absence from fractions L.l.-sT. 4 and L.a.-sT, 8 explains its low content in lupin seeds (less than o.I % of the total seed). This may cause trouble when lupin seeds are used as sheep fodder without sufficient other proteins, as sometimes happens in South Africa during the dry season. AC KNOWLEDGEME NTS
This paper is published with the permission of the South African Council for Scientific and Industrial Research. I wish to thank Dr. F. J. JOUBEgT for the ultracentrifuge experiments, Mr. P. J. HORN for his accurate technical assistance, and Dr. H. M. SCHWARTZfor her interest and criticism. SUMMARY The a m i n o acid c o m p o s i t i o n of t h e t h r e e seed g l o b u l i n s from Lupinus luteus a n d t h e t w o from Lupinus angustilolius h a s b e e n d e t e r m i n e d b y t h e m e t h o d of MOORE AND ST~IN. I t w a s f o u n d t h a t , a l t h o u g h t h e p r o t e i n s w i t h a s e d i m e n t a t i o n c o n s t a n t of 11.6 S.U. from t h e t w o species are v e r y
Re/erences p. 273.
VOL. 22 (1956)
LUP1N SEED PROTEINSIV
273
similar in physical properties, t h e y differ in n i t r o g e n a n d a r g i n i n e c o n t e n t , a n d are t h u s n o t i d e n tical. The a m i n o acid analyses confirm t h 9 physical evidence t h a t t h e c o m p o n e n t w i t h sedimentation c o n s t a n t 7.4 S.U. in L. luteus is n o t the s a m e as t h e dissociation p r o d u c t of the p r o t e i n w i t h s e d i m e n t a t i o n c o n s t a n t 11.6 S.U. The latter contains all the t r y p t o p h a n in the seed, whereas the former is completely devoid of this a m i n o acid. The same was s h o w n for the sT.s a n d the slx.n fraction of L. angusti/olius. Neither the sT.4 c o m p o n e n t of L. luteus nor the s~.s c o m p o n e n t of L. angusti/olius contains t r y p t o p h a n or methionine. A p a r t from this, however, their a m i n o acid compositions are very dissimilar, s u p p o r t i n g the physico-chemical evidence t h a t these p r o t e i n s are not identical. REFERENCES I F. J. JOUBERT, Biochim. Biophys. Acta, 16 (I955) 37 o, 2 F. J. JOUBERT, Biochim. Biophys. Acta, 17 (1955) 444. 3 S. MOORE AND W. S . STEIN, J. Biol. Chem., 192 (1951) 063. 4 E. M. PETRI AND h . J. STAVERMAN, Discussions Faraday Soc., 13 (1953) I 5 i . 5 p. HOLMES, Australian J. Exptl. Biol. ZVIed. Sci., 31 (1953) 595. 6 R. J. BLOCK AND D. BELLING, The Amino Acid Composition o/ Proteins and Foods, 2nd ed., Charles C. T h o m a s , Springfield, IlL, 1951, p. 119, referring to J. L. D. SHAW AND W. D. MACFARLANE, Can. J. Research, 16 ]3 (1938) 361. 7 E. SCHRAM, J. P. DUSTIN, S. MOORE AND E. J. BIGWOOD, Anal. Chim. Acta, 9 (1953) 149. 8 p. C. VAN DER SCHAAF AND T. I-L J. HUISMAN, Biochim. Biophys. Acta, 17 (1955) 81. 9 G. G. PARK AND J. B. SPEAKMAN, Bull. inst. textile France, 3 ° (1952) 255.
Received January 3oth, 1956
I N F L U E N C E D ' U N E CARENCE EN VITAMINE B e SUR LA T E N E U R EN ACIDES TAURO-CONJUGU]~S E T GLYCO-CONJUGUI~S DE LA B I L E DU RAT par
BERNADETTE
BERGERET
ET F E R N A N D E
CHATAGNER
Laboratoire de Chimie biologique de la Facult~ des Sciences, Paris (France)
Des recherches pr6c6dentes ont montr~ qu'une carence en vitamine B 6 provoque, dans le foie et le cerveau du rat, la disparition de l'activit6 d6carboxylante observ~e, in vitro, vis k vis de l'acide eyst6inesulfinique 1 et de l'acide cyst6ique 2, et entraine, in rive, ehez le mBme animal, l'absence de taurinO, * et d'hypotaurinO clans l'urine. On pouvait, k partir de ees r6sultats, envisager que la formation de la taurine 6fair totalement supprim6e par la earence en pyridoxine. I1 6tait done int6ressant de v6rifier dans quelle mesure les acides tauro-conjug6s, qui constituent la plus grande partie des aeides biliaires ehez le rat s, sent touches par eette carence, et le eas 6ch6ant de voir si la diminution en acides tauro-conjugu6s entraine une modification dans la teneur de la bile en acides glyco-eonjugu~s. Le pr6sent travail rend compte des r~sultats obtenus dans la d~termination de la taurine et du glycocolle eonjugu6s aux aeides biliaires, dosages effectu6s parall~lement l'estimation de la teneur en taurine clans l'urine des animaux. Ces r~sultats montrent, c h e z le rat carenc6, une eertaine diminution des aeides tauro-conjugu6s et une forte Bibliographie p. 277.
BIOCHIMICAET BIOPHYSICA.\CTA
269
LUPIN SEED PROTEINS IV. AMINO ACID COMPOSITION OF T H E G L O B U L I N S FROM
L U P I N U S A N G U S T I F O L I U S AND L U P I N U S L U T E U S by TH. GERRITSEN
National Chemical Research Laboratory, South A ]rican Council/or Scientific and Industrial Research, Pretoria (South A ]riea)
In Part I and I I of this series, J O U B E R T 1,2 reported on the isolation and the physicochemical data of the seed globulins of the blue lupin (Lupinus angusti/olius) and the yellow lupin (Lupinus luteus). In the protein fraction of the seed of L. angusti[olius he found two globulins with ultracentrifugal sedimentation constants of 11.6 and 7.8 S.U. (Svedberg Units), and an albumin with a sedimentation constant of 1.6 S.U. 1 These are referred to hereafter as components L.a.-slL 6, L.a.-sT. * and L.a.-sl. e respectively. Afterwards we found t h a t the protein fraction of the seed of L. luteus differed from t h a t of L. angusti[olius in t h a t three components instead of two were precipitated with 85 % saturated a m m o n i u m sulphate. These three globulins had sedimentation constants of 11.6, 7.4 and 2.0 S.U. * and are designated as components L.1.-Sn.s, L.l.-sT. 4 and L.1.-s2. o respectively. The present paper reports the amino acid composition of the five globulins mentioned above, as determined b y the chromatographic method of MOORE AND STEIN3. The albumin fraction L.a.-sL6 was not analysed, as it was impossible to obtain it free from the yellow coloration which washed out during the alcohol and water washings of the blue lupin seed meal. PETRI AND STAVERMAN 4 have determined the amino acids of the total protein of sweet yellow lupin seed b y microbiological assay, while HOLMES5 has done the same for L. pilosis. The amino acids of the individual proteins of lupin seeds have not, however, been determined previously. EXPERIMENTAL
Preparation and purification T h e five globulins were isolated as described in P a r t s I a n d II of this series. E a c h globulin w a s purified b y r e p e a t e d f r a c t i o n a t i o n s u n t i l t h e criteria of p u r i t y , discussed in t h e following p a r a g r a p h , were satisfied. T h e purified p r o t e i n s were finally dialysed free f r o m salt. freeze-dried, a n d stored as d r y w h i t e powders.
Criteria o/ purity T h e u l t r a c e n t r i f u g a l a n d electrophoretic h o m o g e n e i t i e s were u s e d as criteria of p u r i t y . T h e i m p u r ities in t h e final p r o d u c t s were n o t m o r e t h a n 2 - 4 %, e s t i m a t e d f r o m t h e u l t r a c e n t r i f u g e d i a g r a m s (Fig. I). W h e n it was discovered t h a t t r y p t o p h a n was a b s e n t f r o m proteins L.a.-s~. 8, L.l.-s~. 4 a n d
Re/erences p. 273.
27o
VOL. 22 (1956)
T. GERRITSEN
TABLE AMINO ACID ANALYSES NO. of residues = N u m b e r of residues nearest Fraction: L.l.-s,.o* Mol. weight: 27,600 Total N: 16.62% N recovery: 98.20o/0
Amino acids
Fraction: L.l.-sT.4* Mot. weight: z73,ooo Total N: 17.36 o/~ N recovery: 99.o8%
0/0 mean
No. ol residues
lO.O8 I. 19 4.93 39.25 2.53 2.2o 1.3o 1.57 o.45 3.76 11.35 0.82 3.87 0.66 l.O 7 9-44 7.61
21 3 13 74 6 8 4 4 l 8 24 I 7 1 2 15 17
in duplo
Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Histidine Lysine Arginine Cysteine + cystine/2 Tryptophan Amide-NH2-groups
Total * See text.
lO.21 ; 9-94 i. 20 ; i. 18 4-94; 4.92 38.98; 39-53 2.59; 2.47 2.21 ; 2.18 1.29; 1.3o 1.6o; 1.53 0.45 ; 0.45 3.75; 3.77 11.38; 11.32 o.8o; 0.85 3.96; 3.77 o.65 ; o.67 i. I i ; 1.o2 9.26; 9.61 7.4o; 7.82 . . 3.75 : 3.74
. 3.75
.
. (65)
105.83
209**
% in duplo
14.58; 15.25 3.o6 ; 3.03 5.91 ; 5.76 22.97; 23-58 4.30; 4.26 3.2o; 3.27 3.13; 2.98 3.95; 4.27 . . . 5-75; 6.1o 9.33; 9.55 5.58; 5 .I2 5.48; 5.35 i .92 ; 1.64 4.17; 3.62 12.2o; 12.27 1.64; 1.68 . . 2.50; 2.56
mean
14.92 3-o5 5.84 23.28 4.28 3.24 3.06 4 -11 . 5.92 9.44 5.35 5.42 i .78 3.9o 12.24 1.66
198 44 96 274 64 75 60 61
2.53
(274)
78 125 51 57 2o 46 121 24
.
1Io.o2
** A m i d e - N H 2 - g r o u p s not included.
L.l.-s,.o, this was used as an additional criterion of p u r i t y . The purification of these proteins was repeated until t h e y were free from t r y p t o p h a n as estimated colorimetrically according to SHAW AND MACFARLANE 6. Since the accuracy of the d e t e r m i n a t i o n of a m i n o acids b y the m e t h o d of MOORE AND STEIN is only of t h e order of 3 %, the presence of 2 - 4 % of i m p u r i t y in t h e individual proteins should not affect the final results significantly. Fig. I. Sedimentation diagrams of t h e final purification stages of lupin seed proteins. (t = ca. 2o min; r = ca. 60,000 r.p.m.). F r o m 1 --+ r, L u p i n u s l u t e u s fractions s2.0, sT.4 and Sll.6 , L u p i n u s a n g u s t i / o l i u s fractions ST.8 and Sll.6. H y d r o l y s i s o] the p r o t e i n s
Acid hydrolysis was carried o u t in dilute solution, according to SCHRAM et a L L A b o u t 60 m g of protein w a s dissolved in 2oo ml 5 N redistilled hydrochloric acid, and refluxed for 24 hours. The excess hydrochloric.acid waS r e m o v e d i n v a c u o a n d the h y d r o l y s a t e dissolved in distilled w a t e r and m a d e up to io ml. The p H of the solution w a s b e t w e e n i.o and 2.o. T h e solution was used directly or k e p t in the dark at - - 15 o C until required. According to SCHRA~t et al. 7 this m e t h o d of hydrolysis causes no destruction of a m i n o acids. This was b o r n e o u t in t h e p r e s e n t w o r k b y t h e fact t h a t the recovery of a m i n o acids in the h y d r o l y s a t e was n o t less t h a n 97.5 % (Table I). Consequently we did not t h i n k it necessary to follow the elaborate m e t h o d of VAN DER SCHAAF AND HUISMANs for the d e t e r m i n a t i o n of losses during hydrolysis, for these proteins. R e f e r e n c e s p. 273.
No. o#. residues
1,394"*
VOL. 22 (I956)
LUPIN SEED PROTEINS IV
271
*F LUP1N SEED PROTEINS O the calculated n u m b e r per molecule. Fr.~ction.L.l.-sla.8* Mol. weight." 336,00o T~dd N: I7.49% ,V recovery." 99.7 o % %
Fraction: L.a.-sT., ~ Mol. weight." I8Gooo Total N: 27.zo°/o N recovery: 99.47%
in duplo
mean
No. o! residues
13.32; 13.33 3.83; 3.55 4.75; 4.32 26.45; 26.27 5.oo; 4.48 4-65; 4.45 3.43; 3-46 4-77; 4.48 o.48; o.39 5.70; 5.74 8.78; ~.62 3.85; 3.68 5.32; 5.16 2.93; 2.81 4-39; 4 .ol 11.43; ii.46 1.7o; 1.74 1.59; :.61 2.28; 2.32
13.33 3.69 4.54 26.36 4-74 4.55 3.45 4.63 0.44 5.72 8.70 3-77 5.24 2.87 4 .20 11.4. 5 1.72 1.6o 2.30
336 lO 4 146 602 138 204 13o 134 IO 164 224 7° lO6 62 96 222 48 26 (484)
113.3o
Methods
2,822**
% in duplo
mean
13.34; 13.53 2.36; 2.21 5.28; 5.56 25.32; 26.20 3.89; 3.74 3.34; 3.48 2.53; 2-50 3.59; 3.4 °
~3.4, 2.29 5.42
6.00 7.60 4.74 4 .82 2.23 4.43 13.5o 1.68 --2.45
5.95
5.90; 7.51 ;
4-83; 4-84; 2.36; 4 .21; 13.31; 1.66; 2.20;
25.75
3 .82 3.41 2-52 3.50
7.59 4.79 4.83 2.30 4.32 13.41 1.67 2.33
lO7.35
Fraction.. Mol. weight: Total N : N recovery."
L.a.-sta., ~ 336,oo0
I8.39% 97.51 °/o
No. o/ residues
in duplo
mean
183 35 93 316 60 82 51 ,54 -82 IO5 48 53 27 54 I4O 25 -(264)
t3.o2; :2.74 3.65; 3.7 ° 4-85; 4.58 25.39; 25.39 4.76; 4.77 4.24; 4.20 3.38; 3-34 4.32; 4.21 o.56: 0.44 5.6I: ,5.57 8.38 ; 8.49 3.72; 3.72 5.35; 5.32 3.15; 2.9r 3.8o: 3.96 14.37; 14.31 1.74; 1.86 1.6o; 1.63 2.11: 2.14
12.88 3.68 4.72 25.39 4-77 4.25 3.36 4-29 0.50 5.59 8.44 3.72 .5.34 3.03 3.88 14.34 1.8o :.62 2.i 3
1,4o8"*
%
113.73
No. o~ residues
324 lO 4 :52 588 14o 19o 126 124 :2 144 216 7° io8 66 90 278 5° 28 (,48 )
2.8~o**
o/estimation
The q u a n t i t a t i v e c h r o m a t o g r a p h y was performed on columns of Dowex 5 ° according to the technique described b y MOORE AND STEIN s . T r y p t o p h a n was determined on a solution of the protein in lO% sodium hydroxide b y BLOCK AND BOLLING'S modification of the m e t h o d of SHAW AND MACFARLANEe. This fast m e t h o d gives reliable results for p u r e proteins. Cysteine plus cystine was e s t i m a t e d in a 4-hour acid hydrolysate, after reduction with bisulphite b y a slight modification of t h e m e t h o d of PARK AND SPEAKMAN9. Amide nitrogen was estimated b y determining the a m m o n i a released on hydrolysing 3o0 m g protein w i t h 15 ml 2 N HC1 for 2 h o u r s at 12o °. RESULTS
The amino acid compositions of the five proteins are listed in Table I. Duplicate analyses were obtained on two different hydrolysates of each protein. The duplicate values for tryptophan, cysteine plus cystine, and amide nitrogen were obtained on separate portions of protein. The molecular weights of fractions L.a.-s~. 8 and L.a.-s:l.6 are I8i,ooo and 335,ooo:. Those of fractions L.l.-s~. 4 and L.l.-s:Ls were estimated b y Dr. F. J. JOUBERT to be I73,ooo and 336,000. From the sedimentation rate, the molecular weight of fraction L.l.-s~. o must be about 25,ooo. The calculated molecular weight from the R e f e r e n c e s p . 27.3.
272
"r. GERRVrSEN
VOL. 22 (1956)
contents of all amino acids with less than ten residues per molecule gives an average value of 27,600. DISCUSSION
The globulin fractions L.a.-SlL 6 and L.l.-Stl.S show several points of similarity. Their sedimentation constants, molecular weights and behaviour in the electrophoretic field are very much the same. Each can be reversibly dissociated, into an s7.16 component in the case of the blue lupin seed 1, or an sT.,0 component in the yellow*. Thus from their physico-chemical properties, the two proteins would appear to be identical. However, the nitrogen contents of the two fractions are different (17.5% in L.l.-slL 6 and 18.4% in L.a.-sll.~ ), and the two proteins show a significant difference (p < o.oi after five analyses) in the number of arginine residues per molecule (222 in L.l.-SlL 6 and 278 in L.a.-s11.~ ). The other differences in amino acid composition are not significant. Fraction L.a.-s~.,~ has 912 acid, and 882 basic groups while fraction L.l.-Sll.~ contains 938 acid and 864 basic groups, per molecule. These differences are also not significant, so that the similarity in the electrophoretic mobility of the two proteins is explainable. The amino acid analyses confirm JOUBERT'S 1 conclusion that the dissociation product of L.a.-s11.6 is not identical with L.a.-s~. s. The same holds for L.l.-slL ~ and L.l.-s~. 4. The most striking difference in amino acid compositions is that the S~l.6 fractions contain tryptophan, which is completely absent from the lighter fractions. Fractions L.l.-sT. 4 and L.a.-sT. s show certain broad similarities in composition, for example in the absence of tryptophan and methionine. The difference between the amounts of most amino acids present is, however, greater than the experimental error of the determination. This supports the physical evidence that these proteins are not identical. It is interesting to note that in both lupin seeds, tryptophan is present only in the stx.s fractions. The very small amount of methionine in the proteins investigated and in particular its complete absence from fractions L.l.-sT. 4 and L.a.-sT, 8 explains its low content in lupin seeds (less than o.I % of the total seed). This may cause trouble when lupin seeds are used as sheep fodder without sufficient other proteins, as sometimes happens in South Africa during the dry season. AC KNOWLEDGEME NTS
This paper is published with the permission of the South African Council for Scientific and Industrial Research. I wish to thank Dr. F. J. JOUBEgT for the ultracentrifuge experiments, Mr. P. J. HORN for his accurate technical assistance, and Dr. H. M. SCHWARTZfor her interest and criticism. SUMMARY The a m i n o acid c o m p o s i t i o n of t h e t h r e e seed g l o b u l i n s from Lupinus luteus a n d t h e t w o from Lupinus angustilolius h a s b e e n d e t e r m i n e d b y t h e m e t h o d of MOORE AND ST~IN. I t w a s f o u n d t h a t , a l t h o u g h t h e p r o t e i n s w i t h a s e d i m e n t a t i o n c o n s t a n t of 11.6 S.U. from t h e t w o species are v e r y
Re/erences p. 273.
VOL. 22 (1956)
LUP1N SEED PROTEINSIV
273
similar in physical properties, t h e y differ in n i t r o g e n a n d a r g i n i n e c o n t e n t , a n d are t h u s n o t i d e n tical. The a m i n o acid analyses confirm t h 9 physical evidence t h a t t h e c o m p o n e n t w i t h sedimentation c o n s t a n t 7.4 S.U. in L. luteus is n o t the s a m e as t h e dissociation p r o d u c t of the p r o t e i n w i t h s e d i m e n t a t i o n c o n s t a n t 11.6 S.U. The latter contains all the t r y p t o p h a n in the seed, whereas the former is completely devoid of this a m i n o acid. The same was s h o w n for the sT.s a n d the slx.n fraction of L. angusti/olius. Neither the sT.4 c o m p o n e n t of L. luteus nor the s~.s c o m p o n e n t of L. angusti/olius contains t r y p t o p h a n or methionine. A p a r t from this, however, their a m i n o acid compositions are very dissimilar, s u p p o r t i n g the physico-chemical evidence t h a t these p r o t e i n s are not identical. REFERENCES I F. J. JOUBERT, Biochim. Biophys. Acta, 16 (I955) 37 o, 2 F. J. JOUBERT, Biochim. Biophys. Acta, 17 (1955) 444. 3 S. MOORE AND W. S . STEIN, J. Biol. Chem., 192 (1951) 063. 4 E. M. PETRI AND h . J. STAVERMAN, Discussions Faraday Soc., 13 (1953) I 5 i . 5 p. HOLMES, Australian J. Exptl. Biol. ZVIed. Sci., 31 (1953) 595. 6 R. J. BLOCK AND D. BELLING, The Amino Acid Composition o/ Proteins and Foods, 2nd ed., Charles C. T h o m a s , Springfield, IlL, 1951, p. 119, referring to J. L. D. SHAW AND W. D. MACFARLANE, Can. J. Research, 16 ]3 (1938) 361. 7 E. SCHRAM, J. P. DUSTIN, S. MOORE AND E. J. BIGWOOD, Anal. Chim. Acta, 9 (1953) 149. 8 p. C. VAN DER SCHAAF AND T. I-L J. HUISMAN, Biochim. Biophys. Acta, 17 (1955) 81. 9 G. G. PARK AND J. B. SPEAKMAN, Bull. inst. textile France, 3 ° (1952) 255.
Received January 3oth, 1956
I N F L U E N C E D ' U N E CARENCE EN VITAMINE B e SUR LA T E N E U R EN ACIDES TAURO-CONJUGU]~S E T GLYCO-CONJUGUI~S DE LA B I L E DU RAT par
BERNADETTE
BERGERET
ET F E R N A N D E
CHATAGNER
Laboratoire de Chimie biologique de la Facult~ des Sciences, Paris (France)
Des recherches pr6c6dentes ont montr~ qu'une carence en vitamine B 6 provoque, dans le foie et le cerveau du rat, la disparition de l'activit6 d6carboxylante observ~e, in vitro, vis k vis de l'acide eyst6inesulfinique 1 et de l'acide cyst6ique 2, et entraine, in rive, ehez le mBme animal, l'absence de taurinO, * et d'hypotaurinO clans l'urine. On pouvait, k partir de ees r6sultats, envisager que la formation de la taurine 6fair totalement supprim6e par la earence en pyridoxine. I1 6tait done int6ressant de v6rifier dans quelle mesure les acides tauro-conjug6s, qui constituent la plus grande partie des aeides biliaires ehez le rat s, sent touches par eette carence, et le eas 6ch6ant de voir si la diminution en acides tauro-conjugu6s entraine une modification dans la teneur de la bile en acides glyco-eonjugu~s. Le pr6sent travail rend compte des r~sultats obtenus dans la d~termination de la taurine et du glycocolle eonjugu6s aux aeides biliaires, dosages effectu6s parall~lement l'estimation de la teneur en taurine clans l'urine des animaux. Ces r~sultats montrent, c h e z le rat carenc6, une eertaine diminution des aeides tauro-conjugu6s et une forte Bibliographie p. 277.