Pure Renin ISOLATION FROM HOG KIDNEY AND CHARACTERIZATION*

The pressor enzyme renin (EC 3.4.99.19) was isolated in a pure and stable form from hog kidney by affinity chromatography on a pepstatin/agarose gel followed by three addi- tional steps of conventional chromatography. Destruction of the enzyme by proteolysis during isolation was prevented by chemically eliminating proteases in extracts. The pure preparation was used for the characterization of this enzyme. Renin was found to be a glycoprotein containing glucosamine and possessing binding affinity to concanavalin A. Contrary to previous reports, pure renin is stable at neutral pH either at 4 or -20” for 3 to 8 weeks. It has a molecular weight of 36,400 as determined by equilibrium ultracentrifugation, an isoelectric point of 5.2 and E:?,, (280 nm) of 9.1. In contrast to crude preparations, the enzyme activity has a broad pH optimum between pH 5.5 and 7.0 for both hog angiotensinogen and the synthetic octapeptide substrate The rate of formation of angiotensin I from hog angiotensinogen at pH 6.0 and 37” was 267 pg/h/pg of renin, 2000 Goldblatt unitslmg renin. For the synthetic

The pressor enzyme renin (EC 3.4.99.19) was isolated in a pure and stable form from hog kidney by affinity chromatography on a pepstatin/agarose gel followed by three additional steps of conventional chromatography. Destruction of the enzyme by proteolysis during isolation was prevented by chemically eliminating proteases in extracts. The pure preparation was used for the characterization of this enzyme. Renin was found to be a glycoprotein containing glucosamine and possessing binding affinity to concanavalin A. Contrary to previous reports, pure renin is stable at neutral pH either at 4 or -20" for 3 to 8 weeks. It has a molecular weight of 36,400 as determined by equilibrium ultracentrifugation, an isoelectric point of 5.2 and E:?,, (280 nm) of 9.1. In contrast to crude preparations, the enzyme activity has a broad pH optimum between pH 5.5 and 7.0 for both hog angiotensinogen and the synthetic octapeptide substrate benzyloxycarbonyl -Pro -Phe -His-Leu -Leu-Val-Tyr-Se@-naphthylamide.
The rate of formation of angiotensin I from hog angiotensinogen at pH 6.0 and 37" was 267 pg/h/pg of renin, or 2000 Goldblatt unitslmg of renin. For the synthetic fluorogenic octapeptide substrate benzyloxycarbonyl-Pro-Phe-His-Leu-Leu-Val-Tyr-Ser-P-naphthylamide, a K,R of 33 pM and a I/,,, of 0.94 pmol/h/mg of enzyme were obtained at pH 6.5 and 37".
The highly specific proteolytic cleavage of angiotensinogen by renin (EC 3.4.99.19) to produce angiotensin I is the first step in the series of reactions leading to the formation of the major pressor substances angiotensin II and angiotensin III and to the release of aldosterone.
Furthermore, the secretion of renin from the kidney into the circulation is the rate-limiting step regulating the subsequent production of the angiotensins (1 determine some of the molecular and enzymological properties of renin.

MATERIALS AND METHODS
Renin Activity -Renin activity was determined by the radioimmunoassay of angiotensin I generated after incubation with partially purified hog angiotensinogen (plasma renin substrate, Miles Laboratories) at pH 6.0 for 1 h at 37". The assay was carried out according to the method of Haber et al. (23) using an assay kit (New England Nuclear).
The Goldblatt unit value of the specific activity was determined in reference to standardized hog renin kindly provided by Dr. Erwin Haas of Cleveland (preparation no. 119, standardized in March, 1976). Activity of purified renin preparations free from contaminating proteases was determined by the fluorometric method of Reinharz and Roth (24) using benzyloxycarbonyl-Pro-Phe-His-Leu-Leu-Val-Tyr-Ser-p-napthylamide as substrate (25). Polyacrylamide Gel Electrophoresis -Polyacrylamide gel electrophoresis was run in a 6-cm gel containing 7.5% polyacrylamide with 2.5% cross-linkage at pH 9.5 with a constant current of 2 mA/gel for 1 h by the method of Davis (261,but  Co.) in the solution section of a sector cell with the dialysate b&g added to the solvent sector. Equilibrium scans were made at 6" at a rotor velocity of 13,000 rpm. In calculating the weight average molecular weight, the partial specific volume of protein was estimated to be 0.734 using the amino acid composition (35). The molecular weight was also estimated by gel filtration on a calibrated column (2.5 x 110 cm) of Sephadex G-75, and by electrophoresis in a 10% polyacrylamide gel (IO cm long) with 2.7% cross-link in 0.1% SDS* with and without 0.25% dithiothreitol (36, 37).

RESULTS
The outline and result of the purification of renin from hog kidneys are summarized in Table I. A 133,000-fold purification was obtained with a remarkably high yield of 19%. Two milligrams of pure renin can be obtained from 20 kg of hog kidney. The final product obtained after chromatography on CM-cellulose was used for subsequent characterization studies reported in this paper. The preparation after DEAE-cellulose chromatography already had a very high specific activity, almost comparable with that of the final product. The use of the mixture of protease inactivators seems to reduce multiple peaks, thus facilitating purification. The product obtained with the protease inactivators produced a clean band on polyacrylamide gel electrophoresis, whereas omission of the inactivators caused great difficulties in obtaining a pure product.
Purity -Electrophoresis of the purified hog renin on polyacrylamide gel at pH 9.5 (Fig. 5A) gave a discrete single band. Likewise, electrophoresis in SDS-polyacrylamide in the absence (Fig. 5B) and presence (Fig. 5C) of dithiothreitol gave single bands. Isoelectric focusing on a polyacrylamide gel also produced a single band (Fig. 6). These results along with other observations cited below can be considered as a strong evidence for homogeneity of the purified renin. The preparation obtained after DEAE-cellulose chromatography already gave 2 The abbreviations used are: SDS. sodium dodecvl sulfate: iPr,P-F, diisopropylphosphorofluoridate; PhCH,SO,F, phenylmethanesulfonyl fluoride; Mops, 3-(N-morpholinofpropanesulfonic acid: Mes. 2-(N-morpholino)ethanesulfonic-acid; -Tri^cine, N-Tris-(hydroxymethyl)methylglycine. single bands upon disc electrophoresis and electrofocusing although the bands were less discrete.
Stability-The enzyme solution (0.2 mg/ml) was stored at various pH values in the presence of 0.02 M pyrophosphate either at 4" or frozen at -20". No loss of activity was noted when stored at 4" for 8 weeks at pH 5.0 and 7.0, or at -20" for 8 weeks at pH 7.0. However, at acidic pH values, it lost activity at a noticeable speed. At pH 3.3 and 4", 40 to 50% of activity was lost in 20 min. Repeated freezing and thawing at pH 5.0 caused precipitation of the enzyme protein with a concomitant irreversible loss of renin activity.
Isoelectric Point -Electrofocusing of approximately 10 pg of the enzyme protein produced a single discrete band at a position corresponding to a p1 of 5.2 as shown in Fig. 6.
Extinction Coefficient -An E:Ts of 9.1 was obtained at 280 nm. This value was used throughout the characterization studies.
Molecular Weight-Molecular weight was determined at 6" with a renin solution, 0.3 mg/ml in 0.05 M sodium/pyrophosphate buffer, pH 6.2, containing 0.1 M KCl. The molecular weight estimated from the least squares slope of the plot of the natural logarithm of the absorbance at 280 nm against the square of the radius was 36,400. The plot was linear over the entire cell and did not give any sign of aggregation at the bottom of the cell. Passage of the enzyme solution through a calibrated column of Sephadex G-75 (2.5 x 110 cm) gave a molecular weight estimate of 42,000 (Fig. 7). Electrophoresis  in the same buffer, as shown in Fig. 9. These observa-intended to minimize the proteolytic destruction and subsequent generation of multiple isoenzymes of renin in view of the reported observation of multiplicity upon acidification (44). Nevertheless, the presence or the generation of minor components was observed in the elution pattern of ion exchange chromatographic steps ( Fig.  3 and 4). In the present investigation, no attempts were made to purify these minor components.
tions indicate that hog renin contains carbohydrate residues at nonreducing termini which have a specific affinity to concanavalin A (39, 40)." Renin Activity -The pH dependence of the rate of hydrolysis of the synthetic octapeptide substrate of Roth and Reinharz (25) was investigated at 37" at a fixed substrate concentration of 50 mM. A broad pH optimum ranging from pH 5.5 to 7 was observed, as shown by the solid curve in Fig. 10 (38), renin from kidney has never been available in a pure and stable form because of its very low concentration in kidney and also because of its alleged instability in the pure state, which seems to be due to contaminating proteases. In the present studies, by circumventing these difficulties through the use of affinity chromatography and protease inactivators, we were able to obtain a pure and stable renin preparation from the kidney for the first time since the discovery of this enzyme in 1898 (43). This preparation allowed us to determine properties that can be obtained only with a pure substance.
Thus this study will provide a frame of reference for many studies conducted with crude preparations of renin. The 300-fold purification attained at the affinity chromatographic step was no doubt the single most important factor contributing to the completion of the present purification studies with an overall yield of 19% and 133,000-fold purification. However, it must be pointed out that the affinity chromatography alone could not produce a pure preparation in one step.