Retinol-binding protein: the transport protein for vitamin A in human plasma Masamitsu Kanai, … , Amiram Raz, DeWitt S. Goodman J Clin Invest. 1968;47(9):2025-2044. https://doi.org/10.1172/JCI105889. Research Article Vitamin A circulates in human plasma as retinol bound to a specific transport protein. This protein differs from the known low and high density plasma lipoproteins and has a hydrated density greater than 1.21. In order to study this protein, volunteers were injected intravenously with retinol-15-14C. Plasma was collected 1-3 days later, and the purification of retinol-binding protein (RBP) was monitored by assaying for 14C and also by following the fluorescence of the protein- bound retinol. Purification of RBP was effected by the sequence: Cohn fractionation, chromatography on columns of Sephadex G-200 and diethylaminoethyl (DEAE)-Sephadex, preparative polyacrylamide gel electrophoresis, and finally chromatography on Sephadex G-100. These procedures resulted in a preparation of RBP which was at least 98% pure and which had been purified more than 1500-fold. Purified RBP has α1 mobility on electrophoresis and has a molecular weight of approximately 21,000-22,000. There appears to be one binding site for retinol per molecule of RBP. Solutions of RBP are fluorescent (characteristic of retinol) and have ultraviolet absorption spectra with peaks at 330 mμ (resulting from the bound retinol) and at 280 mμ. There are no fatty acid or fatty acyl chains present in purified RBP. The usual concentration of RBP in plasma is of the order of 3-4 mg/100 ml. In plasma, RBP apparently circulates as a complex, together with another, […] Find the latest version: https://jci.me/105889/pdf Retinol-Binding Protein: the Transport Protein for Vitamin A in Human Plasma MAsAmrrsu KANAI, AMIRAM RAZ, and DEWrrr S. GOODMAN From the Department of Medicine, Columbia University College of Physicians and Surgeons, New York 10032 A BSTRA CT Vitamin A circulates in human fatty acyl chains present in purified RBP. The plasma as retinol bound to a specific transport usual concentration of RBP in plasma is of the protein. This protein differs from the known low order of 3-4 mg/100 ml. In plasma, RBP ap- and high density plasma lipoproteins and has a hy- parently circulates as a complex, together with drated density greater than 1.21. In order to study another, larger protein with prealbumin mobility this protein, volunteers were injected intravenously on electrophoresis. The RBP-prealbumin complex with retinol-15-14C. Plasma was collected 1-3 days remains intact during Cohn fractionation and dur- later, and the purification of retinol-binding pro- ing chromatography on Sephadex and on DEAE- tein (RBP) was monitored by assaying for 14C and Sephadex columns. The complex dissociates dur- also by following the fluorescence of the protein- ing gel electrophoresis, permitting the isolation bound retinol. Purification of RBP was effected by and subsequent purification of each of the com- the sequence: Cohn fractionation, chromatography ponents. The complex is again formed by mixing on columns of Sephadex G-200 and diethylamino- together solutions of the separated RBP and of ethyl (DEAE)-Sephadex, preparative polyacryl- prealbumin. Retinol transport in plasma thus ap- amide gel electrophoresis, and finally chromatog- pears to involve both a lipid-protein (retinol- raphy on Sephadex G-100. These procedures re- RBP) interaction and a protein-protein (RBP- sulted in a preparation of RBP which was at least prealbumin) interaction. 98%o pure and which had been purified more than 1500-fold. Purified RBP has al mobility on elec- INTRODUCTION trophoresis and has a molecular weight of ap- proximately 21,000-22,000. There appears to be It is well established that vitamin A mainly cir- one binding site for retinol per molecule of RBP. culates in plasma as retinol (vitamin A alcohol), Solutions of RBP are fluorescent (characteristic apparently associated with a transport protein of of retinol) and have ultraviolet absorption spectra hydrated density greater than 1.21 (1-3). This with peaks at 330 m~m (resulting from the bound protein differs from serum albumin in man (1) retinol) and at 280 mu. There are no fatty acid or and has been reported to have a, mobility (2) or a2 mobility (4) in the rat. Recently, Alvsaker, This paper is No. 8 in the series "Vitamin A and Haugli, and Laland (5) have suggested that this carotenoids" from the laboratory of DeW. S. Goodman. protein is identical with the tryptophane-rich pre- An abstract of this work was presented to the Seventh International Congress of Biochemistry in Tokyo, Japan, albumin in human serum. Detailed information in August 1967 (39), and the work was presented, in about the transport of retinol in plasma is, how- part, at the 11th International Conference on the Bio- ever, not available. We now report the purification chemistry of Lipids, Jerusalem, Israel, August 1967, and and partial characterization of retinol-binding pro- at the 1968 annual meeting of the American Society for tein (RBP), the specific protein to which retinol Biological Chemists (40). Masamitsu Kanai's permanent address is Shinshu Uni- is bound in human plasma. RBP has-a, mobility versity Hospital, Matsumoto City, Japan. and differs from all previously identified plasma Received for publication 6 February 1968. proteins. The Journal of Clinical Investigation Volume 47 1968 2025 METHODS of 1 mamp per tube at room temperature and was termi- reported here were carried out nated just when the tracking dye ran out of the gel. Plasma. The studies For preparative purposes, the Buchler2 "polyprep" ap- with fresh blood, collected in acid citrate dextrose (ACD) similar to that described adult donors. The iso- paratus was used in a manner anticoagulant, from healthy young by Jovin, Chrambach, and Naughton (9). The prepared lation of RBP was carried out three times: first with cm was prerun for 1 hr at from one donor, second with 1700 ml gel column, about 9.5 high, 280 ml of plasma 30 mamps before the sample was applied. In the usual of plasma from six donors, and third with 5 liters of a of 30-60 mg used in the first of procedure, carried out at 2°-5°C, sample plasma from 16 donors. The plasma of protein was dissolved in 4.5 ml of upper buffer, 0.5 ml these preparations, and 300 ml of the plasma used in the of 40% sucrose solution was added, and the mixed sample second preparation, was derived from subjects previously was then applied to the top of the gel. Electrophoresis injected with "C-labeled retinol. was begun at a current of 15-20 mamps. After 30 min Assay. The purification of RBP was quantitatively 30-35 and was main- each puri- the current was adjusted to mamps monitored by assaying the fractions from tained at this level until the voltage reached 500 volts, fication procedure for protein-bound retinol. In the was continued at a con- carried out by after which time electrophoresis initial stages of this work this assay was stant voltage of 500 volts. The elution rate was 30-40 the extraction of plasma or plasma fractions with CHCI3- ml/hr. The complete procedure usually required 8-10 hr CHaOH, 2:1 (v/v), followed by the chromatography time. During gel electrophoresis the effluent stream was of the total lipid extracts on small columns of deacti- monitored continuously for absorption of light at wave- vated alumina (6) in order to isolate retinol. The retinol length 280 m,.3 The effluent stream from most column was then assayed spectrophotometrically (in our labora- chromatographic runs was similarly monitored. toryEing = 1625 at 330 mg& for retinol in benzene). in most of this work, however, a simpler assay was Paper electrophoresis was performed glycine-ace- During tate buffer, pH 8.6, ionic strength 0.15 (10). Zone elec- used, in which the protein-bound retinol was first labeled Dr. then monitored trophoresis on Pevikon was kindly performed by with '4C, and the purification of RBP was Jane H. Morse, with barbital buffer, pH 8.6, ionic by radioassay, for '4C, of the total lipid extracts. To 0.1 At the end of 1 cm this end, normal volunteers were injected intravenously strength (11). electrophoresis, strips of Pevikon were collected and eluted. The eluates with retinol-15-"'C dispersed in their own plasma. The were analyzed for protein and for 'C (after CHC13- dispersion was achieved by adding, via a microsyringe and under sterile conditions, 0.5 ml of acetone contain- CH30H extraction). ing 12 of retinol-15-"C (specific radioactivity 29.6 Analytical ultracentrifugation. Sedimentation equi- gc librium and velocity analyses were kindly carried out by Ac/mg) to 20 ml of freshly collected plasma. Plasma was E ultra- collected 1-3 days after "C-retinol injection and was Dr. Herbert S. Rosenkranz in a Spinco model was estimated for RBP purification. The radioactivity ("C) in centrifuge. The molecular weight of RBP used method as these plasma samples was shown by extraction and chro- by the sedimentation equilibrium (Archibald) described by Schachman (12). Analyses were carried matography to reside almost entirely in retinol. The "4C ml in 0.05 assay procedure for protein-bound retinol was validated out on a solution of 10 mg of RBP per M po- for each purification method by showing that comparable tassium phosphate buffer, pH 7.2. Centrifugation was con- ducted at 10.6°C at two speeds (9341 and 15,220 rpm), results were obtained by "4C assay and by spectrophoto- Pho- metric assay for retinol. In addition, RBP purification and photographs were taken with Schlieren optics. tographs taken at each speed at bar angles of 70 and of was also qualitatively monitored by observing under ul- with traviolet light the visible, green fluorescence of protein- 80° were measured in a microcomparator, together bound retinol.