A method was previously developed in this Laboratory for estimating the enzyme renin in rabbit plasma (Lever, Robertson & Tree, 1963 a, b,c, 1964. The present paper describes the adaptation of the method to the measurement of renin in the plasma of man. Preliminary accounts of this work have been published (Brown, Davies, Lever & Robertson, 1963a, b, 1964a.
METHODSThe technique consists of an estimation of renin concentration by determining the initial velocity of angiotensin formation under standard conditions of incubation with substrate. Angiotensinase-free ox-serum substrate, as prepared for the estimation of rabbit renin (Lever et al. 1964), was used throughout the present studies. The technique of incubation and assay, and the tests for contamination with angiotensinase and endogenous substrate, were also as described by Lever et al. (1964). The methods of preparation of standard human renin, and of extracting renin from human plasma, however, differed from those used with the rabbit.Preparation of standard human renin. Human kidneys (4.5 kg.) were obtained poet mortem, and excess of fat and fibrous tissue was removed. The kidneys were then minced and allowed to stand in 101. of water at 8°for 36 hr. The mince was filtered through muslin, and 10% (w/v) trichloroacetic acid was added to the filtrate at 80, with constant stirring, to give pH 2-9. Then NaCl (52 g. to each litre) was added slowly with stirring; the pH was checked after 15 min., and if necessary readjusted to pH 2-9 by adding 10% trichloroacetic acid or N-NaOH. The solution was then filtered at 80 overnight through Whatman no. 50 paper and the filtrate adjusted to pH 5-0 with N-NaOH. Then 2 1. batches of this solution were dialysed in Visking cellophan sacs (28/32 in.) against three 15 1. changes of water at 80 over 48 hr. (unpublished work) during the purification of pig renin. The 2 1. batches of renin solution were dialysed in Visking cellophan sacs (28/32 in.) against three 151. changes of 5 mM-sodium phosphate buffer, pH 7 0 (0-0240% NaH2PO4; 0-1074% Na2HPO4,2H20), over 48 hr. at 80. This solution was then applied at room temperature to a column (65 cm. x 2-5 cm.; dry wt. 100 g.) of lightly-packed DEAE-cellulose equilibrated with the 5 mM-sodium phosphate buffer, pH 7-0. Renin was adsorbed during this application. The column was washed with 31. of 0 03M-sodium phosphate buffer, pH 7*0 (0-140% NaH2PO4; 0-653% Na2HPO4,12H20), and the eluted protein discarded. Renin was then eluted with 600 ml. of 0 35M-phosphate-saline buffer, pH 6.0 (0-05M-Na2HPO4; 0 3M-NaCl, adjusted to pH 6-0 with 6N-HCI), containing neomycin sulphate (0-01 %).The eluate was dialysed against three 101. changes of glycine-HCl-saline buffer, pH 3-0 (0 IM-glycine; 0-9M-NaCl; 0 018N-HCI), at room temperature over 24 hr. The precipitate that then formed was removed by filtration
