Comp. Biochem. Physiol., 1972, Vol. 42B, pp. 57 to 64. Pergamon Press. Printed in Great Britain

PORCINE RETINOL BINDING PROTEIN*

C. C. H U A N G , R. E. H O W A R T H t and B. D. O W E N

Department of Animal Science, University of Saskatchewan, Saskatoon, Saskatchewan

(Received 20 September 1971)

Abstract--1. The retinol binding protein (RBP) in porcine serum was partially purified and characterized.

2. Purification was by Cohn fractionation, filtration on Sephadex G-200, chromatography on DEAE Sephadex A-50 and preparative polyacrylamide electrophoresis.

3. Porcine RBP has cq-globulin mobility on polyacrylamide gel electro- phoresis, a sedimentation coefficient of 4"5 S and a molecular weight of approxi- mately 47,000.

4. Porcine RBP differs from human RBP in having a larger molecular weight and sedimentation coefficient and no affinity for prealbumin.

INTRODUCTION

THE MECHANISM of vitamin A transport in plasma has been studied by a number of investigators. Krinsky et al. (1958) established that vitamin A circulated in human plasma predominantly as retinol, associated with a protein of density greater than 1.21 g/ml. This observation was later supported by Garbers et al. (1960) and Goodman et al. (1965) for the rat. More recently Kanai et al. (1968) have isolated from human plasma a specific retinol binding protein (RBP). This protein has ~t-mobility on electrophoresis, a molecular weight of 21,000-22,000 and apparently circulates in plasma as a complex with a prealbumin protein.

We have used a purification procedure similar to that of Kanai et al. (1968) and obtained highly purified porcine RBP. Partial characterization indicates that porcine RBP has properties different from human RBP.

MATERIALS AND METHODS Serum preparation

Six pigs (Sus domesticus) weighing approximately 95 kg were used in the development of the purification procedure. Characterization studies were conducted on two of these. Each animal was given 1"5 million I.U. of retinyl palmitate, fasted for 24 hr and exsanguinated.

Blood was collected in citric acid, dextrose anticoagulant and held at 4°C for 3 hr prior to centrifugation. Bovine thrombin was added to plasma (3 I.U./ml) and after stirring for 2 hr at room temperature, fibrin was removed by filtration.

* Supported by a grant from the National Research Council of Canada. f Present address: Department of Veterinary Physiology, University of Saskatchewan,

Saskatoon.

57

58 C.C. HUANC, R. E. HOWARTH AND B. D. OWEN

Purification procedure Purification involved a sequential application of: Cohn fractionation (Cohn et al.,

1950), gel filtration (Gelotte, 1964) on Sephadex G-200, anion exchange chromatography (Pharmacia, 1968) on DEAE Sephadex A-50, electrophoresis (Jovin et al., 1964) in 7'5% polyacrylamide gels. These procedures were conducted at 4-6°C. Precautions taken to avoid oxidation of retinol included protection from light and flushing buffer solutions with nitrogen.

Fractionation of the serum by Cohn Method 10 (Cohn et al., 1950) removed most of the albumin and gamma globulin. Cohn Fraction III had a ninefold higher retinol : protein ratio than other fractions and was selected for further purification.

Apparatus and buffers for preparative polyacrylamide gel electrophoresis were as described by Jovin et al. (1964). Electrophoresis was in 80 ml of 7"5% polyacrylamide gel at a constant current of 10 mA until the protein entered the gel and 30 mA thereafter. A buffer flow rate of 25-30 ml/hr was maintained. A 2-hr prerun at 30 mA was essential to avoid ammonium persulfate oxidation of retinol.

Monitoring and characterization procedures Elution profiles were monitored by absorbance at 280 m/,. Protein fractions obtained at

each stage of purification were subjected to analytical polyacrylamide gel electrophoresis using a modified procedure of Ornstein & Davis (1962). Potassium ferricyanide and the concentrating gel layer were omitted. A current of 5 mA per tube was used. Gels were stained with Coomassie brilliant blue (Chamback et al., 1967) in 12% trichloroacetic acid and washed with 10% trichloroacetic acid.

The fluorescence of each protein fraction, measured in a Turner Model 110 fluorometer, served as a guide in locating the elution position of retinol. Positive identification of retinol was by thin-layer chromatography on 0"5-mm plates of silica gel-G. Application of samples and development of plates (1% v/v ethanol in chloroform) was under N2 and minimum light. Retinol was detected by spraying with 50~o v/v trifluoroacetic acid in chloroform. Quantitative retinol analyses were according to the method of Neeld & Pearson (1963).

Analytical ultracentrifugation studies were performed using a Spinco Model E analytical ultracentrifuge. Sedimentation coefficients were determined at 20°C and 68,000 rev/min in 0-1 M sodium chloride with and without pH 7"0 phosphate buffer. Observed sedimenta- tion coefficients were corrected to standard conditions (S°20, buffer)"

RESULTS

Total vitamin A levels in fasting pigs sera ranged from 40 to 60/zg/100 ml and agreed well with those reported by Thompson et al. (1950). Chromatography on alumina (Thompson et al., 1949) demonstrated that 90-95 per cent of this total was retinol; the balance was retinyl ester(s). Additional preliminary investigation in- volving preparative ultracentrifugation showed that all of the retinol was associated with proteins of density greater than 1.21 g/ml. Retinyl ester could be detected only in the chylomicra and lipoprotein fractions. Krinsky et aL (1958) reported similar observations on human plasma.

Distribution o f retinol in Cohn fractions

The Cohn procedure yielded four fractions. All contained retinol (Table 1) with fraction I I I containing more than the other major fractions. The overall recovery of serum retinol was approximately 65 per cent. Probably some ethanol

PORCINE RETINOL BINDING PROTEIN 59

T A B L E 1 - - D I S T R I B U T I O N OF PROTEIN AND RETINOL IN THE C O H N FRACTIONS OF PIG SERUM

Protein Retinol Colin fraction (%) (%)

I 5 0.3 II 10 4.8

III 17 65-5 IV+V+VI 69 29-5

denaturation of transport protein occurred, with subsequent loss of retinol during dialysis.

Analytical polyacrylamide gel electrophoresis demonstrated Cohn Fraction I I I to be a heterogeneous mixture of proteins with a much reduced content of albumin and gamma globulin, compared to the original serum.

Gel filtration on Sephadex G-200

Cohn Fraction I I I proteins from approximately 1.5 1. of serum were applied onto Sephadex G-200 and upon elution yielded three peaks (Fig. 1). The third

d O

0 ~3o

g2o

o ,b

TT

20 30 4 0 50 60 70 80 90 Fraction number (20 ml/tube)

o

150 I00 50

FIG. 1. Gel filtration of Cohn Fraction III on Sephadex G-200. Cohn Fraction III was dissolved in 40-50 ml of 0"05 M sodium phosphate buffer, pH 7"6 contain- ing 0-2 M sodium chloride. The same buffer was used to dialyze the protein, equilibrate the column (5.5 x 85 cm) and elute the proteins. The elution rate was 25 ml/hr. Heights of the cross hatched areas indicate amounts of retinol in each

fraction.

peak contained about 60 per cent of the total eluted retinol; the remaining 40 per cent was distributed in the other two peaks in proportion to protein concentration. The presence of retinol in peaks I and II was believed to be due to incomplete separation, since when peaks I and II were subsequently examined on DEAE Sephadex, retinol appeared in the same elution position as for peak III.

60 C .C . HUANG, R. E. HOWARTH AND B. D. OWEN

DEAE Sephadex chromatography Peak I l l from the gel filtration procedure was dialyzed, lyophilized, dissolved

in 0-02 M phosphate buffer and chromatographed on DEAE Sephadex A-50. Seven distinct peaks were obtained (Fig. 2), with all of the retinol appearing in the

"/v

o m N 1.5 ~. 0.6

1.0 0.4

,~o.5 02 ~

0 I0 20 30 4 0 5 0 6 0 70 80 Fraction number (2Oral/tube)

FIc. 2. Chromatography of Sephadex G-200, fraction I I I on DEAE-Sephadex. Sephadex G-200 fraction I I I was dialyzed, lyophilized and applied to the column (2"5 x 100 cm) in 40-50 ml of 0"02 M sodium phosphate, pH 7"6. Elution was with a linear NaC1 gradient (0-0"6 M) in 0"02 M sodium phosphate, pH 7"6. Elu-

tion rate was 20--40 ml/hr. The shaded area indicates the position of retinal.

fourth peak. Peak IV, which overlapped with albumin (peak V), was shown by analytical polyacrylamide gel electrophoresis to contain five or six distinct proteins. These were located in the albumin, al-, a2- and fl-globulin regions. No prealbumin was observed in peak IV but was contained in peak VI.

Kanai et al. (1968) obtained a similar elution profile from human serum. How- ever human retinal binding protein was located in the fraction corresponding to peak VI (Fig. 2) which also contained prealbumin. These observations were the first evidence for a difference between the retinal transportation protein of the two species.

Preparative polyacrylamide gel electrophoresis DEAE Sephadex peak IV was dialyzed, lyophilized and fractionated by pre-

parative electrophoresis (Fig. 3). Electrophoresis yielded three peaks; only peak I I I contained retinal. This fraction, subjected to a second preparative electro- phoresis under identical conditions (Fig. 4) yielded highly purified retinal trans- port protein which migrated as a major band with c~l-globulin mobility when subjected to analytical polyacrylamide gel electrophoresis (Fig. 5). The analytical gels showed a second minor component, with some streaking, ahead of the major band and with albumin mobility.

The purification steps and yields are summarized in Table 2. A yield of 90 mg RBP, obtained from 1.5 1. serum, corresponds to 6 mg RBP per 100 ml; a value comparable to 3-4 mg RBP/100 ml human serum (Kanai et al., 1968).

P O R C I N E R E T I N O L B I N D I N G P R O T E I N 61

Bands of material with absorbance at 280 m/z appeared in gels prepared from aged, stock acrylamide solutions (Fig. 3, peak I and Fig. 5, No. 4). They occurred even in the absence of added protein and may be avoided by use of fresh, acrylamide solutions (2 weeks or less) or recrystallization of stock acrylamide reagent (Gordon & Louis, 1967).

o ~.7~ (I) o ~ .5C

I

I0 20 30 40 50 60 70. 80 90 Fraction number (Sml/fube)

FIG. 3. Preparative polyacrylamide gel electrophoresis of DEAE Sephadex, peak IV purified retinol binding protein. Sample size was 60 mg protein in 4 ml Tris buffer. Peak I is the material relating to the use of aged solution gel. Peak I I I

(shaded area) contained retinol.

O

O IJ r-

g ~o.t

I0 20 30 40 50 60 70 80 90 Frecflon number (5 ml/fube)

FIG. 4. Preparative polyacrylamide gel electrophoresis of retinol binding protein. The sample was retinol containing peak (III) from the first preparative polyacryl- amide gel electrophoresis (Fig. 3). The shaded area indicates the tubes which

contained retinol.

Properties of porcine retinol binding protein Sedimentation velocity studies (Fig. 6A) demonstrated that the transport

protein has a sedimentation coefficient (S°20. butler) of 4"5 S. This value, which was concentration dependent, was obtained by extrapolation to zero protein concen- tration and expressed as standard conditions. The value of 4.5 S differs markedly from the 2.26 S reported by Kanai et al. (1968) for human retinol binding protein

62 C. C. HUANG, R. E. HOWARTH AND B. D. OWEN

TABLE 2--PURIFICATION OF PORCINE RETINOL BINDING PROTEIN

Protein Fraction (g)

Whole serum 102 Cohn Fraction III 17'35 Sephadex G-200, peak I I I 1 "04 DEAE-Sephadex A-50, peak IV 0.287 First preparative electrophoresis, peak I I I 0" 109 Second preparative electrophoresis 0'090

(RBP). The preparation also contained slower sedimenting material of which the sedimentation velocity could not be measured.

To study the possibility of complex formation between the retinol binding protein and prealbumin, such as reported for human RBP (Kanai et al., 1968), porcine prealbumin was prepared from fraction VI of the proteins recovered from DEAE Sephadex chromatography. Prealbumin, as shown in Fig. 6B, migrated as a single peak with a sedimentation coefficient (S°~0, buffer) of 3"8 at 0.8% concentra- tion in 0.1 M sodium chloride. A mixture of the transport protein and prealbumin also yielded a single peak with a sedimentation coefficient not measurably different from prealbumin alone. Since there was no measurable increase in prealbumin sedimentation coefficient in the presence of RBP, it was concluded that no complex had formed, and that the single peak of the mixture on the Schlieren diagram re- flected a similarity of sedimentation coefficients for the two proteins at the concen- trations chosen.

The molecular weight of porcine retinol binding protein was estimated by gel filtration (Whitaker, 1963). Sephadex G-100 (1.7x90 cm) was employed and elution was with 0.05 M sodium phosphate buffer, pH 7.5, containing 0.2 M NaCI. Void volume (Vo), determined with blue dextran, was 48 ml and RBP elution volume (Ve) was 72 ml. Molecular weight (M) of 47,000 was estimated (Determann & Michel, 1966) from the relationship log M = 5.941 -0.847 x (VJ V0). In contrast, the molecular weight of human RBP was reported to be 21,000-22,000 (Kanai et al., 1968).

Of interest here is the comparative study by Baumstark (1968) in which he found general similarity in molecular weights of human and swine serum proteins. Some exceptions were swine transferrin which had a molecular weight lower than the human while the converse was true for albumin and a-globulin.

DISCUSSION Like other lipid materials, vitamin A is transported in blood bound to plasma

proteins. Retinyl esters account for 5-10 per cent of total plasma vitamin A and are found in chylomicrons and lipoprotein fractions. Retinol accounts for 90-95 per cent of total plasma vitamin A and has been shown to bind specifically to an RBP

FIG. 5. Analytical polyacrylamide gel electrophoresis. 1. Whole pig serum (5 ~1). 2. Retinol binding protein after the first preparative polyacrylamide gel electro- phoresis. 3. Purified retinol binding protein after the second preparative poly- acrylamide gel electrophoresis. 4. Purified retinol binding protein on a gel column

prepared of aged polyacrylamide solution.

FIG. 6. Schlieren patterns of purified proteins. A. Purified retinol binding protein after centrifugation at 68,300 rev/min for 24 min at 20°C in 0.1 M NaCl. The protein concentration was 0.4% (Soeo,bti~r = 4.6 S). B. Purified porcine prealbumin after centrifugation at 68,250 rev/min for 36 min at 20°C in 0.1 M

NaCl. The protein concentration was 0.8% (So,,, btier = 3.8 S).

PORCINE RETINOL BINDING PROTEIN 63

in human plasma. The experiments reported here have demonstrated the presence of RBP in swine serum.

A highly purified preparation of RBP has been obtained. Although RBP was the major component in the final preparation it was apparent that the final prepara- tion contained a minor component. This material had a lower molecular weight than the RBP as indicated by its location at the leading edge of RBP in polyacryl- amide gels (Fig. 5) and its slower sedimentation in the centrifugation studies (Fig. 6A). Examination of the elution profiles from preparative electrophoresis shows that retinol binding protein overlapped with the preceding peak. This preceding peak is probably the minor component in the final RBP preparation but it is impos- sible to state whether the minor component represents heterogeneity of protein composition or dissociation of a homogeneous protein. The presence of the minor component does not invalidate our conclusion that the major protein component is RBP.

Porcine RBP has at-mobility, an approximate molecular weight of 47,000, a sedimentation coefficient (S°20. bu t l e r ) of 4"5 S and a hydrated density of greater than 1-21 g/ml. Human RBP has ~l-mobility, a molecular weight of 21,000-22,000, a sedimentation coefficient of 2.26 S and apparently circulates in plasma as a complex with prealbumin. Unlike human RBP, porcine RBP eluted from DEAE Sephadex in a fraction separate from prealbumin. Nor did porcine RBP complex with purified porcine prealbumin.

It is concluded that porcine retinol binding protein differs distinctly from human retinol binding protein. Although both have ~l-globulin mobility, the former protein is a larger molecule, and does not appear to complex with pre- albumin.

Acknowledgements--We gratefully acknowledge the assistance of Drs. D. E. Eveleigh, S. L. Mackenzie, R. A. Miller and J. E. Watkin and Mr. A. S. Sieben of National Research Council, Saskatoon, Saskatchewan, for their assistance in various phases of this work.

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Key Word Index--oq-Globulin; binding protein; electrophoretic mobility; human; molecular weight; plasma; porcine; prealbumin; sedimentation coefficient; transport; vitamin A; Sus domesticus.