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Structural properties of umecyanin
246
BIOCHIMICAET BIOPHYSICAACTA
BBA 3583Z STRUCTURAL P R O P E R T I E S OF UMECYANIN A COPPER P R O T E I N FROM H O R S E R A D I S H ROOT
TORGNY ST[GBRAND
D@arlme*Tt of Che*nislry, Section of Physiological Chemistry, University of Umed, Umed (Sweden) (Received December iSth, ~97 o)
SUMMARY
I. Umecyanin, a novel intensely blue copper protein, has an s20,~, of 1. 9 • IO-13 sec, corresponding to a molecular weight of 14 6oo as determined from sedimentation velocity and equilibrium centrifugation studies. The frictional ratio fifo has been determined to be I.Z2, indicating a spherical molecule. 2. The N-terminal amino acid is glutamic acid, and according to the amino acid distribution, there are I25 amino acid residues per molecule. The isoelectric point is 5.85. 3. The carbohydrate content is very low compared to other Cu-containing proteins. Umecyanin contains only three glucosamine residues per molecule. Umecyanin behaves similarly to azurin, stellacyanin and Pseudomonas blue protein in with respect to its lack of enzymatic activity towards some substrates that are oxidized by Cu-containing oxidases (ceruloplasmin, laccase, ascorbic acid oxidase, mono- and diamino oxidase).
INTRODUCTION
Umecyanin, a novel intensely blue protein from horseradish root, was recently isolated in this laboratory1, e. On the basis of gel filtration, its molecular weight was estimated at 14 6oo, and it was found to contain o.46~o copper, corresponding to I Cu/molecule. Its general properties resemble those of stellacyanin a, azurinL and Pseudomonas blue protein s, all of which possess I copper atom per molecule. These proteins still lack a reasonable function. Recently there has been a great deal of interest in intensely blue proteins that function as oxidases, such as ceruloplasmin, laccase and ascorbate oxidase 6. Electron paramagnetic resonance has been used to describe the properties of the cuprous and cupric atoms in each of these proteins. All these proteins contain more than z Cu/molecule, occurring in different forms within the moleculeL Before these Cu-proteins can be satisfactorily compared, it is necessary to further characterize umecyanin. Ydiochim. t3ioj)hys, dcta, 236 (I97 I) 246-252
NOVEL BLUE COPPER PROTEIN
247
MATERIALS AND METHODS
Umecyanin was prepared from horseradish root as described in a previous paper ~. Ultracentrifugation and sedimentation velocity studies of umecyanin were performed in a Spinco Model-E ultracentrifuge equipped with phase plate Schlieren and interference optics. Sedimentation velocity experiments were performed in a standard aluminium I2-mm 4 ° sector cell or in a synthetic boundary cell at 59 78o rev./min at 2o °. The protein concentration was 5 mg/ml in 3° mM acetate, pH 5.7 o. The molecular weight was estimated by the method of Archibald as modified by TRAUTMAN1°. From gel filtration data and the sedimentation coefficient, the molecular weight was also determined by the method of SIEGEL AND 3JONTY11. Reference proteins were ribonuclease, myoglobin, cytochrome c and pepsin. Data for the molecular radii were taken from LAURENT AND KILLANDER1~. The frictional ratio was calculated as described by SCHACHMAN1~. Equilibrium centrifugation was performed in the synthetic boundary cell at 50 780 rev./min and 31 41o rev./min. From these data the molecular weight was determined according to YPHANTIS14. Amino acid analyses were performed in a Beckman 12o C automatic amino acid analyzer with an Infotronics integrator CRS-Ioo A. Samples were hydrolyzed in 6 M HC1 at IiO ° in sealed tubes for 24 and 72 h. Norleucine (Calbiochem AB, Lucerne, Switzerland) was dried over silica gel and used as internal standard. Ammonia and basic amino acids were separated on the sulphonate styrene copolymer Beckman PA-35, other amino acids and hexosamines being separated on the similar Beckman PA-28 resin. The values in Table II are extrapolated to zero time for Thr, Ser, Tyr, NH~, and Glc/N; for Pro and Leu the mean value, and for the other amino acids the maximum value, has been used. Tryptophan was determined spectrophotometrically on native enzyme samples in o.I M NaOH as described by GOODWIN AND MORTON15. N-termi~¢al amino acids were determined as phenylthiohydantoin derivatives by the method of Edman as modified by BLOMBACK et al. ~7. The chromatographic systems ot; S J 6 Q U I S T is w e r e used for identification and quantitative determination of the phenylthiohydantoin derivatives. Carbohydrate content was determined by the anthrone method as modified by SHIELDS AND BURNETT16 for protein-bound carbohydrates with a I : I mixture of D-galactose and D-mannose as reference. The anthrone solution was allowed to react with the protein at 92o for 8 min. Isoelectric focussing was performed in the LKB column 81Ol, IiO ml (LKB Stockholm, Sweden) with 10(7 Ampholine according to L K B ' s suggestions and with a sucrose density gradient. The pH gradient covered the interval 3-1o or 5-7Umecyanin was determined spectrophotometrically by measuring the absorption at 280 and 61o nm. Enzymatic activity was tested with ascorbic acid, as described by DAWSON et al. 2°, with p-phenylenediamine as described by PEISACH et al. 3, and with tyramine, histamine and spermidine as described by ORELAND21. The procedures given by these authors were followed. Biochim. Biophys. _dcta, 236 (1971) 246-252
248
T. STIGBRAND
Fig. I. S c h l i e r e n p a t t e r n s for s e d i m e n t a t i o n v e l o c i t y e x p e r i m e n t on u n l e c y a n i n . T h e p r o t e i n s o l u t i o n c o n t a i n e d 5 m g / m l in 3 ° m M s o d i u m a c e t a t e , p H 5.7. P i c t u r e s w e r e t a k e n a t (a) lO9 rain, (b) 83 m i n , (c) 5 T rain, a t 59 78o r e v . / m i n .
RESULTS
Ultracentrifugal analysis The protein sedimented in the ultracentrifuge as a completely homogeneous component (Fig. I). The sedimentation coefficient was determined with a 0.5% protein solution, s20,w was found to be 1. 9 . I o ~ sec. B y the m e t h o d of Archibald modified b y T R A U T M A N 1°, the molecular weight was calculated to 15 ooo. From the results of amino acid analysis (below), the partial specific volume was calculated as described by COHN AND EDSALL19 to be 0.74. TABLE
1
GEL FILTRATION DATA FOR DETERMINATION OF THE MOLECULAR RADIUS OF UMECYANIN A c o l u m n w i t h 9oo n m l × 26 m m S e p h a d e x G-75 in 3 ° m M s o d i u m a c e t a t e w a s used. T h e t o t a l v o l u m e , VT, was 158 ml a n d t h e v o i d v o l u m e , V o, 51 ml. Kav (Ve - - V 0 ) / ( I " T - - Vo). D a t a for t h e m o l e c u l a r r a d i i w e r e t a k e n f r o m LAURENT AND ] ( I L L A N D E R 12.
Ribonuclease Myoglobin Cytochrome c Peroxidase Umecyanin
V~
K~v
v / ~ K~v
r~
99.6 94,2 lO3.2 69.2 98. 4
o.455 o.4o2 o.486 o. 168 0.46
o.89 o.95 0.85 1.33 o. 88
19- 2 [ 9-7 I6.4 3 o. 2 -
From the gel c h r o m a t o g r a p h y data on the reference proteins and their molecular radii (taken from LAI~RENT AND K I L L A N D E R 12) (Table I) the molecular radius for umecyanin was determined to be 16.6 A (Fig. 2). This value could also be used according to Stoke's formula to determine the molecular weight which was then calculated to be 13 8oo. The frictional ratio fifo 13 was found to be 1.12, and the above figure would be a reasonable molecular weight if the protein is a rather compact sphere. Equilibrium sedimentation showed that all material had sedimented to the cell b o t t o m at 5o 78o rev./min. At 31 41o rev./min, however, an equilibrium was found for the smallest particle in the solution. The molecular weight determined by the method of YPHANTIS14 was 2 9 O0O (Fig. 3), indicating t h a t the protein polymerizes at higher concentrations in the medium used (3o mM sodium acetate, p H 5.7o). No Biochim. t3iophys. Acla, 236 (197 I) 2 4 6 - 2 3 2
NOVEL BLUE COPPER PROTEIN
249
2.C
Z
I
,
r
r
I
I
2.0
1.C
t
I
0
I
I
I
I
I
10 Iqodiu~
~
t
I
b 20
I
I
I
I
I
. .
(~ )
.0 I r2
Fig. 2. Plot of V/~n Kav against radius ()k) for determination of the molecular radius of unlecyanin. Reference proteins were ribonuclease (19.2), nlyoglobin (I9.7), cytochronle c (16.4), peroxidase I I I b (30.2). See t e x t and Table I for f u r t h e r explanations. Fig. 3. Plot of In c against r ~ from equilibrium sedimentation of u m e c y a n i n at a r o t a t i o n speed of 31 41o rev./min, 2o °, in 3 ° mM sodium acetate, p H 5.7 o. TABLE I l M O L E C U L A R
W E I G H T
D E T E R M I N A T I O N S
OF
U M E C Y A N I N
Details are given in MATERIALS AND METHODS.
Method
Mol. wt.
Ref.
Cu-analysis (;el filtration (Sephadex G-75 ) S e d i m e n t a t i o n centrifugation Method according to Stoke Equilibrium eentrifugation
13 800 14 600 15 ooo 13 800 29 ooo
2 i This work This w o r k This work
tendencies to inhomogeneity were seen. The molecular weight determinations are summarized in Table II.
Amino acid analysis The amino acid composition is shown in Table I I I . The remarkably high recovery of amino acids (99%, lO2%) based on dry weight determinations of the entire material shows a very low content of carbohydrate or other material. Three preparations of umecyanin gave the same recoveries and the same amino acid distribution. The number of residues per molecule was calculated on the basis of a molecular weight of 14 600. This value is consistent with the results of amino acid analysis, since the calculated numbers of residues per molecule were close to whole numbers. The determination of tyrosine spectrophotometrically and by the amino acid analyzer both indicated that there are 4 tyrosine residues per molecule. In umecyanin there is an excess of 4 basic amino acid residues over acid residues (arginine + histidine + lysine = 16; aspartic acid + glutamic acid -- NH 3 = 12). The origin of the 13 molecules of ammonia has not yet been determined. The faintly acid character of umecyanin, I.P. 5.85 (Fig. 4), suggests that not all ammonia stems from amide groups. The N-terminal amino acid was found to be glutamic acid. No signs of impurities were seen. Biochim. Biophys. Acta, 236 (1971) 246-252
T. STIGBRAND
250
.4, 0,25
~
6
.
pH
0
5.0 .4.0 3,0
0:1~ 15 20 Tube No.
25
Fig. 4. I s o e l e c t r i c focussing of u m e c y a n i n . 2.0 m g p r o t e i n w a s a s s a y e d in t h e p H r a n g e 5 7 a t 4 o o V for 4 8 h . Q - - O , p H ; , ~ - - ( ) , A2~onm; ~ - - • ~ A~10 n in.
Carbohydrate content The amino acid analysis predicted a low carbohydrate content, and this was indeed confirmed. The carbohydrate content was determined to be 1.2%, corresponding to one hexose sugar per molecule. The anthrone solution used is known to react slowly with glucosamine and this explains the too low figure. The amino acid analysis gave a value corresponding to 3.7% carbohydrate.
TABLE HI AMINO
ACID
COMPOSITION
OF UMECYANIN
o.5346 m g u m e c y a n i n was a n a l y s e d .
Amino acid
iirnoles/mg
Asp*
1.o44 0.636 0.280 0.503 o.4oo 0.809 0.484 0.251 0.632 0.250 0.372 0.226 0.226 o.273 -o.224 o.6oo o.21o o.8ot 0.208
Thr
Ser Glu** Pr o ( ;ly Ala Cyg Val Met ile Leu Tyr Phe Trp*** GlcN Lys His NH a Arg
Residues per r4 6oo
17. 3 lO.6 4.7 8.3 6.7 13.4 8.0 3.0 lO.4 4.2 6.2 3.8 3.8
4.6 4 .0 3.7
Residues per Cu
16. 4 io.I 4-5 7.9 6.4 12.7 7.6 2.9 9.9 4.0 5-9 3.6 3.6 4-4 4 .2 3.5
17 ii 5 8 7 13 8 3 IO 4 6 4 4 5 4 3
9"5
IO
iO.O
3.5 i3.3§ 3.4 129.6
Residues per molecule
3.3 12.6§ 3.3 123.7
3 I3§ 3 128
* I n c l u d e s Asn. ** I n c l u d e s Gln. *** D e t e r m i n e d s p e c t r o p h o t o m e t r i c a l l y on i n t a c t prot e i n. § N o t i n c l u d e d in t h e sum.
Biochim. Biophys. Acta, 236 (1971) 246-252
NOVEL BLUE COPPER PROTEIN
251
DISCUSSION
The data presented above clearly indicate that umecyanin is not identical to any known copper protein and also that it has been isolated in pure form. The molecular weight determinations are in slight disagreement due to tt~e different procedures used. The value 14 6oo determined earlier is in good agreement with the centrifugation data 15 ooo, 13 8oo, and 14 5oo. The value of 14 6oo has been used as the molecular weight in further calculations. The formation of at least dimers at high concentrations of umecyanin in the cell during sedimentation equilibrium is noteworthy, indicating a tendency to polymerize under certain conditions. The frictional ratio fifo, 1.12, indicates that the protein is a rather compact sphere with a molecular radius of 16.6 A. Umecyanin proved to have an extremely low carbohydrate content compared with other members of the intensely blue copper protein group, which all have been classified as nmco- or glycoproteins. I t has also been suggested 3 that the blue colour is due to a copper carbohydrate complex; this apparently is not the case. Amino acid analysis showed all common amino acids to be present including valine and methionine which are lacking in stellacyanin, and tryptophan which is lacking in Pseudomonas blue protein. Calculation of total amino acids on the basis of a molecular weight of 14 6oo indicated that there are 125 amino acid residues, which, along with the low carbohydrate content, suggests that it would be feasible to determine the amino acid sequence in umecyanin. There is one N-terminal amino acid, glutamic acid, which suggests that there m a y be but a single protein chain. The function of umecyanin is still unknown. It has been suggested that the intensely blue proteins containing I Cu/molecule function in electron transfer reactions in contrast to those containing several copper atoms per molecule, which have oxidase or enzymatic activity. The absence of activity towards ascorbic acid, p-phenylenediamine and several common "classical" substrates for copper proteins suggests that umecyanin belongs to the first group. ACKNOWLEDGEMENTS
Skilful technical assistance has been given by Mrs. Kerstin Hjortsberg. I also wish to express my gratitude to Dr. HSkan Pertoft (Department of medical chemistry, Uppsala) for carrying out the ultracentrifugal analysis and Prof. John SjSquist (The Wallenberg laboratory, Uppsala) for determination of the N-terminal amino acid. The study was supported by a grant from the Swedish Natural Science Research Council (320-7, 7926 K). REFERENCES K. G. PAUL AND T. STIGBRAND,Acta Chem. Scan&, 24 (197 o) 3607 . K. G. PAUL AND T. STIGBRAND, Biochim. Biophys. Acia, 221 (197 o) 255. J. PEISACH, W. G. LEVlNE AND VvT. E. BLUMBERG, ./r. Biol. Chem., 242 (1967) 2847. E. HAMMARSTEN, W. PALMSTIERNA AND E. MEYER, J . Biochem. Microbiol. Technol. Eng., i (1959) 273. 5 M. L. COVAL, T. HORRO, AND M. D. KAMEN, Biochim. Biophys. Acta, 51 (1961) 246. 6 J. PEISACH, P. AISEN AND W. E. BLUMBERG, The Biochemistry of Copper, A c a d e m i c Press, New York , London, 1966, p. 305 .
i 2 3 4
Biochim. Biophys. Acta, 236 (1971) 246-252
252 7 8 9 io II 12 13 14 15 16 17 18 19 20 2i
T. STIGBRAND
R. MALKIN AND B. G. MALMSTROM, Advan. Enzymol., 33 (197 o) 177. H. S. MASON, Biochem. Biophys. Res. Commun., i o (1963) i i . T. OMURA, J. Biochem. Tokyo, 5 ° ( i 9 6 i ) 391. I{, TRAUTMAN, J. Am. Chem. Soc., 8I (1959) 4o3 • L. M. SIEGEL AND K. J. MONTE, Biochim. Biophys. Acta, 112 (1966) 346. T. LAURENT AND J. KILLANDER, J. Chromatog., 14 (1964) 317 . H. J~. SCHACHMAN,Ultracentrifuge Biochem., 2 (1963) 887. D. A. YPHANTIS, Biochemistry, 3 (19641 297. T. \V. GOODWIN AND R. A. MORTON, Biochem. J., 4 ° (1946) 628. ]~. SHIELDS AND V~T. BURNETT, Anal. Chem., 32 (196o) 885. g . BLOMB~,CK, M. BLOMB'~CK, P. ]~DMAN AND B. HESSEL, Biochim. Biophys. Acta, 115 (19661 371 • J. SJOQUIST, Biochim. Biophys. Acta, 41 (196o) 2o. E. J. COHN AND J. T. EDSALL, in Proteins, Amino Acids, and Peptides as Ions and Dipolar Ions, R e i n h o l d , New York, 1943 p. 372. C. R. ]_)AWSON AN[) 1{. J. MAGEE, in S. P. COLOWlCK AND N. O. I~APLAN, Methods in Enzwnology, Vol. 11, A c a d e m i c Press, New York, 1955, p. 831. L. ORELAND, to be p u b l i s h e d .
Biochim. Biophys. Acta, 236 (1971) 246-252
BIOCHIMICAET BIOPHYSICAACTA
BBA 3583Z STRUCTURAL P R O P E R T I E S OF UMECYANIN A COPPER P R O T E I N FROM H O R S E R A D I S H ROOT
TORGNY ST[GBRAND
D@arlme*Tt of Che*nislry, Section of Physiological Chemistry, University of Umed, Umed (Sweden) (Received December iSth, ~97 o)
SUMMARY
I. Umecyanin, a novel intensely blue copper protein, has an s20,~, of 1. 9 • IO-13 sec, corresponding to a molecular weight of 14 6oo as determined from sedimentation velocity and equilibrium centrifugation studies. The frictional ratio fifo has been determined to be I.Z2, indicating a spherical molecule. 2. The N-terminal amino acid is glutamic acid, and according to the amino acid distribution, there are I25 amino acid residues per molecule. The isoelectric point is 5.85. 3. The carbohydrate content is very low compared to other Cu-containing proteins. Umecyanin contains only three glucosamine residues per molecule. Umecyanin behaves similarly to azurin, stellacyanin and Pseudomonas blue protein in with respect to its lack of enzymatic activity towards some substrates that are oxidized by Cu-containing oxidases (ceruloplasmin, laccase, ascorbic acid oxidase, mono- and diamino oxidase).
INTRODUCTION
Umecyanin, a novel intensely blue protein from horseradish root, was recently isolated in this laboratory1, e. On the basis of gel filtration, its molecular weight was estimated at 14 6oo, and it was found to contain o.46~o copper, corresponding to I Cu/molecule. Its general properties resemble those of stellacyanin a, azurinL and Pseudomonas blue protein s, all of which possess I copper atom per molecule. These proteins still lack a reasonable function. Recently there has been a great deal of interest in intensely blue proteins that function as oxidases, such as ceruloplasmin, laccase and ascorbate oxidase 6. Electron paramagnetic resonance has been used to describe the properties of the cuprous and cupric atoms in each of these proteins. All these proteins contain more than z Cu/molecule, occurring in different forms within the moleculeL Before these Cu-proteins can be satisfactorily compared, it is necessary to further characterize umecyanin. Ydiochim. t3ioj)hys, dcta, 236 (I97 I) 246-252
NOVEL BLUE COPPER PROTEIN
247
MATERIALS AND METHODS
Umecyanin was prepared from horseradish root as described in a previous paper ~. Ultracentrifugation and sedimentation velocity studies of umecyanin were performed in a Spinco Model-E ultracentrifuge equipped with phase plate Schlieren and interference optics. Sedimentation velocity experiments were performed in a standard aluminium I2-mm 4 ° sector cell or in a synthetic boundary cell at 59 78o rev./min at 2o °. The protein concentration was 5 mg/ml in 3° mM acetate, pH 5.7 o. The molecular weight was estimated by the method of Archibald as modified by TRAUTMAN1°. From gel filtration data and the sedimentation coefficient, the molecular weight was also determined by the method of SIEGEL AND 3JONTY11. Reference proteins were ribonuclease, myoglobin, cytochrome c and pepsin. Data for the molecular radii were taken from LAURENT AND KILLANDER1~. The frictional ratio was calculated as described by SCHACHMAN1~. Equilibrium centrifugation was performed in the synthetic boundary cell at 50 780 rev./min and 31 41o rev./min. From these data the molecular weight was determined according to YPHANTIS14. Amino acid analyses were performed in a Beckman 12o C automatic amino acid analyzer with an Infotronics integrator CRS-Ioo A. Samples were hydrolyzed in 6 M HC1 at IiO ° in sealed tubes for 24 and 72 h. Norleucine (Calbiochem AB, Lucerne, Switzerland) was dried over silica gel and used as internal standard. Ammonia and basic amino acids were separated on the sulphonate styrene copolymer Beckman PA-35, other amino acids and hexosamines being separated on the similar Beckman PA-28 resin. The values in Table II are extrapolated to zero time for Thr, Ser, Tyr, NH~, and Glc/N; for Pro and Leu the mean value, and for the other amino acids the maximum value, has been used. Tryptophan was determined spectrophotometrically on native enzyme samples in o.I M NaOH as described by GOODWIN AND MORTON15. N-termi~¢al amino acids were determined as phenylthiohydantoin derivatives by the method of Edman as modified by BLOMBACK et al. ~7. The chromatographic systems ot; S J 6 Q U I S T is w e r e used for identification and quantitative determination of the phenylthiohydantoin derivatives. Carbohydrate content was determined by the anthrone method as modified by SHIELDS AND BURNETT16 for protein-bound carbohydrates with a I : I mixture of D-galactose and D-mannose as reference. The anthrone solution was allowed to react with the protein at 92o for 8 min. Isoelectric focussing was performed in the LKB column 81Ol, IiO ml (LKB Stockholm, Sweden) with 10(7 Ampholine according to L K B ' s suggestions and with a sucrose density gradient. The pH gradient covered the interval 3-1o or 5-7Umecyanin was determined spectrophotometrically by measuring the absorption at 280 and 61o nm. Enzymatic activity was tested with ascorbic acid, as described by DAWSON et al. 2°, with p-phenylenediamine as described by PEISACH et al. 3, and with tyramine, histamine and spermidine as described by ORELAND21. The procedures given by these authors were followed. Biochim. Biophys. _dcta, 236 (1971) 246-252
248
T. STIGBRAND
Fig. I. S c h l i e r e n p a t t e r n s for s e d i m e n t a t i o n v e l o c i t y e x p e r i m e n t on u n l e c y a n i n . T h e p r o t e i n s o l u t i o n c o n t a i n e d 5 m g / m l in 3 ° m M s o d i u m a c e t a t e , p H 5.7. P i c t u r e s w e r e t a k e n a t (a) lO9 rain, (b) 83 m i n , (c) 5 T rain, a t 59 78o r e v . / m i n .
RESULTS
Ultracentrifugal analysis The protein sedimented in the ultracentrifuge as a completely homogeneous component (Fig. I). The sedimentation coefficient was determined with a 0.5% protein solution, s20,w was found to be 1. 9 . I o ~ sec. B y the m e t h o d of Archibald modified b y T R A U T M A N 1°, the molecular weight was calculated to 15 ooo. From the results of amino acid analysis (below), the partial specific volume was calculated as described by COHN AND EDSALL19 to be 0.74. TABLE
1
GEL FILTRATION DATA FOR DETERMINATION OF THE MOLECULAR RADIUS OF UMECYANIN A c o l u m n w i t h 9oo n m l × 26 m m S e p h a d e x G-75 in 3 ° m M s o d i u m a c e t a t e w a s used. T h e t o t a l v o l u m e , VT, was 158 ml a n d t h e v o i d v o l u m e , V o, 51 ml. Kav (Ve - - V 0 ) / ( I " T - - Vo). D a t a for t h e m o l e c u l a r r a d i i w e r e t a k e n f r o m LAURENT AND ] ( I L L A N D E R 12.
Ribonuclease Myoglobin Cytochrome c Peroxidase Umecyanin
V~
K~v
v / ~ K~v
r~
99.6 94,2 lO3.2 69.2 98. 4
o.455 o.4o2 o.486 o. 168 0.46
o.89 o.95 0.85 1.33 o. 88
19- 2 [ 9-7 I6.4 3 o. 2 -
From the gel c h r o m a t o g r a p h y data on the reference proteins and their molecular radii (taken from LAI~RENT AND K I L L A N D E R 12) (Table I) the molecular radius for umecyanin was determined to be 16.6 A (Fig. 2). This value could also be used according to Stoke's formula to determine the molecular weight which was then calculated to be 13 8oo. The frictional ratio fifo 13 was found to be 1.12, and the above figure would be a reasonable molecular weight if the protein is a rather compact sphere. Equilibrium sedimentation showed that all material had sedimented to the cell b o t t o m at 5o 78o rev./min. At 31 41o rev./min, however, an equilibrium was found for the smallest particle in the solution. The molecular weight determined by the method of YPHANTIS14 was 2 9 O0O (Fig. 3), indicating t h a t the protein polymerizes at higher concentrations in the medium used (3o mM sodium acetate, p H 5.7o). No Biochim. t3iophys. Acla, 236 (197 I) 2 4 6 - 2 3 2
NOVEL BLUE COPPER PROTEIN
249
2.C
Z
I
,
r
r
I
I
2.0
1.C
t
I
0
I
I
I
I
I
10 Iqodiu~
~
t
I
b 20
I
I
I
I
I
. .
(~ )
.0 I r2
Fig. 2. Plot of V/~n Kav against radius ()k) for determination of the molecular radius of unlecyanin. Reference proteins were ribonuclease (19.2), nlyoglobin (I9.7), cytochronle c (16.4), peroxidase I I I b (30.2). See t e x t and Table I for f u r t h e r explanations. Fig. 3. Plot of In c against r ~ from equilibrium sedimentation of u m e c y a n i n at a r o t a t i o n speed of 31 41o rev./min, 2o °, in 3 ° mM sodium acetate, p H 5.7 o. TABLE I l M O L E C U L A R
W E I G H T
D E T E R M I N A T I O N S
OF
U M E C Y A N I N
Details are given in MATERIALS AND METHODS.
Method
Mol. wt.
Ref.
Cu-analysis (;el filtration (Sephadex G-75 ) S e d i m e n t a t i o n centrifugation Method according to Stoke Equilibrium eentrifugation
13 800 14 600 15 ooo 13 800 29 ooo
2 i This work This w o r k This work
tendencies to inhomogeneity were seen. The molecular weight determinations are summarized in Table II.
Amino acid analysis The amino acid composition is shown in Table I I I . The remarkably high recovery of amino acids (99%, lO2%) based on dry weight determinations of the entire material shows a very low content of carbohydrate or other material. Three preparations of umecyanin gave the same recoveries and the same amino acid distribution. The number of residues per molecule was calculated on the basis of a molecular weight of 14 600. This value is consistent with the results of amino acid analysis, since the calculated numbers of residues per molecule were close to whole numbers. The determination of tyrosine spectrophotometrically and by the amino acid analyzer both indicated that there are 4 tyrosine residues per molecule. In umecyanin there is an excess of 4 basic amino acid residues over acid residues (arginine + histidine + lysine = 16; aspartic acid + glutamic acid -- NH 3 = 12). The origin of the 13 molecules of ammonia has not yet been determined. The faintly acid character of umecyanin, I.P. 5.85 (Fig. 4), suggests that not all ammonia stems from amide groups. The N-terminal amino acid was found to be glutamic acid. No signs of impurities were seen. Biochim. Biophys. Acta, 236 (1971) 246-252
T. STIGBRAND
250
.4, 0,25
~
6
.
pH
0
5.0 .4.0 3,0
0:1~ 15 20 Tube No.
25
Fig. 4. I s o e l e c t r i c focussing of u m e c y a n i n . 2.0 m g p r o t e i n w a s a s s a y e d in t h e p H r a n g e 5 7 a t 4 o o V for 4 8 h . Q - - O , p H ; , ~ - - ( ) , A2~onm; ~ - - • ~ A~10 n in.
Carbohydrate content The amino acid analysis predicted a low carbohydrate content, and this was indeed confirmed. The carbohydrate content was determined to be 1.2%, corresponding to one hexose sugar per molecule. The anthrone solution used is known to react slowly with glucosamine and this explains the too low figure. The amino acid analysis gave a value corresponding to 3.7% carbohydrate.
TABLE HI AMINO
ACID
COMPOSITION
OF UMECYANIN
o.5346 m g u m e c y a n i n was a n a l y s e d .
Amino acid
iirnoles/mg
Asp*
1.o44 0.636 0.280 0.503 o.4oo 0.809 0.484 0.251 0.632 0.250 0.372 0.226 0.226 o.273 -o.224 o.6oo o.21o o.8ot 0.208
Thr
Ser Glu** Pr o ( ;ly Ala Cyg Val Met ile Leu Tyr Phe Trp*** GlcN Lys His NH a Arg
Residues per r4 6oo
17. 3 lO.6 4.7 8.3 6.7 13.4 8.0 3.0 lO.4 4.2 6.2 3.8 3.8
4.6 4 .0 3.7
Residues per Cu
16. 4 io.I 4-5 7.9 6.4 12.7 7.6 2.9 9.9 4.0 5-9 3.6 3.6 4-4 4 .2 3.5
17 ii 5 8 7 13 8 3 IO 4 6 4 4 5 4 3
9"5
IO
iO.O
3.5 i3.3§ 3.4 129.6
Residues per molecule
3.3 12.6§ 3.3 123.7
3 I3§ 3 128
* I n c l u d e s Asn. ** I n c l u d e s Gln. *** D e t e r m i n e d s p e c t r o p h o t o m e t r i c a l l y on i n t a c t prot e i n. § N o t i n c l u d e d in t h e sum.
Biochim. Biophys. Acta, 236 (1971) 246-252
NOVEL BLUE COPPER PROTEIN
251
DISCUSSION
The data presented above clearly indicate that umecyanin is not identical to any known copper protein and also that it has been isolated in pure form. The molecular weight determinations are in slight disagreement due to tt~e different procedures used. The value 14 6oo determined earlier is in good agreement with the centrifugation data 15 ooo, 13 8oo, and 14 5oo. The value of 14 6oo has been used as the molecular weight in further calculations. The formation of at least dimers at high concentrations of umecyanin in the cell during sedimentation equilibrium is noteworthy, indicating a tendency to polymerize under certain conditions. The frictional ratio fifo, 1.12, indicates that the protein is a rather compact sphere with a molecular radius of 16.6 A. Umecyanin proved to have an extremely low carbohydrate content compared with other members of the intensely blue copper protein group, which all have been classified as nmco- or glycoproteins. I t has also been suggested 3 that the blue colour is due to a copper carbohydrate complex; this apparently is not the case. Amino acid analysis showed all common amino acids to be present including valine and methionine which are lacking in stellacyanin, and tryptophan which is lacking in Pseudomonas blue protein. Calculation of total amino acids on the basis of a molecular weight of 14 6oo indicated that there are 125 amino acid residues, which, along with the low carbohydrate content, suggests that it would be feasible to determine the amino acid sequence in umecyanin. There is one N-terminal amino acid, glutamic acid, which suggests that there m a y be but a single protein chain. The function of umecyanin is still unknown. It has been suggested that the intensely blue proteins containing I Cu/molecule function in electron transfer reactions in contrast to those containing several copper atoms per molecule, which have oxidase or enzymatic activity. The absence of activity towards ascorbic acid, p-phenylenediamine and several common "classical" substrates for copper proteins suggests that umecyanin belongs to the first group. ACKNOWLEDGEMENTS
Skilful technical assistance has been given by Mrs. Kerstin Hjortsberg. I also wish to express my gratitude to Dr. HSkan Pertoft (Department of medical chemistry, Uppsala) for carrying out the ultracentrifugal analysis and Prof. John SjSquist (The Wallenberg laboratory, Uppsala) for determination of the N-terminal amino acid. The study was supported by a grant from the Swedish Natural Science Research Council (320-7, 7926 K). REFERENCES K. G. PAUL AND T. STIGBRAND,Acta Chem. Scan&, 24 (197 o) 3607 . K. G. PAUL AND T. STIGBRAND, Biochim. Biophys. Acia, 221 (197 o) 255. J. PEISACH, W. G. LEVlNE AND VvT. E. BLUMBERG, ./r. Biol. Chem., 242 (1967) 2847. E. HAMMARSTEN, W. PALMSTIERNA AND E. MEYER, J . Biochem. Microbiol. Technol. Eng., i (1959) 273. 5 M. L. COVAL, T. HORRO, AND M. D. KAMEN, Biochim. Biophys. Acta, 51 (1961) 246. 6 J. PEISACH, P. AISEN AND W. E. BLUMBERG, The Biochemistry of Copper, A c a d e m i c Press, New York , London, 1966, p. 305 .
i 2 3 4
Biochim. Biophys. Acta, 236 (1971) 246-252
252 7 8 9 io II 12 13 14 15 16 17 18 19 20 2i
T. STIGBRAND
R. MALKIN AND B. G. MALMSTROM, Advan. Enzymol., 33 (197 o) 177. H. S. MASON, Biochem. Biophys. Res. Commun., i o (1963) i i . T. OMURA, J. Biochem. Tokyo, 5 ° ( i 9 6 i ) 391. I{, TRAUTMAN, J. Am. Chem. Soc., 8I (1959) 4o3 • L. M. SIEGEL AND K. J. MONTE, Biochim. Biophys. Acta, 112 (1966) 346. T. LAURENT AND J. KILLANDER, J. Chromatog., 14 (1964) 317 . H. J~. SCHACHMAN,Ultracentrifuge Biochem., 2 (1963) 887. D. A. YPHANTIS, Biochemistry, 3 (19641 297. T. \V. GOODWIN AND R. A. MORTON, Biochem. J., 4 ° (1946) 628. ]~. SHIELDS AND V~T. BURNETT, Anal. Chem., 32 (196o) 885. g . BLOMB~,CK, M. BLOMB'~CK, P. ]~DMAN AND B. HESSEL, Biochim. Biophys. Acta, 115 (19661 371 • J. SJOQUIST, Biochim. Biophys. Acta, 41 (196o) 2o. E. J. COHN AND J. T. EDSALL, in Proteins, Amino Acids, and Peptides as Ions and Dipolar Ions, R e i n h o l d , New York, 1943 p. 372. C. R. ]_)AWSON AN[) 1{. J. MAGEE, in S. P. COLOWlCK AND N. O. I~APLAN, Methods in Enzwnology, Vol. 11, A c a d e m i c Press, New York, 1955, p. 831. L. ORELAND, to be p u b l i s h e d .
Biochim. Biophys. Acta, 236 (1971) 246-252