A novel purification method of human renin

Life Sciences, Vol. 32, pp. 1591-1598 Printed in the U.S.A.

A NOVEL

PURIFICATION

Pergamon Press

METHOD

OF HUMAN

RENIN

Jitsuo Higaki, Sigehisa Hirose, *Toshio Ogihara, Nobuyuki Imai Masatsugu Kisaragi, Kazuo Murakami and *Yuichi K u m a h a r a Institute of Applied Biochemistry, University of T s u k u b a Ibaraki-ken 305 and *Department of Medicine and Geriatrics Osaka University Medical School, Osaka 553, Japan (Received in final form December 22, 1982) Summary H u m a n renal renin was isolated from a partially purified preparation, Haas' preparation step 5 material, with a remarkably high yield of 28% by a newly developed method. This method consisted of only four steps including affinity chromatography on pepstatin-aminohexyl-agarose, chromatofocusing, gel filtration and DEAE-chromatography after the initial extraction and concentration. This method enabled us to obtain pure h u m a n renin without another affinity column used by other investigators. Pure h u m a n renin had a molecular weight of 40,000 daltons as estimated by gel filtration and sodiumdodecylsulfate-polyacrylamide gel electrophoresis. The pI of purified renin was 5.6. Renin (EC 3.4.23) is an endopeptidase that hydrolyzes renin substrate in the blood stream to release the decapeptide angiotensin I which is subsequently converted to angiotensin II, the potent vasoconstrictor and stimulator of aldosterne secretion. The purification of h u m a n renin was first attempted in 1979 by Haas et al. (i). Although homogeneity was not achieved, these authors achieved a 500 fold purification by the limited classical methods which consisted of successive salt and solvent fractionation. In 1979, Galen et al. reported complete purification of h u m a n renin from a renin secreting tumor (2). Purification of only 40 fold was needed to obtain homogeneous renin because the tumor contained a tremendous amount of renin. In 1980, Yokosawa et al. (3) reported a complete purification of h u m a n renin from a partially purified preparation, Haas' preparation step 5 material (i), which had been prepurified 40 fold from normal h u m a n kidneys. In the first step of their method, destructive protease in the Haas' preparation had to be removed by hemoglobin-agarose. Even after this treatment, renin was inactivated in some degree by the elution under acidic condition from pepstatinaminohexyl-agarose column. Later in 1981, Slater and Strout reported a complete purification of h u m a n renin by two methods (4). In their method, renin was eluted from pepstatin-aminohexyl-agarose by lithium bromide and further purified by an affinity column including either the synthetic octapeptide renin inhibitor (D-Leu s) or antirenin immunoglobulin as ligand. These n e w affinity columns seemed to be effective but difficalt to obtain. Moreover, Slater and Strout obtained two major forms of renin, which differ in both apparent size and charge. The larger form of h u m a n renin (M=50,000) (4) is different from renin isolated by Yokosawa et al. (3). This disparity is most likely to be attributed to differences ~n isolation method or characterization techniques. 0024-3205/83/141591-08503.00/0 Copyright (c) 1983 Pergamon Press Ltd.

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Pure renin in sufficient yield is needed for further characterization of renin and for establishment of direct radioimmunoassay of h u m a n renin which introduces a new tool into studies on the pathophysiology of high blood pressure. In this paper, we report a novel purification method of h u m a n renin with higher yield. Materials and Methods Materials---Diisopropyl phosphorofluoridate (DFP) was obtained from Sigma Chemical Company. Polybuffer-74 for chromatofocusing and DEAE-cellulose were from Pharmacia Fine Chemicals. Ultrogel A c A 44 was obtained from L K B . All other reagent were of reagent grade. Pepstatin-aminohexyl-agarose was prepared according to our method (5). Renin Assay---Renin was measured as the rate of angiotensin I formation at 37°C from partially purified hog substrate (i0 rag) in 0.1 M phosphate buffer, p H 6.5, containing 5 m M E D T A and 5raM D F P in a total volume of 0.5 ml (6). Angiotensin I was measured by the radioimmunoassay method of Haber et a]. (7). Protein concentration was estimated from sample absorbances assuming that 1 absorbance unit at 280 n m is 1 m g protein/ml. Startin~ Material---Partially purified renal renin of h u m a n subject was kindly provided by Dr. E. Haas. This preparation, step 5 material in Haas' preparation, has a renin activity of 0.08 Goldblatt unit/mg protein (i). Purification Steps---Step I: Affinity Chromatography

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Purification of the partially purified renin, step 5 in Haas' preparation by a pepstatin-aminohexyl-agarose. The elution buffers were changed at arrows as described in "Materials and Methods". *i: 0.05 M Tris buffer, p H 7.5, *2: 0.15 M Tris buffer, p H 7.5.

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F o r t y - n i n e grams (3,920 G o l d b l a t t u n i t s ) of the p a r t i a l l y p u r i f i e d r e n i n , s t e p 5 in H a a s ' p r e p a r a t i o n ( i ) were d i s s o l v e d i n 730 ml of 0.02 M N a - a c e t a t e b u f f e r , pH 6.5, c o n t a i n i n g i M NaCl a n d 0.2 mM DFP, a n d a p p l i e d to a p e p s t a t i n - a m i n o h e x y l a g a r o s e column (2.5 x i0 cm), p r e v i o u s l y e q u i l i b r a t e d with t h e same b u f f e r . A f t e r sample a p p l i c a t i o n , t h e p r o t e i n s a d s o r b e d in the a f f i n i t y column were e l u t e d s t e p wise with 300 ml of 0.05 M T r i s - a c e t a t e b u f f e r , pH 7.5. A l t h o u g h a small amount of r e n i n was l e a k e d out from the column with 0.05 M T r i s - a c e t a t e b u f f e r pH 7.5, most of r e n i n was e l u t e d with 0,15 M T r i s - a c e t a t e b u f f e r pH 7.5 a n d s e p a r a t e d from o t h e r p r o t e i n s . Step 2: Chromatofocusing

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FIG. 2 P u r i f i c a t i o n of r e n i n f r a c t i o n s from a f f i n i t y column (Fig. 1) b y c h r o m a t o f o c u s i n g . A d o t t e d line shows the pH c h a n g e d u r i n g chromatofocusing. The renin-containing fractions (bracket, Fig. i) were pooled and concentrated by ultrafiltration using Amicon filter PM-10 and dialyzed against 0. 025 M imidazolechloride buffer p H 6.5. Dialyzed sample was applied to chromatofocusing column (0.9 x 30 cm) previously equilibrated with the same buffer as used in dialysis. The column is eluted with 200 ml of Polybuffer-74 adjusted to p H 4.0 by addition of 6 N HCI. Renin was eluted between p H 5.5 and 4.5 according to its isoelectric point. Step 3: Gel F i l t r a t i o n (Fig. 3) R e n i n f r a c t i o n s ( b r a c k e t , Fig. 2) were pooled a n d c o n c e n t r a t e d b y Amicon f i l t e r PM-10 a n d S a r t o r i u s collodion b a g 25. T h e c o n c e n t r a t e d sample was a p p l i e d to a p r e v i o u s l y c a l i b r a t e d Ultrogel AcA 44 column (3 x 100 cm) i n 0.01 M Nap y r o p h o s p h a t e b u f f e r , pH 6.5 c o n t a i n i n g 0.1 M NAG1. R e n i n was e l u t e d as a s i n g l e p e a k c o r r e s p o n d i n g to molecular weight of 40,000 d a l t o n s . T h e r e n i n a c t i v i t y p e a k s ( b r a c k e t , Fig. 3) were pooled a n d d i a l y z e d a g a i n s t 0.02 M Nap h o s p h a t e b u f f e r , pH 6.2. Step 4: D E A E cellulose c h r o m a t o g r a p h y (Fig. 4) T h e d i a l y z e d sample from s t e p 3 was c h r o m a t o g r a p h e d on a DEAE-cellulose column

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FRACTION NUMBER FIG. 3 Purification of renin fractions from chromatofocusing on Ultroge] A c A 44.

(Fig. 2) by a gel filtration

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FIG. 4 Purification of renin fractions from the gel filtration (Fig. 3) by a D E A E cellulose column.

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(0.6 x 20 cm)equilibrated with the dialysis buffer. Renin was eluted by a linear NaCl gradient elution, which consisted of the initial buffer and the buffer containing 0.2 M NaCl. T w o major peaks of renin activity were obtained. Fractions under the second peak (bracket, Fig. 4) had higher specific activity and were used for characterization. Results A protocol employed to purify human renin and the results are presented in Table i. The final step of purification on DEAE-cellulose separated renin into two peaks. The specific activity of the second peak was 940 Goldblatt units/mg. This peak fraction gave a single protein band on the polyacrylamide disc-gel electrophoresis (Fig. 5). Starting with 49 g of partially purified materials which was derived from 35 kg of human kidney, i. 3 m g of pure renin (the second peak on DEAE-cellulose) was obtained with a remarkable high yield of 28%. Since the starting material has been prepurified 40 fold (I), the overall purification was 480,000-fold. B y analytical gel isoelectric focusing, the purified enzyme exhibited a single band which has a pI of 5.6. Gel filtration on Ultrogel A c A 44 and polyacrylamide gel electrophoresis in sodiumdodecyl sulfate (SDS-PAGE, Fig. 6) indicated that the purified h u m a n renin has a molecular weight of 40,000. The lower molecular weight bands in Fig. 6 may represent the fragments derived from nicked renin molecules; nicking of renin appears to have occurred at the acidification step employed by Haas et al. (i). It is unlikely that the lower molecular weight species are impurities since the contaminating low molecular weight proteins were removed by gel filtration (step 3).

TABLE

1

Purification of Renin from H u m a n Kidney Purification Step

Haas, step 5 Pepstatin Column Chromatofocusing Ultrogel A c A 44 DEAE-cellulose

Total Protein mg 49,000 95 25 9.9 1.3

Specific Activity*

Purification

0.08 31 i00 260 940

I( 40) 390(16000) 1300(52000) 3300(130000) 12000(480000)

Yield % 100(90) 74(67) 67(60) 66(54) 31(28)

*Goldblatt unit per m g of protein

Discussion Affinity chromatography on pepstatin-aminohexyl-agarose has played a central role in the purification of renin from various mammalian kidneys (3,4, 813). The present report describes a simplified procedure for purifying h u m a n renal renin, which consists of three conventional column chromatography and only one affinity chromatography; the previous methods (3, 4) for isolating renin from the normal h u m a n kidney employs two affinity columns: a combination of pepstatinaminohexyl-agarose and another gel coupled with hemoglobin (3), the synthetic octapeptide renin inhibitor (D-Leu6), or antirenin antibody (4). The novel aspects of the present purification procedure are i) the introduction of chromato-

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1 cm

! FIG. 5 Polyacrylamide disc-gel electrophoresis of h u m a n renin obtained under the second peak on DEAE-cellulose chromatography (Fig. 4). Electrophoresis was performed at p H 9.5 according to Davis (14). Approximately I0 lJg was electrophoresed and protein was stained by Coomassie Blue.

Mr(x1000 ) 100

50

19

4u38'~ 20 1714.5

~~A EA Lysozyrne

10

, 1 cm

FIG. 6 Densitometric scan of a SDS-polyacrylamide gel. Purified h u m a n renin (1.5 p g) was electrophoresed on 10% slab gels according to the method of Laemmli (15). Protein was visualized using a Bio-Rad Silver Stain kit. Molecular weight standards used were bovine serum albumin (BSA, Mr=68,000), egg albumin (EA, Mr=45,000), ~-chymotrypsinogen (CTg, Mr=24,500), and lysozyme (Mr=13,500).

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focusing and ii) the differential elution of renin from the pepstatin column, which is carried out under neutral conditions. Adoption of this stepwise elution allowed a 390 fold purification with a 74% recovery in one step. Slater and Strout (4) have also used neutral conditions (a linear LiBr gradient) for elution. Although the recovery of their pepstatin affinity chromatographic step (78%) is excellent and comparable to that of our present method (74%), the purification they achieved was about 50 fold. Yokosawa et al. (3) have used acid elution which resulted in a more than 700 fold purification and a recovery of 58%. Acid elution of renin from a pepstatin column, therefore, seems to have merit in terms of the degree of purification; however, exposing samples to acidic conditions poses a serious problem since contaminating cathepsin D destroys renin molecules at a low pH. Thus, the above mentioned improvement of the pepstatin affinity chromatography and the introduction of chromatofocusing made possible the development of a method for complete purification of h u m a n renin without using acid conditions and affinity gels other than pepstatin-aminohexyl-agarose, that are not generally available. The present purification begins with Haas' preparation. This is mainly because of the limited availability of h u m a n kidney. We believe that our purification protocol can be applicable to crude h u m a n kidney extracts since the pepstatin affinity column works well even with the crude extracts. The characteristics of the purified renin, such as specific activity (940 G U / mg), pI (5.6), and the molecular weight estimated by gel filtration (Mr=40,000), agree very well with previous reports (2-4). However, our preparation showed a marked difference in the molecular weight determined by S D S - P A G E (Mr=40,000); the preparations of Galen et al. and Slater & Strout (4) exhibited a major band of Mr=50,000 on S D S - P A G E . This discrepancy may be partly due to that we used the Haas' preparation as starting material which received, during its initial purification, extensive exposure to acid p H where the proteolytic degradation or the deletion of small fragments from renin molecules is likely to occur. Without further information on such as the pulse-chase experiments, however, definitive conclusions as to the precise molecular weight of mature renin in the h u m a n kidney would be premature. Acknowledgements We wish to thank especially Dr. E. Haas for his kind gift of the partially purified h u m a n renin preparation. We are greatly indebted to Dr. T. Inagami for his encouregement during this work. This study was supported by grantsin-aid for Scientific Research from the Ministry of Education, Science and Culture, Japan. References i. E. Haas. H. Goldblatt and E. C. Gipson, Arch. Biochem. Biophys. Ii0 534543 (1965). 2. F. -X. Galen, C. Devaux, T. Guyenne, J. Menard and P. Corvol, J. Biol. Chem. 254 4848-4855 (1979). 3. H. Yokosawa, L. A. Holladay, T. Inagami, E. Haas and K. Murakami, J. Biol. Chem. 255 3498-3502 (1980). 4. E. E. Slater and H. V. Strout, J. Biol. Chem. 256 8164-8171 (1981). 5. E. Murakami and T. Inagami, Biochem. Biophys. Res. C o m m u n . 62 757-763 (1975). 6. K. Murakami, S. Takahashi, F. Suzuki, S. Hirose and T. Inagami, Biomed. Res. 1 392-399 (1980). 7. E. Haber, T. Koerner, L. B. Page, B. Kliman and A. Purnode, J. Clin. Endocrinol. Metab. 29 1349-1355 (1969). 8. E. Haber and E. E. S--later, Circ. Res. 40 Suppl. I 1-36-40 (1977). 9. T. Inagami and K. Murakami, J. Biol. Chem. 252 2978-2983 (1977).

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I0. C. Devaux, J. Menard, P. Sicard and P. Corvol, Eur. J. Biochem. 64 621627 (1976). ii. P. Corvol, C. Devaux, T. Ito, P. Sicard, J. Ducloux and J. Menard, Cir. Res. 41 616-622 (1977). 12. T. Matoba, K. Murakami and T. Inagami, Biochem. Biophys. Acta 526 560571 (1978). 13. V. Dzau, E. E. Slater and E. Haber, Biochemistry 18 5224-5228 (1979). 14. B. J. Davis, Ann. N. Y. Acad. Sci. 121 404-427 (1964). 15. U. K. Laemmli, Nature 227 680-685 (1970).