Biochimica et Biophysica Acta, 434 (1976) 452--461

© Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands BBA 37357 BIOCHEMICAL STUDIES ON H U M A N C E R U L O P L A S M I N

MICHAEL L. McCOMBS* and BARBARA H. BOWMAN Department of Human Biological Chemistry and Genetics, The University of Texas Medical Branch, Galveston, Texas 77550 (U.S.A.)

(Received October 6th, 1975)

SUMMARY Ceruloplasmin from nephrotic urine, ascites fluid and plasma has been partially characterized. All ceruloplasmin preparations were found to be comprised of two light and two heavy polypeptide subunits. Characterization of the purified subunits indicated that the a chain had a mol. wt. of 16 000 and had N-terminal valine while the /3 chain had a mol. wt. of 59 000 and had N-terminal lysine. All carbohydrate resided in the /3 subunit. Incomplete cleavage of the 5-methionine residues of the a chain enabled a preliminary ordering of the CNBr fragments. Automated sequence analysis of the a chain was carried out and the sequence determined was V a l - P h e - A s x - P r o - A r g - A r g - L y s - L e u - G l x - P h e - A l a - L e u - L e u - P h e - L e u Val-Phe-Asx-Glx-Asx-Glx.

INTRODUCTION Ceruloplasmin is a copper-containing serum a2-globulin which was first described by Holmberg and Laurell in 1948 [1]. Starch gel [2] and acrylamide electrophoresis [3] provided evidence that this protein is genetically polymorphic in the human population. Although the principle function of ceruloplasmin is yet unknown, it has clinical interest because of its marked reduction in the serum of patients with Wilson's disease [4]. Kasper and Deutsch [5], Poillon and Bearn [6], and Simons and Bearn [7] have examined the subunit structure of human ceruloplasmin and found evidence for two unique polypeptide chains. Simons and Bearn [7] presented evidence that ceruloplasmin was comprised of a and fl chains of tool. wts. 15 900 and 58 900 and Nterminal residues of valine and lysine, respectively. The study described here was undertaken to examine the ceruloplasmin subunit structure from nephrotic urine, ascites fluid and plasma and to characterize the Nterminal region of the a chain of ceruloplasmin in each. A preliminary ordering of the CNBr fragments of the beta chain is also presented. " Present address: Pan Scientific Corporation, 8132 MacArthur Blvd., Oakland, Calif. 94605, U.S.A.

453 MATERIALS AND METHODS

Ceruloplasmin purification. Ceruloplasmin used in this study was isolated from human ascites fluid from patients with ovarian carcinoma, from urine of a single patient with nephrotic syndrome, and from out-dated frozen human plasma. The procedure for purification of ceruloplasmin was modified from Deutsch et al. [8]. Volumes of 250 ml of either ascites fluid, serum or concentrated urine were dialyzed at 4 °C against 0.03 M sodium acetate buffer, pH 4.7. The insoluble proteins were removed by centrifugation at 2 °C. The supernatant was applied to a 5 x 20 cm DEAE-cellulose column equilibrated with the same buffer. The column was eluted with a stepwise gradient of 0.3 M sodium acetate buffer, pH 5.6, and 0.5 M sodium acetate, pH 5.6. Ceruloplasmin was concentrated in the first protein peak. Ceruloplasmin was concentrated to 10-12 ml by ultrafiltration in dialysis tubing (Union Carbide, size 8). The ceruloplasmin solution was subjected to starch-block electrophoresis [9]. Ceruloplasmin could be visualized as a light blue area in the starch block. The ceruloplasmin solution concentrated by ultrafiltration to 10 ml was dialyzed against 0.05 M Tris/HC1, pH 7.6, and subjected to gel filtration on a column of Sephadex G-200. The purity of each preparation was checked by immunoelectrophoresis [10] with antiserum to whole human serum and by electrophoresis on 6.0 polyacrylamide gels visualized by protein stain [11] or oxidase stain [3]. Ceruloplasmin a-chain isolation. Purified ceruloplasmin was reduced and alkylated according to the procedure of Gordon et al. [12]. Reduced and alkylated ceruloplasmin was pumped onto a Sephadex G-200 column. The elution profile of reduced and alkylated ceruloplasmin is shown in Fig. 1. Tubes containing a chain and /3 chain were p3oled separately, dialyzed exhaustively against 10 ~ acetic acid, and lyophilized. Ceruloplasmin a-chain analysis. Molecular weight determination: An estimation of the molecular weight of ceruloplasmin a chain was made by the method of Andrews [13] on a guanidine Sephadex G-200 column, with molecular weight references of al chain (9300), the a2 chain (16 100), and the/3 chain (42 600) of haptoglobin. Molecular weight estimation was also made by the sodium dodecyl sulfate (SDS)polyacrylamide gel technique modified by Shapiro et al. [14]. Gels were stained with 0.01~ Coomassie Brilliant Blue in 10~ acetic acid. Reference proteins included human immunoglobulin G, albumin, haptoglobin/3 chain, and haptoglobin a2 chain. N-Terminal amino acid determination: Determination of the N-terminal amino acid was performed with 1-dimethylaminonaphthalene-5-sulphonyl (dansyl) chloride technique of Morse and Horecker [15] on polyamide layers (Cheng Chin Trading Co., Taipei, Taiwan). Solvent systems 1 and 2 described by Woods and Wang [16] were used. Amino acid analysis. Aliquots, representing 6.67 nmol of ceruloplasmin a chain, were lyophilized in vacuum hydrolysis tubes. After adding 0.5 ml 6 M HC1 to each sample, the tubes were evacuated and flushed twice with nitrogen before they were sealed under a final high vacuum. Hydrolysis was carried out at 110 °C for 18, 24, 50 and 72 h. Analyses were performed on a Beckman Model 116 amino acid analyzer modified for analysis in the 2-15 nmol range for each amino acid. Duplicate analyses were run on each sample, with a total of four analyses for each timed period.

454

CNBr cleavage of ceruloplasmin a chain: CNBr fragment isolation. 2 #mol (32 mg) of ceruloplasmin a chain were cleaved with CNBr as described by Gross [17]. The CNBr fragments were dissolved in 30 ~ acetic acid and separated on a Sephadex G-50 column, equilibrated with 30 ~ acetic acid. Approx. 6 ~ of the effluent stream was diverted to an automatic peptide analyzer for alkaline hydrolysis and reaction with ninhydrin [18]. Fragments were pooled and labeled with Roman numerals according to their elution position from Sephadex G-50. Aliquots of 100 #1 were taken from each pooled CNBr fraction for amino acid analysis. Amino acid compositions were based on the recovery of homoserine lactone being unity. Automated sequence determination of the N-terminal region of ceruloplasmin a chain. The sequence of the amino-terminal region of ceruloplasmin a chain was determined by automated sequence analysis [19] in a Beckman Sequencer, Model 890. The Beckman Quadrol Double Cleavage Program D was used. Phenylthiazolinone amino acid derivatives were removed from the sequencer and dissolved in ethyl acetate. 3 0 ~ of each sample was taken for conversion to phenylthiohydantoin derivatives for gas chromatographic analysis. The remaining 70 ~ was used for amino acid analysis after conversion to parent amino acids by the hydriodic acid method of hydrolysis [20]. Gas chromatography analysis of the phenylthiohydantoin-amino acids was by the single-column method of Pisano and Bronzert [21, 22]. RESULTS AND DISCUSSION

Ceruloplasm& purification and a chain isolation The purification of ceruloplasmin from three different human sources (serum, ascites and urine) yielded identical results. Eight ceruloplasmin a chain preparations were used in this study. Table I documents the experiments in which each preparation was used. Before reduction and alkylation ceruloplasmin was eluted on gel filtration in the molecular weight range of 100 000-200 000. After reduction and alkylation, ceruloplasmin a and fl chains were completely separated on the guanidine/HC1 Sephadex G-200 column (Fig. 1). Elution profiles were identical for ceruloplasmin derived from urine, ascites fluid, or plasma. The profile is similar to that observed by Simons and Bearn [7].

TABLE I PROCEDURES PERFORMED ON CERULOPLASMIN ct CHAIN PREPARATIONS Procedure

Amino acid analysis N-Terminal analysis N-Terminal sequence CNBr reduction

Urine

Ascitesfluid

Plasma

I

II

III

IV

V

VI

x ×

x x

x

x

x x

x

x

x

x ×

VII

VIII

× x

455

Amino acid analysis Table II presents the a m i n o acid c o m p o s i t i o n o f the c e r u l o p l a s m i n a chain f r o m p r e p a r a t i o n V. The a chain analysis is c o m p a r e d to the c o m p o s i t i o n o f an a-fl s u b u n i t as d e t e r m i n e d by K a s p e r a n d D e u t s c h [5]. The a chain c o n t a i n e d 153 a m i n o acid residues, excluding t r y p t o p h a n . A m i n o acid analyses o f c e r u l o p l a s m i n a chain f r o m urine (Prep. I), ascites fluid (Preps. I I - I V ) , and p l a s m a (Preps. V - V I ) were identical, within the technical r e p r o d u c i b i l i t y o f the a m i n o acid analyzer. T i m e d hydrolysis analysis was p e r f o r m e d only on Prep. V. Present in the a chain are a total o f 12 lysine and arginine residues. A t least 13 tryptic p e p t i d e s could result from trypsin d i g e s t i o n o f the a chain. Five m e t h i o n i n e residues were d e t e r m i n e d for the a chain. The C N B r r e a c t i o n with the methionine residues w o u l d b r e a k the a chain into six fragments. The a chain c o n t a i n e d only one half-cystinyl residue, which, in the native c e r u l o p l a s m i n molecule, m u s t be connected t h r o u g h a disulfide linkage to the fl chain since the chains can be s e p a r a t e d after r e d u c t i o n and alkylation. TABLE II AMINO ACID COMPOSITION OF CERULOPLASMIN a CHAIN N.D., not determined. Summary of timed hydrolysesa

Lysine Histidine Arginine CM-Cysteine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Tryptophan

18 h

24 h

50 h

72 h

22.4 30.0 16.8 3.5 N.D. 33.4 24.6 ~ 56.4 20.0 43.0 15.1 34.0 16.4 23.0 40.6 16.4 31.9

21.8 28.1 15.3 4.4 64.4 30.6 21.5 49.6 19.9 36.5 13.6 29.1 14.4 20.8 37.2 14.9 28.3

21.1 26.9 15.0 4.6 65.9 29.8 18.5 45.3 16.6 35.9 12.8 27.2 12.9 19.7 35.1 15.5 26.9

19.6 25.8 14.8 4.4 58.5 28.4 16.2 45.0 15.0 33.9 12.3 24.6 12.0 17.4 31.5 14.4 24.8

Residues/ a chain

Residues/ ct-fl¢

Residues/ fl chain

7 9 5 1 21 10 8 17 7 13 5 10 5 7 13 5 10 ÷d

34 22 14 8 78 43 35 65 29 43 28 34 13 28 40 36 27

27 13 9 7 57 33 27 48 22 30 23 24 8 21 27 31 17

a Values represent averaged nmol recoveries of 4 analyses, 2 analyses per duplicate sample of 6.67 nM ceruloplasmin u chain. b Corrected 6 ~ for 18-h destruction in 6 N HCI, 110 °C. c Kasper, C. B. and H. F. Deutsch (1963) J. Biol. Chem. 238; 2325. d Ehrlich stain.

Ceruloplasmin a chain analysis Molecular weight determination. T h e m o l e c u l a r weight o f c e r u l o p l a s m i n a chain was a p p r o x . 16 000 as d e t e r m i n e d b y gel filtration on the guanidine/HC1 S e p h a d e x G-200 column. The a b s o r b a n c e p e a k for c e r u l o p l a s m i n a chain, fraction D

456 (Fig. 1), was at 215 ml effluent volume, while haptoglobin a2 chain (16 100 mol. wt.) emerged at 210 ml effluent volume. The molecular weight by gel filtration was consistent with the 15 900 determined by Simons and Bearn [7]. Employing SDSpolyacrylamide gel electrophoresis the molecular weight of ceruloplasmin a chain was estimated at 15 000. The molecular weights determined for fractions A, B, and C were 122 000, 58 000, and 30 000, respectively.

A I

B

C

II

I

I

D I

I

I

0.5

~

0.4 0.3

~,

o.2

i

0.1 0,0

_

i

i

i

i

i

i

i

i

100

120

140

160

180

200

220

240

E F F L U E N T (ML)

Fig.. 1 Elution profile of reduced and alkylated ceruloplasmin from a Sephadex G-200 column, 2.5 × 60 cm, equilibrated with 5 M guanidine/HCl/0.05 M Tris/HCl, pH 7.75. A./3 chain dimer. B. fl chain. C. a chain dimer. D. cl chain.

N-Terminal amino acid determination. The N-terminal amino acids were analyzed on chain preparations I, II, and V and found to be lysine for the/3 chain (fractions A and B) and valine for the a chain (fractions C and D). The amino acids are identical to those found in the separated ceruloplasmin chains [7] and to those found in native ceruloplasmin [23]. Furthermore, these findings substantiate the possibility that fraction A is a dimer of fraction B and that fraction C is a dimer of fraction D. CNBr fragment isolation and analysis Although six CNBr fragments were expected for the ceruloplasmin a chain based on the methionine concentration, eight fragments were eluted from the Sephadex G-50 column. The fragments were labeled according to their elution position as shown in Fig. 2. Fragments I and II contained methionine residues which were not cleaved by CNBr. The N-terminal amino acid for each fragment, determined by the dansyl chloride technique [15], were CNBr-I, glycine; CNBr-II, valine; CNBr-III, valine; CNBr-IV, glycine; CNBr-V, phenylalanine; CNBr-VI, leucine; CNBr-VII, glutamic acid or glutamine; and CNBr-VIII, phenylalanine. Of the six fragments which contained homoserine lactone (CNBr I I I - V I I I ) , CNBr-III was the only fragment with a valyl amino-terminus. Therefore, CNBr-III is most probably the Nterminal CNBr fragment. CNBr-VII is the C-terminal fragment since it contained neither methionine nor homoserine lactone. With the N-terminal determination and the results of the amino acid corn-

457

0.4

0,3

i

r

0.2

=~

i

VIII VII

0.1

i

150

I

i

180

240

210

i

i

i

i

270

300

330

360

i

i

390

420

EFFLUENT(ml)

Fig. 2. Elution profile of ceruloplasmin a chain CNBr fragments from a Sephadex G-50 column, 2.5 × 120 cm, equilibrated with 30~ acetic acid. position for each fragment, it was possible to position the fragments in a presumed alignment in the native chain. Two of the fragments resulted from incomplete cleavage by CNBr. The amino acid composition gave sufficient information as to which smaller fragments comprised the larger ones. Fig. 3 is the diagram of the CNBr fragment alignment. Val

NH3-

Phe

I

III

I-

Phil I Gly V

it

Leu IV

I

GIx Vl

I

-COOH

Vll I

Fig. 3. Sequence alignment of the CNBr fragments of the ceruloplasmin a chain. N-terminal amino acids are indicated above each fragment.

Amino acid analysis of CNBr fragments. The amino acid composition of the CNBr fragments is shown in Table III. Composition data for CNBr fragments II, Ill, IV, V, VI and VIII were calculated on the basis of homoserine lactone recovery equal to 0.54 residue per fragment, a correction factor empirically derived by Barnett [18]. The composition of CNBr-VII, the C-terminal fragment, was calculated on the basis of 1.00 lysine residue per fragment. The amino acid analysis of CNBr-I did not yield a measurable quantity of homoserine lactone. Its amino acid composition was determined after trial and error inspection to reveal the nmol value which represented one amino acid per fragment I and gave the nearest integral recoveries for every amino acid. CNBr fragments I and II contained 1.0 and 0.6 methionine residues, respectively. CNBr fragment alignment The incomplete cleavage of a chain preparations by CNBr was used to advantage. The complete alignment of the CNBr fragments could be deduced with

458 TABLE III A M I N O ACID COMPOSITION OF CNBr F R A G M E N T S OF CERULOPLASM1N a CHAIN Amino acid

I

11

llI

IV

V

Vl

VII

Lysine Histidine Arginine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Half-cystine ~ Valine Homoserine lactone Methionine Isoleucine Leucine Tyrosine Phenylalanine Tryptophan b

5 3 4 9 6 5 11 4 7 4 -5 -2 5 4 4 4 .

4 6 3 9 5 4 7 4 5 2 1 4

3 2 2 8 2 3 6 2 1 1 -2

3 2 2 3 3 2 4 2 3 1

1 1 1 4 2 1 3 1 3 1

1

3

-4 1 1 1 1 1 2 3 1 1 1

1

2 2 2 3 1 --1

1

1

1

1

1

--

Total

83

73

45

39

N-terminus

Gly

Val

Val

Gly

1 4 6 2 5 .

-

.

.

2 4 2 4 .

.

.

2 2 3 3 .

.

1 2 1 1

1 2 1 1

25

25

15

11

Phe

Leu

Glu

Phe

.

.

1 -2 1

. 2 l -

2 2 -. 3

.

Vlll

.

1 ÷

a As carboxymethylcysteine. b As Ehrlich stain. d a t a f r o m a m i n o a c i d c o m p o s i t i o n , N - t e r m i n a l analyses, a n d p a r t i a l a u t o m a t e d s e q u e n c e analysis o f s o m e f r a g m e n t s . C N B r - I I I was c o n f i r m e d as the N - t e r m i n a l C N B r f r a g m e n t by a u t o m a t e d s e q u e n c e analysis since t h e first t w e l v e r e s i d u e s of C N B r - I I I w e r e i d e n t i c a l to t h e a m i n o - t e r m i n a l s e q u e n c e o f i n t a c t a chain. C N B r - I I c o n t a i n e d a m e t h i o n i n e r e s i d u e as d e t e r m i n e d by a m i n o a c i d analysis. T h i s i n d i c a t e d t h a t it was c o m p r i s e d o f t w o C N B r f r a g m e n t s . C N B r - I I I , the N t e r m i n a l f r a g m e n t , was o n e o f t h e s u s p e c t e d c o m p o n e n t s o f C N B r - I I , since e a c h c o n t a i n e d a v a l i n e N - t e r m i n u s . T h e s e q u e n c e o f t h e first six r e s i d u e s o f C N B r - I I was d e t e r m i n e d w i t h t h e S e q u e n c e r a n d was i d e n t i c a l to the a m i n o - t e r m i n a l s e q u e n c e of C N B r - I I I a n d t h e i n t a c t a chain. T h e o t h e r c o m p o n e n t o f C N B r - I I is C N B r - V , since b o t h C N B r - I I a n d C N B r - V c o n t a i n e d a c a r b o x y m e t h y l c y s t e i n e residue. A m i n o a c i d analysis d e m o n s t r a t e d o n l y a single h a l f - c y s t i n e p e r a chain. F u r t h e r m o r e , t h e t o t a l a m i n o a c i d c o m p o s i t i o n o f C N B r - I I I a n d C N B r - V is v e r y s i m i l a r to t h a t o f C N B r - I I , as s h o w n in T a b l e IV. T h e r e f o r e , C N B r - I I is t h o u g h t to be c o m p r i s e d o f C N B r - I I I and CNBr-V. By a m i n o a c i d analysis, C N B r - I c o n t a i n e d o n e m e t h i o n i n e r e s i d u e w h i c h i n d i c a t e d t h a t , like C N B r - I I , it was c o m p r i s e d o f t w o c y a n o g e n b r o m i d e f r a g m e n t s . H o w e v e r , as w i l l be d e m o n s t r a t e d later, C N B r - I c o n t a i n e d t h r e e f r a g m e n t s . T h e p r e s e n c e in C N B r - I o f t w o f r a g m e n t s , C N B r - I V a n d C N B r - V I I , is i m m e d i a t e l y suggested. C N B r - I a n d C N B r - I V h a v e t h e s a m e N - t e r m i n a l a m i n o acid, glycine. M o r e o v e r , t h e first six r e s i d u e s o f b o t h C N B r - I a n d C N B r - I V w e r e a n a l y z e d b y a u t o m a t i c

459 TABLE IV COMPARISON OF THE AMINO ACID COMPOSITIONS OF CNBr FRAGMENTS I AND II AND THEIR COMPONENT FRAGMENTS

Lysine Histidine Arginine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Half-cystine" Valine Methionine~ Isoleucine Leucine Tyrosine Phenylalanine Total

III

V

Total

II

IV

VI

VII

Total

I

3 2 2 8 2 3 6 2 1 1 -2 1 2 4 2 4 45

-4 1 1 1 1 1 2 3 1 1 1 1 2 2 3 25

3 6 3 9 3 4 7 4 4 2 1 3 2 4 6 2 7 70

4 6 3 9 5 4 7 4 5 2 1 4 2 4 6 2 5 73

3 2 2 3 3 2 4 2 3 1

1 1 1 4 2 1 3 1 3 1 1 1 1 2 1 1 25

1 --2 2 2 3 -1 --1 --

5 3 3 9 7 5 10 3 7 2 0 5 2 3 6 5 4 79

5 3 4 9 6 5 11 4 7 4 0 5 2 5 5 4 4 83

3 1 2 2 3 3 39

2 1 -15

* As carboxymethylcysteine. b As methionine or homoserine lactone. sequence analysis a n d f o u n d to be identical. The absence of homoserine lactone in C N B r - I indicates that C N B r - I also contains CNBr-VII, the C - t e r m i n a l fragment. However, the total a m i n o acid c o m p o s i t i o n of C N B r - I V a n d C N B r - V I I is insufficient to a c c o u n t for the c o m p o s i t i o n of CNBr-I, thereby suggesting that a n o t h e r fragment is also present. The molecular weight o b t a i n e d for C N B r - I by gel filtration o n Sephadex G-50 w o u l d also suggest the presence of a n a d d i t i o n a l fragment besides C N B r - I V and CNBr-VII. C N B r - V I I I is n o t present in CNBr-I, or any larger fragment, since it is the only f r a g m e n t which contains t r y p t o p h a n . For the c o m p o s i t i o n of C N B r - I only one c o m b i n a t i o n o f fragments is possible, CNBr-IV, CNBr-VI, a n d CNBr-VII. The total a m i n o acid c o m p o s i t i o n of these three fragments is very similar to the c o m p o s i t i o n o f C N B r - I (Table IV). Therefore, it was concluded that C N B r - I is comprised of CNBr-IV, C N B r - V I , a n d C N B r - V I I . The finding of b u t one m e t h i o n i n e residue in C N B r - I after acid hydrolysis is neither u n u s u a l n o r unexpected. The failure to o b t a i n q u a n t i t a t i v e yields of m e t h i o n i n e after acid hydrolysis of proteins is well k n o w n , especially if all oxygen is n o t excluded f r o m the hydrolysis vessel. C N B r - I I also lost m e t h i o n i n e d u r i n g acid hydrolysis, for the analysis revealed 0.6 residue per fragment. Depicted in Fig. 3 is the tentative a l i g n m e n t o f the C N B r peptides of the c e r u l o p l a s m i n a chain.

Amino-terminal sequence of the ceruloplasmin a chain A u t o m a t e d a m i n o acid sequence analysis was performed o n a chain prepar a t i o n s III, IV, VI, a n d VIII. Approx. 500 n m o l (8 mg) o f a c h a i n were used in each

460 sequencer run. Automated sequence analysis of a chain obtained from preparation VIII established the N-terminal 21 residues, as shown below: Val-Phe-Asx-Pro-Arg-Arg-Lys-Leu-Glx-Phe-Ala-Leu-Leu-Phe-Leu-Val-Asx-Glx-Asx-Glx The sequence of the N-terminal 21 residues was compared with published sequences of various proteins [24] in search of homology. No readily detectable homology was noted. The N-terminal sequence of the human ceruloplasmin a chain is reported here for the first time and represents consistent results from preparations obtained from serum and ascites fluid. The preliminary ordering of the CNBr peptides were made possible by incomplete cleavage resulting in overlapping fragments in addition to the six expected fragments. Subunit characterization of ceruloplasmin confirms the work of Simons and Bearn [7] in which two types of polypeptide chain pairs were found to comprise the ceruloplasmin molecule. Ryd6n [25] has presented evidence that the molecular weight of ceruloplasmin prepared from fresh serum was that of a single chain of 120 000 daltons. The results of our study do not have bearing on Ryd6n's conclusions [25] since the material and methods used for preparing ceruloplasmin were not the same. Our results do, however, indicate the consistent existence of two unique chains comprising human ceruloplasmin prepared from three different sources. ACKNOWLEDGMENTS We thank Dr. Julian Smith, Department of Gynecology, M. D. Anderson Hospital, Houston, for ascites fluid, Drs. Tong-Ho Lee and Don R. Barnett for serum fractions and the American Red Cross for ceruloplasmin fractions. This research was supported in part by a grant from the Robert A. Welch Foundation, H-378, and a grant from the National Institutes of Health, H D 03321. Excellent technical help was contributed by Mr. Horace Kelso and Mr. Billy Touchstone. REFERENCES 1 Holmberg, C. G. and Laurell, C. B. (1948) Acta Chem. Scand. 2, 550-556 2 Shreffler, D. C., Brewer, G. J., Gall, J. C. and Honeyman, M. S. (1967) Biochem. Genet. 1, 101115 3 McCombs, M. L. and Bowman, B. H. (1969) Tex. Rep. Biol. Med. 27, 769-772 4 Beam, A. G. and Cleve, H. (1966) in The Metabolic Basis of Inherited Disease (Stanbury, J. B., Wyngaarden, J. B. and Fredrickson, D. S., eds.), 2nd edn., pp. 1321-1342, McGraw-Hill, New York 5 Kasper, C. B. and Deutsch, H. F. (1963) J. Biol. Chem. 238, 2343-2350 6 Poillon, W. N. and Beam, A. G. (1966) Biochim. Biophys. Acta 127, 407-427 7 Simons, K. and Beam, A. G. (1969) Biochim. Biophys. Acta 175, 260-270 8 Deutsch, H. F., Kasper, C. B. and Walsh, D. A. (1962) Arch. Biochem. Biophys. 99, 132-135 9 Bowman, B. H. and Beam, A. G. (1965) Proc. Natl. Acad. Sci. U.S. 53, 722-729 10 Hirschfeld, J. (1960) Sci. Tools 7, 18-20 11 Raymond, S. and Weintraub, L. (1959) Science 130, 711 12 Gordon, S., Cleve, H. and Beam, A. G. (1968) Proc. Soc. Exp. Biol. Med. 127, 52-59 13 Andrews, P. (1965) Biochem. J. 96, 595-606 14 Shapiro, A. L., Vinuela, E. and Maizel, Jr., J. V. (1967) Biochem. Biophys. Res. Comm. 28, 815820

461 15 Morse, D. and Horecker, B. L. (1966) Anal. Biochem. 14, 429-433 16 Woods, K. R. and Wang, K.-T. (1967) Biochim. Biophys. Acta 133, 369-370 17 Gross, E. (1969) in Methods in Enzymology (Hirs, C. H. W., ed.), Vol. XI, pp. 238-255, Academic Press, New York 18 Barnett, D. R. (1969) Isolation of and Partial Chemical Characterization of Some Tryptic Peptides of the Beta Chain of Human Haptoglobin, Thesis, The University of Texas Medical Branch, Galveston, Texas. 19 Edman, P. and Begg, G. (1967) Eur. J. Biochem. 1, 80-91 20 Smithies, O., Gibson, D., Fanning, E. M., Goodfliesh, R. M., Gilman, J. G. and Ballantyne, D. L. (1971) Biochemistry 10, 4912-4921 21 Pisano, J. J. and Bronzert, T. J. (1969) J. Biol. Chem. 244, 5597-5607 22 Pisano, J. J. and Bronzert, T. J. (1970) Fed. Proc. 29, 916 23 Kasper, C. B. (1967) Biochemistry 6, 3185-3196 24 Dayhoff, M. O. (ed.), (1969) in Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, Silver Spring, Md. 25 Ryd6n, L. (1972) Eur. J. Biochem. 26, 380-386

Biochemical studies on human ceruloplasmin.

Biochimica et Biophysica Acta, 434 (1976) 452--461 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands BBA 37357 BIOCHE...
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