Purification of human transcobalamin II-cyanocobalamin by affinity chromatography using thermolabile immobilization of cyanocobalamin

40

Biochimica et Biophysica Acta, 579 (1979) 40--51

@)Elsevier/North-Holland Biomedical Press

BBA 38224 PURIFICATION OF HUMAN TRANSCOBALAMIN II-CYANOCOBALAMIN BY A F F I N I T Y CHROMATOGRAPHY USING THERMOLABILE IMMOBILIZATION OF CYANOCOBALAMIN

J. LINDEMANS,J. VAN KAPEL and J. ABELS Institute of Hematology, Erasmus University, Rotterdam (The Netherlands)

(Received December 7th, 1978) Key words: Transcobalamin H; Cyanocobalamin; Isoprotein; Vitamin B-12-binding protein; (Affinity chromatography)

Summary Transcobalamin II-cyanocobalamin was isolated from Cohn fraction III of pooled human plasma by affinity chromatography on cyanocobalamin-Sepharose and some conventional separation methods. The affinity ligand cyanocobalamin was coupled to AH-Sepharose by a thermolabile linkage. The unsaturated binding protein was absorbed at 4°C and eluted from the column at 37°C as transcobalamin II-cyanocobalamin complex. The final preparation had a specific cyanocobalamin-binding capacity of 0.98 mol cyanocobalamin/mol transcobalamin II, the yield was 55% and the purification index amounted to 1.1 • 1 0 6 . In dodecyl sulphate polyacrylamide gel electrophoresis one major protein band was observed at a molecular weight of 37 000 and a faint band at a molecular weight of 29 000. In polyacrylamide gel isoelectric focusing the pure preparation turned out to be heterogeneous with isoelectric points ranging from pH 6.2 to 6.8, possibly by the occurrence of isoproteins.

Introduction The isolation of the vitamin B-12-binding protein transcobalamin II, is hampered by the very low concentration of this protein in blood plasma. Puutula and Gr~isbeck [1] obtained a one-million fold purification with conventional methods, starting with Cohn fraction III from human plasma. Their final a m o u n t of transcobalamin II was about 0.3 mg of protein material (yield 2.2%). Allen and Majerus [2] used affinity chromatography and reached a

41 2-million fold purification with a yield of 31%. A modification of this m e t h o d was described by Savage et al. [3], who obtained a purification index o f 3.15 • l 0 s and a yield of 46%. Nex¢ described affinity chromatography for the isola,tion of cobalophilines and intrinsic factor [4] and rabbit transcobalamin II [5], in which denaturation of the binding proteins by guanidine as used by Allen et al. [ 2], was avoided. Hydroxycobalanlin was attached to a solid matrix by a temperature-labile linkage with 3,3'-diaminodipropylamine coupled to Sepharose 4B. Cobalamin-binding proteins were retained by the hydroxocobalamin-Sepharose at 4°C and were eluted from the column at 37°C carrying thee hydroxocobalamin with them. The latter m e t h o d proved to be inadequate because of the high a m o u n t of other plasma proteins which were absorbed by the hydroxocobalamin-Sepharose column. In this study a simple, effective modification of the m e t h o d of Nex~b is presented, in which hydroxocobalamin, attached to the Sepharose, is converted to cyanocobalamin. The advantages of this procedure for the purification of human transcobalamin II are demonstrated. Materials and Methods Chromatography materials were obtained from Pharmacia Fine Chemicals, Uppsala, Sweden; [ s7Co] cyanocobalamin, with specific activities ranging from 180 to 220 Ci/g, was obtained from Philips-Duphar, Petten, Holland or from the Radiochemical Centre, Amersham, England. Hydroxocobalamin was purchased from Sigma, St. Louis, U.S.A. Cohn fraction III from pooled human plasma was kindly supplied by the Central Laboratory of the Netherlands Bloodtransfusion Service. All other reagents were of analytical grade purity.

Preparation of the affinity column 1 g AH-Sepharose 4B was swollen in 200 ml 0.5 M NaC1 for 15 min and washed with another 300 ml 0.5 M NaC1. The swollen Sepharose was directly incubated with hydroxocobalamin in about 2 ml 0.1 M NaHCO3, 0.5 M NaC1, pH 8.2. The a m o u n t of hydroxocobalamin, which can be coupled to 1 g AH-Sepharose ranges from 3 up to 1000 nmol. The coupling efficiency was generally 20% at all concentrations of hydroxocobalamin. About 75% of the immobilized cobalamin was available for the absorption of cobalamine-binding proteins. For the detection o f transcobalamin II in further purification steps the Sepharose was incubated with hydroxo [ STCo] cobalamin of an appropriate specific activity. Hydroxo[SVCo]cobalamin was prepared from cyano[STCo]cobalamin according to Mahoney et al. [6] by exposure to a 60 W tungsten lamp at a distance of 15 cm for 18 h in 8 mM HC1. The cuvette was thermostatistically chilled to 4°C. The Sepharose suspension was incubated with hydroxo[SVCo]cobalamin for about 18 h at room temperature in a rotary mixed, cooled on ice for 30 min and poured into a precooled glass column (1.5 × 5.5 cm). The Sepharose was washed with 50 ml 20 mM sodium phosphate, 1 M NaC1, 10 mM KCN, buffer pH 8.2, by which the hydroxo[S7Co]cobalamin was converted to dicyano[STCo]cobalamin. The excess of [STCo]cobalamin in the effluent was

42 measured spectrophotometrically to establish the coupling efficiency. KCN was removed with 200 ml of the phosphate-NaC1 buffer without KCN and at the same time the dicyanocobalamin was converted to monocyanocobalamin.

Purification o f human transcobalamin II A representative purification procedure is described in detail. 80 1 of 10 mM sodium phosphate, 0.1 M NaC1, buffer pH 5.2 was added to 19 kg of Cohn fraction III, and the suspension was stirred continuously for 12 h at 4°C. Insoluble material was allowed to settle and the supernatant was filtered over nylon gauze (63 pm). The filtrate was stirred for another 12 h with 100 g of dry CM-Sephadex C-50. The Sephadex was collected by filtration over nylon gauze and washed first on the gauze and afterwards on a glass filter with 10 1 of 10 mM sodium phosphate, 0.05 M NaC1, buffer pH 5.2. Transcobalamin II was eluted from the Sephadex by stirring the suspension in 1.5 1 of 0.2 M Tris, 1 M NaC1 pH 8.2. The Sephadex was washed with another 400 ml Tris buffer. The total filtrate, about 3300 ml, was centrifuged for 2 h at 105 000 X g. The supernatant was passed through filter paper (S&S 5893) to remove floating lipid material. The binding capacity for cobalamin was measured and the total volume was applied to an affinity column which was prepared as described above with 1 g AH-Sepharose, 1 mg hydroxocobalamin and 10 pCi hydroxo[STCo]cobalamin. The total volume was 3 ml and the column contained 210 pg of immobilized cyanocobalamin with a specific activity of 19 175 cpm/ pg. The flow rate was 100 ml/h. The column was washed with 400 ml cold 20 mM sodium phosphate, 1 M NaC1, 0.02% NAN3, pH 7.2 and during this washing the Sepharose was resuspended and allowed to settle in order to remove particulate material. Elution of the transcobalamin II-cyanocobalamin took place after incubation of the column for 10 h at 37°C and washing with about 5 ml of warm phosphate/NaCl buffer. After another 10 h at 37°C the remainder of the transcobalamin II-cyanocobalamin and free cobalamin were eluted with 4 ml of warm buffer. The combined eluates were applied to a Sephacryl S 200 column (2.6 X 90 cm) and elution t o o k place with the same phosphate-NaC1 buffer. The fractions containing transeobalamin II-cyano[STCo]cobalamin were pooled and concentrated by ultrafiltration on a YM 10 membrane (Amicon). The concentrate was diluted with 50 ml 50 mM Tris-HC1, pH 8.25, and concentrated again. This procedure was repeated twice in preparation for the next purification step, the DEAE-Sepharose ion exchange chromatography. The final concentrate was put on a 0.9 × 4 cm column of DEAE-Sepharose Tris-HC1 and eluted with a linear gradient from 0 to 225 mM NaC1 in 50 mM Tris/HC1 pH 8.25 with a total volume of 200 ml. The concentrated fractions containing transcobalamin II-cyanocobalamin were again brought in 50 mM Tris-HC1 pH 8.25 and applied to a second DEAESepharose column. A concave gradient from 0--150 mM NaC1 was created by a two-pump system in which the efflux from the mixing chamber was 14 times the influx of the high salt solution into the mixing chamber [7]. The total volume of the gradient was 200 ml. The fractions containing transcobalamin II-cyanocobalamin were pooled and concentrated on a YM-10 ultrafiltration membrane. The different steps of purification were followed by measurements of the

43 cyanocobalamin-binding capacity or the cyanocobalamin content and the amount of protein in order to establish the specific cyanocobalamin-binding c~ipacity of the isolated material.

Determination of cyanocobalamin-binding capacity Solutions of cyanocobalamin were standardized spectrophotometrically (e lcm 3 6 8 n m = 30 800) in 0.1 M NaOH, 10 mM KCN. The cyanocobalamin-binding capacity was measured according to Gottlieb et al. [8]. A mixture of 0.02 pCi cyano[STCo]cobalamin with an amount of unlabeled cyanocobalamin equal to 1.5--2 times the expected binding capacity was incubated in 50 mM sodium phosphate, 0 1 5 M NaC1, 0.6 mM KCN pH 7.4 with the appropriate amount of sample for 15 min in a total volume of 1.2 ml. 0.3 ml of hemoglobin-dextran coated charcoal (0.05% Hb, 0.25% dextran in 5% charcoal s u s p e n s i o n ) w a s added to separate the free from the b o u n d cyanocobalamin. After centrifugation the b o u n d vitamin was measured in 1 ml of the supernatant and the radioactivity was compared with a 100% value, obtained without charcoal absorption. In the CM-Sephadex eluate, charcoal absorption was inadequate and the binding capacity was determined by means of gelfiltration. The sample was mixed with an excess of radioactive cyanocobalamin of a suitable specific activity, incubated, applied to a Sephadex G50 column (1.6 X 35 cm) and eluted with 20 mM sodium phosphate, 1 M NaC1 buffer, pH 7.4. The radioactivity in the 2-ml fractions was measured and the partition between free and b o u n d cyanocobalamin was used to calculate the cobalamin binding capacity of the sample.

Determination of cobalamin Cobalamin concentrations were determined b y a modification of the radioassay m e t h o d of Lau et al. [9]. Cyanocobalamin solutions were standardized as described above and both the standard solutions and the samples containing transcobalamin II were boiled in cobalamin-free serum, which was diluted with 4 volumes of 0.044 M L-glutamic acid, 0.6 mM KCN buffer, pH 4.1. Hog intrinsic factor was used as the binding protein and separation of b o u n d and free cobalamin was carried o u t with Hb-dextran coated charcoal.

Further analytical methods Protein was measured according to Lowry et al. [10], using bovine serum albumin or human serum albumin protein standard (Kabi, Stockholm) as a reference. Both standards gave identical results. Dodecyl sulphate polyacrylamide gel electrophoresis was carried o u t essentially according to Fairbanks et al. [11]. Electrophoresis was performed at 2.5 mA/gel until the tracking dye, pyronine Y, reached the b o t t o m of the electrophoresis tube. The gels were fixed and stained with Coomassie brilliant blue. Thin layer polyacrylamide gel isoelectric focusing was performed with a modification of the m e t h o d of Vesterberg [12]. The gel was composed of 13.5 ml 30.5% acrylamide, 15 ml 1% N,N'-methylene bisacrylamide, 27 ml H~O, 0.65 g Triton X-100, 7.5 g sucrose, 1.0 ml Ampholine pH 5--7, 1.0 ml Ampholine pH 6--8, 1 ml Ampholine pH 7--9, 1 ml 0.0055% riboflavin and polymeri-

44

zation was induced by illumination with a daylight tube lamp for 45 min at a distance of 15 cm. The samples were applied in small (5 × 10 × 1 mm) basins in the gel. The cathode solution was 0.1 M NaOH, the anode solution 0.1 M H3PO4. Focusing t o o k place on a LKB multiphor apparatus for 90 min at 4°C. At the end of the focusing time the pH gradient was measured with an Ingold Surface electrode t y p e 104033.104. The proteins were fixed in 50% methanol/ 7.5% trichloroacetic acid and stained with Coomassie brilliant blue. Results

Preliminary observations Our improvement of the affinity chromatography technique, was based on the results of purifications of transcobalamin II from rat plasma with the unmodified m e t h o d of Nex~b [4]. When rat plasma with a total specific cyanocobalamin-binding capacity of 0.16 nmol was applied to an hydroxocobalaminSepharose column, 59 nmol of hydroxocobalamin b o u n d to protein were recovered in the 3 7 a c eluate. This means that in addition to transcobalamin II a large amount of other plasma proteins had been retained by the hydroxocobalamin in the column. Because it is known that for instance serum albumin readily binds hydroxocobalamin b u t not cyanocobalamin [13], a cyanocobalamin-Sepharose column was prepared from hydroxocobalamin-Sepharose by successive rinses with a CN--containing and a CN--free buffer, converting hydroxocobalamin to successively dicyanocobalamin and monocyanocobala-

051 0.4

.~ 0.3.

-,,,

250

a;o

y

,;o

,,

5io

6;o

wavelength (nm) F i g , 1. S p e c t r a o f a f f i n i t y c o l u m n e l u a t e s . , hydroxocobalamin from an unconverted column; ...... , dicyanocobalamin from a converted column with CN- in the elution buffer; , cyanocobalamin from a converted column which was previously rinsed with CN- free buffer. All samples were diluted in the respective elution buffers and the same buffer was used as a reference. The spectra were recorded on a Perkin Elmer 124 sPectrophotometer.

45 min. This conversion is illustrated by the absorption spectra of the 37°C eluates in each of the three stages of preparation (Fig. 1). When the rat serum was applied to such a cyanocobalamin-Sepharose column all cyanocobalamin bound to protein in the eluate was bound to transcobalamin II, which means that the non-specific binding to the affinity ligand was completely eliminated.

Purification o f human transcobalamin H With this improved affinity chromatography technique the purification of transcobalamin II was started with Cohn fraction III from human plasma. The total cyanocobalamin-binding capacity of the Coh'n fraction solution was 170 pg. The insoluble material retained some binding capacity, but a second extraction was useless because the yield and the specific binding activity were low. The cyanocobalamin-binding capacity of the first supernatant was defined as 100% for the calculation of the yield of the purification. Using batchwise CM-Sephadex elution the transcobalamin II was recovered from the supernatant in a volume of 3.3 1, with a 17-fold purification and a loss of binding capacity of only 10%. The eluate from the affinity column contained a mixture of free cyanocobalamin and of cyanocobalamin bound to protein. The specific cyanocobalamin binding capacity was calculated from the total amount of protein in the eluate and the amount of protein-bound cobalamin in the eluate of the Sephacryl S 200 column. The elution profile of the Sephacryl S 200 column is given in Fig. 2. The specific binding capacity after gel filtration was 16.3 tzg vitamin B-12/mg protein which suggests a purity of about 50%. Further purification was carried out with DEAE-Sepharose CL-6B ion exchange chromatography and the elution profile with a linear salt gradient is presented in Fig. 3. The shoulder of non-transcobalamin II protein before the transcobalamin II peak was eliminated by DEAE-Sepharose chromatography with a concave salt gradient (Fig. 4). Measurement of protein and vitamin B-12 in the final product gave a specific binding activity of 34.8 pg/mg or 0.98 mol cyanocobalamin per mol tran*co-

radioactivlty cpm 10-3 240-

A280 nm

200-

0,1

160-

'0,06

1208040-

.0,02 J 4O

8O

120

160

200

240

fraction number

Fig. 2. E l u t i o n p r o f i l e o f t h e a f f i n i t y c o l u m n e l u a t e o n a S e p h a c r y l S 2 0 0 c o l u m n (2.6 X 9 0 c m ) equilib r a t e d w i t h 2 0 m M s o d i u m p h o s p h a t e , 1 M NaCI b u f f e r p H 7.2. , A280; ...... 0 S7Co r a d i o a c t i v i t y . F r a c t i o n v o l u m e w a s 2.8 m l and t h e e l u t i o n v e l o c i t y w a s 1 0 m l / h .

46 radioactivity cpm x I0 -3

conductivity mmho

'2°t

f~

i

I ,

1°° 1

8o4

I

6o/1

..~/

=t/1

---" ! 10

20

30

40

50

i

J

', , L/

/

/'"

/ I0. 0.1 A280 8,

\t

0,05

\',

60

4' 2"

70

80

90

100

fraction number F i g . 3. E l u t i o n p a t t e r n o f t h e f i r s t D E A E - S e p h a r o s e C L - 6 B c o l u m n , T h e c o l u m n (0.9 X 4 e m ) w a s equilib r a t e d w i t h 50 m M Tris-HCl p H 8 . 2 5 a n d e l u t i o n t o o k p l a c e w i t h a l i n e a r salt g r a d i e n t f r o m 0 t o 2 2 5 mM NaCI with a volume of 200 ml. ---, A280; ...... , 57Co radioactivity; ..... , conductivity. F r a c t i o n v o l u m e w a s 2 m l and t h e e l u t i o n v e l o c i t y w a s 1 5 . 8 m l / h . '

balamin II (molecular weight transcobalamin II, 38 000), which means that the preparation was almost pure. The purification factor was 1.1 • 106 with a yield of 55%. A summary of the results of the purification procedure is given in Table I.

Characterization of the purified transcobalamin H The pure transcobalamin II-cyanocobalamin was subjected to dodecyl sulphate polyacrylamide gel electrophoresis under reducing and non-reducing conditions (Fig. 5). In electrophoresis without a reducing agent a single major band was observed at a molecular weight of 37 000 and a faint band, less than 5% in densitometric analysis, with a molecular weight of about 29 000. Addition of 1% ~-mercaptoethanol, 10 mM or 50 mM dithiothreitol resulted equally radloacHvHy cpm 10-3

Conductivity mmho

/ /f

ioo,

I ;">..k"

oo.

L.i

...--

,o

10

30

50

°°'

'q

T, t\'I 70

90

110

fracHon number F i g . 4. E l u t i o n p a t t e r n o f t h e s e c o n d D E A E - S e p h a r o s e C L - 6 B c o l u m n . T h e c o l u m n (0.9 × 4 c m ) w a s e q u i llbrated w i t h 50 m M Tris-HC1 p H 8 . 2 5 a n d e l u t i o n t o o k place w i t h a Concave g r a d i e n t f r o m 0 t o 1 5 0 m M NaCI with a total volume of 200 ml. - - , A280; ...... , 57Co radioactivity; ..... , conductivity. F r a c t i o n v o l u m e w a s 2 . 0 m l and t h e e l u t i o n v e l o c i t y 1 5 . 2 m l / h .

I

95 000 3 300 11.5 9.0 7.0 9.0

170 153 120 95.2 91.0 93.0

(~g)

Total cobalaminbinding capacities

II-CYANOCOBALAMIN

Cohn fraction III (solution) CM-Sephadex batchwise elution Aff. chromatography on CN-Cbl-Sepharose S e p h a c r y l $ 2 0 0 gel f i l t r a t i o n DEAE-Sepharose CL 6B linear gradient DEAE-Scpharose CL 6B concave gradient

TRANSCOBALAMIN Volume (ml)

OF HUMAN

Step

PURIFICATION

TABLE

5.36 • 106 0.27 • 106 31 5.84 3.93 2.67

Total protein (mg)

3.17 • 10 -5 5.66 • 10 .4 3.87 16.3 23.2 34.8

/~g/rng

8.9 • 10 -7 0.16 • 10 -4 0.108 0.46 0.65 0.98

tool/tool

Spec. cobalamin-binding capacity

1 18 1.22" 5.14 • 7.32 " 1.10"

10 s l0 s 10 s 106

Purification factor

100 90 71 56 54 55

Yield (%)

48

F i g . 5. D o d e c y l s u l p h a t e p o l y a c r y l a m i d e gel e l e c t r o p h o r e s i s o f 1 2 . 5 # g o f t r a n s c o b a l a m i n n (A a n d B) a n d o f a m i x t u r e o f a l b u m i n , a l d o l a s e a n d m y o g l o b i n (C, 1 0 # g e a c h ) . P r e p a r a t i o n a n d e l e c t r o p h o r e s i s o f t h e s a m p l e s i n A a n d C w e r e c a r r i e d o u t i n t h e p r e s e n c e o f 5 0 m M d i t h i o t h r e i t o l . T h e c h a r a c t e r s a, b , c, d, a n d e indicate the various polypeptides chains with molecular weights of 37 000, 29 000, 24 000, 18 000 and 13 000 respectively.

in a 50% decrease of the a m o u n t of 37 000 molecular weight material, an increase of the 29 000 band and the appearance of some faint bands at 24 000, 18 000 and 13 000. The molecular weights were estimated by comparison'with the relative mobilities of three marker proteins, bovine serum albumin, aldolase and myoglobin. From these data it may be concluded that about 50% of the transcobalamin II dissociates into smaller subunits under reducing conditions. No difference in electrophoresis pattern was observed whether preincubation was carried out for 2 h at 37°C or 3 min at 100°C. Polyacrylamide gel isoelectric focusing of the pure transcobalamin II-cyanocobalamin was initially unsuccessful, because the protein formed large precipitates during the procedure even before it reached the area of its isoelectric point. This phenomenon was probably a result of the poor solubility of transcobalamin II at low ionic strength. The addition of 1% Triton X-10u improved

49 5

~H

5

\. ~

~ ~ii~ ~

~ii! ~ ~i~i!~

\

ii

\

\

~~il~ ii i~i!ii~~I~i~ii~ilii

\ \ \ \ \

\ \

\

4

I

I

5

6

I

pH

7

8

gel i s o e l e c t r i c f o c u s i n g o f 4 5 # g o f pure t r a n s c o b a l a m i n the course of the pH over the gradient from anode to cathode.

Fig. 6. Polyacrylamide

II. The

diagram

indicates

the solubility. Fig. 6 shows an IEF-pattern of the pure transcobalamin II preparation in a pH gradient from 4.9 to 7.4. The protein is concentrated in an area from pH 6.2 to 6.8, in which two double bands around pH 6.30 and 6.45 are visible. Discussion

Transcobalamin II-cyanocobalamin was isolated from Cohn fraction III of pooled human plasma utilizing an affinity chromatography technique. The affinity ligand cyanocobalamin was immobilized by a thermolabile linkage to the solid matrix. This method has the advantage that the dissociation of the binding protein from the solid matrix is effected by a rise in the temperature of the column and not by a strong protein denaturing agent, as used by Allen and Majerus [2], which may damage the protein irreversibly. Weiss et al. [14] e.g. have reported that human intrinsic factor, which was purified by the method of Allen et al. [2] had a decreased affinity for the intestinal receptor, and denaturation of cobalophilin with this method was suggested by Stenman [15].

50 The method, described by Nex¢, made use of immobilized hydroxocobalamin. However, hydroxocobalamin binds to several peptides and proteins [16,17,18] and in our experience this affinity ligand lacked the specificity which is needed for an efficient isolation of cobalamin-binding proteins. It proved to be possible to increase the specificity by conversion of the hydroxocobalamin, attached to the solid matrix, to cyanocobalamin without loss of the thermolabile linkage. Human transcobalamin II from Cohn fraction III was purified to homogeneity by successively CM-Sephadex ion exchange chromatography, affinity chromatography, gelfiltration and DEAE-ion exchange chromatography. The use of isoelectric precipitation for the final purification as was reported by Savage et al. [3] appeared to be irreproducible. Excellent separation of transcobalamin II from contaminating proteins was obtained with the Sephacryl 8-200 column. Another important improvement concerning the yield of the overall procedure was the use of the Amicon YM-10 ultrafiltration membrane for which transcobalamin II has only very low affinity in contrast to UM or PM-type membranes. The final product was virtually pure on the basis of the specific binding activity, which was calculated to be 0.98 mol cyanocobalamin/mol transcobalamin II. A slightly lower specific binding activity, 0.87 mol/mol transcobalamin II, was obtained when the a m o u n t of cyanocobalamin in the final preparation was calculated on the basis of the specific radioactivity of the hydroxocobalamin solution, which had been used for the preparation of the affinity column. A possible explanation for this small discrepancy is, that the hydroxo[STCo] cobalamin, as a result of the conversion by light in an acid environment, contained derivatives, which still adhered to the AH-Sepharose but had lost their binding affinity for transcobalamin II. Unlabeled hydroxocobalamin, which was prepared from cyanocobalamin by this procedure was spectrophotometrically indistinguishable from normal hydroxocobalamin. The purity of the material was further established by means of sodium dodecyl sulphate polyacrylamide gel electrophoresis. The small protein band at a molecular weight of 29 000 does not seem to be a contamination, but rather a subfraction of transcobalamin II, because this band intensified considerably when preincubation and electrophoresis were carried out in the presence of reducing agents. The molecular weights of the other minor components suggest that besides native transcobalamin II with a molecular weight of 37 000, two sets of complementary polypeptide chains occur with molecular weights of either 29 000 and 13 000 or 18 000 and 24 000. Similar observations have been described by Allen et al. [2]. Their initial hypothesis that the molecular weight of transcobalamin II is about 60 000, and that the transcobalamin II molecule is composed of two subunits of 38 000 and 25 000 molecular weight, was revised later when they confirmed the more conventional molecular weight of 38 000 [19]. They suggested that the smaller polypeptide chains had arisen by an internal cleavage of the native polypeptide chain. We have tried to prevent proteolysis during the purification by the addition of diisopropylfluorophosphate at the start of the purification, but this precaution had no influence on the electrophoresis pattern. Because diisopropylfluorophosphate does not inhibit all proteolytic enzymes, it remains unclear when and how proteolysis may have occurred.

51

Polyacrylamide gel isoelectric focusing of the pure transcobalamin II is extremely difficult because of the strong tendency of transcobalamin II to precipitate at low ionic strength. Recently Marcoullis et al. reported that they were unable to establish a typical electrofocusing pattern in a sucrose gradient [20]. Transcobalamin II concentrated in a rather broad area between pH 6.2 and 6.8 in which at least four protein bands were discerned. These protein bands may represent transcobalamin II molecules in the different polypeptide chain configurations, which were demonstrated by the dodecyl sulphate polyacrylamide gel electrophoresis, but another explanation is the existance of isoprotein forms. Genetic polymorphism of transcobalamin II has been reported by Frater-SchrSder et al. [21] and by Daiger [22]..Because of the evidence that transcobalamin II is synthesized by different tissues [23,24] it is tempting to speculate that these tissues may produce slightly different transcobalamin IImolecules. The presented affinity chromatography technique yields the saturated protein (holotranscobalamin II). In this regard it does not distinguish itself from other purification procedures [1--4]. The specific STCo-radioactivity in the final product is determined by the specific radioactivity of the immobilized cyanocobalamin. Cyanocobalamin can be removed from the complex by dialysis against 7.5 M guanidine-HC1 and renaturation in the presence of cobalamin with high specific radioactivity, but this procedure introduces the possibility of structural damage to the binding protein. Otherwise, the isolated holotranscobalamin II is suitable for e.g. physiochemical analysis or functional studies regarding cellular uptake after radioactive labeling with radio-iodine. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

Puutula, L. and Gr~/sbeck, R. (1972) Biochim. Biophys. Acta 263, 734--746 Allen, R.H. and Majerus. P.W. (1972) J. Biol. Chem. 247, 7709--7717 Savage, C.R., Jr., Meehan, A.M. and Hall, C.A. (1976) Preparative Biochern. 6, 99--111 Nex¢, E. (1975) Biochim. Biophys. Acta 379, 189--192 Nex¢, E., Olesen, H.0 Bucher, D. and T h o m s o n , J. (1977) Biochim. Biophys. Acta 4 9 4 , 3 9 5 - - 4 0 2 Mahoney, M.J. and Rosenberg, L.E. (1971) J. Lab. Clin. Med. 7 8 , 3 0 2 - - 3 0 8 Lakshrnanan, T.K. and Lieberrnan. S. (1954) Arch. Biochern. Biophys. 5 3 , 2 5 8 - - 2 8 1 Gottlieb, C., Lau, K.-S., Wasserman, L.R. and Herbert. V. (1965) Blood 25,875---884 Lau, K.-S., Gottlieb, C., Wasserman, L.R. and Herbert. V. (1965) Blood 26, 202--214 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 1 9 3 , 2 6 5 - - 2 7 5 Faixbanks, G., Steck, Th.L. and Wallach, D.F.H. (1971) Biochemistry 10, 2 6 0 6 - - 2 6 1 7 Vesterberg, O. (1972) Biochim. Biophys. Acta 257, 11--19 Bauried~l, W.R., Picken, J.C. and Underkofler, L.A. (1956) Froc. Soe. Exp. Biol. Med. 9 1 , 3 7 7 - - 3 8 1 Weiss, J,P., Rothenberg, S.P. and Cotter, R. (1977) FEBS Lett. 78, 275--278 Sten man , U.H. (1975) Scand. J. Clin. Invest. 35, 147--15 5 Taylor, R.T. and Hanna, M.L. (1970) Arch. Biochern. Biophys. 1 4 1 , 2 4 7 - - 2 5 7 Heathcote, J.G., Moxon, G.H. and Slifkin, M.A. (1971) Spectrochim. Acta, 27A, 1391--1407 Lien, E.L. and Wood, J.M. (1972) Biochirn. Biophys. Acta 264, 530--537 Allen, R.H. (1975) Frog. Hematol. 9, 57--84 Marcoullis, G., Salonen, E.M. and Gr~isbeck, R. (1977) Biochim. Biophys. Acta 495, 336--348 Frater-Schr~ider0 M., Vitins, P., Hitzig, W.H. and H~'kkinen, A.K. (1977) Abstract 6th Meeting of Europ. Soc. Ped. Haernatol. Irnrnunol. 22 Daiger, S.P., Labowe, M.L., Parsous, M., Wang, L. and Cavalli-Sforza, L.L. (1978) Am. J. Hum. Genet. 30, 202--214 23 Tan, C.H. and Hansen, H.J. (1968) Proc. Soc. Exp. Biol. Med. 127, 740--744 24 Rachrnflewitz, B., Rachmilewitz, M., Chaouat, M. and Schlesinger. M. (1977) Biomedicine 27, 213 --214