Anaphylatoxin inactivator of human plasma: its isolation and characterization as a carboxypeptidase Viktor A. Bokisch, Hans J. Müller-Eberhard J Clin Invest. 1970;49(12):2427-2436. https://doi.org/10.1172/JCI106462. Research Article The failure of human serum to give rise to anaphylatoxin activity could be attributed to the presence of a potent inactivator of anaphylatoxin in human serum. The inactivator was isolated and characterized as an α-globulin with a molecular weight of approximately 310,000. It was found to abolish the activity of both anaphylatoxins, which are derived respectively from the third and the fifth component of complement, and of bradykinin. Inactivation of C3-derived anaphylatoxin and of bradykinin was accompanied by release of C-terminal arginine from these peptides. The anaphylatoxin inactivator was shown to hydrolyze the synthetic substrates hippuryl-L-arginine and hippuryl-L-lysine and to be inhibited by ethylenediaminetetraacetate (EDTA) or phenanthroline. These observations indicate that the anaphylatoxin inactivator constitutes a metal-dependent enzyme resembling in specificity pancreatic carboxypeptidase B. Find the latest version: https://jci.me/106462/pdf Anaphylatoxin Inactivator of Human Plasma: Its Isolation and Characterization as a Carboxypeptidase V=IOR A. BoiascH and HANs J. MUWTI-EBEmIARD From the Department of Experimental Pathology, Scripps Clinic and Research Foundation, La Jolla, California 92037 A B S T R A C T The failure of human serum to give rise molecular weight fragments, called C3a' and C5a, which to anaphylatoxin activity could be attributed to the arise during the complement reaction through the ac- presence of a potent inactivator of anaphylatoxin in hu- tion of complement enzymes. Both peptides have the man serum. The inactivator was isolated and charac- capacity to contract smooth muscle and to increase capil- terized as an a-globulin with a molecular weight of ap- lary permeability. These activities are primarily due to proximately 310,000. It was found to abolish the activity release of histamine from cellular elements and are of both anaphylatoxins, which are derived respectively therefore inhibitable by antihistamines. from the third and the fifth component of complement, Since anaphylatoxin could be produced from isolated and of bradykinin. Inactivation of C3-derived anaphyla- human complement proteins but not from whole human toxin and of bradykinin was accompanied by release of serum, it was postulated that human serum contains an C-terminal arginine from these peptides. The anaphyla- inactivator of anaphylatoxin (2). The presence of a heat- toxin inactivator was shown to hydrolyze the synthetic labile inactivator could subsequently be demonstrated in substrates hippuryl-L-arginine and hippuryl-L-lysine and the a-globulin fraction of human serum (3). The pur- to be inhibited by ethylenediaminetetraacetate (EDTA) pose of this communication is to report the isolation of or phenanthroline. These observations indicate that the the anaphylatoxin inactivator (AI), its enzymatic anaphylatoxin inactivator constitues a metal-dependent specificity, and its effect on both anaphylatoxins and on enzyme resembling in specificity pancreatic carboxy- bradykinin. Emphasis will be placed on studies of the peptidase B. action of the inactivator on C3a anaphylatoxin, since this fragment to date has been more extensively charac- terized than C5a and has recently been found to exert INTRODUCTION strong biological activity in skin tests in humans (2).' It has long been known that anaphylatoxin activity can- not be generated in human serum, whereas this activity METHODS is readily produced in a variety of animal sera, including Preparation of the anaphylatoxins from C3 and rat, guinea pig, and hog serum. The question arose C5 of human complement therefore as to whether human serum lacks the anaphyla- C3a anaphylatoxin was produced and isolated from highly toxin precursor or contains an inhibitor of anaphylatoxin. purified C3 after treatment with the complement enzyme C3 With the isolation and identification of complement pro- convertase, trypsin, or the C3 inactivator complex according teins from human serum, it was possible in recent years 'The symbols used have been recommended by the WHO to demonstrate that two distinct anaphylatoxins can be Committee on Complement Nomenclature (1968). C2, C3, derived from the isolated third and fifth components of C4, and C5 denote, respectively, the second, third, fourth, human complement (1, 2). Both anaphylatoxins are low and fifth components of the complement system. C4,2 desig- nates the activating enzyme of C3, which is also called C3 This is publication No. 418 from the Department of Ex- convertase. EAC4,2,3 refers to a complex consisting of perimental Pathology, Scripps Clinic and Research Founda- erythrocytes, antibody and C4, C2, and C3. C3a and C5a tion, La Jolla, Calif. 92037. refer to the biologically active, low molecular weight cleav- Dr. Bokisch is a Fellow of the Helen Hay Whitney Foun- age products of C3 and C5, respectively. dation. 'Wuepper, K. D., V. A. Bokisch, H. J. Muller-Eberhard, Received for publication 27 May 1970. and R. B. Stoughton. In preparation. The Journal of Clinical Investigation Volume 49 1970 2427 to methods described previously (3). C5a anaphylatoxin was Rabbit antisera produced from highly purified C5 by the action of trypsin or EAC4,2,3, as reported earlier (4). Anti-AI was produced by injecting 20 pg of AI in complete Freund's adjuvant into the popliteal lymph nodes Isolation of the anaphylatoxin inactivator from of rabbits. 4 wk later these animals were boosted three human serum times at weekly intervals by injecting the same dose intra- muscularly. The antisera obtained reacted strongly with AI Preparation of pseudoglobulin. Usually 500-ml batches forming a precipitin band on "Ouchterlony and immuno- of fresh human serum were processed in the following electrophoretic analysis. In order to test whether the anti- fashion. The conductance was lowered to 2.5 mmho/cm by body was capable of inhibiting AI activity, a dose of AI dilution with approximately 2 liters of distilled water, and sufficient to inactivate S ,ug of C3a was incubated for 30 the pH was adjusted to pH 7.0 with 1 N HCG. After standing min at 37°C with either 0.3 ml of antiserum or the equiva- overnight at 4VC, the resulting precipitate was removed by lent amount of 'y-globulin isolated from this antiserum by centrifugation. Pevikon block electrophoresis. The antisera were heated for Triethylaminoethyl (TEAE) cellulose chromatography. 30 min at 56°C before use in order to abolish anaphylatoxin The supernatant (approximately 2.5 liters) was applied to inactivator activity present in rabbit serum. a 5 X 80 cm column containing approximately 1600 ml of Antisera to a2-macroglobulin, av-lipoprotein, and hapto- packed TEAE cellulose which had been equilibrated with globin were purchased from Behringwerke AG, Marburg/ 0.02 M phosphate buffer, pH 7.0 (starting buffer I). Follow- Lahn, Germany. ing the application of the sample, the column was washed with 4 liters of starting buffer. The protein was then eluted Molecular weight determinations with a NaCl concentration gradient. A 3 liter beaker, which served as mixing chamber and contained 2500 ml of The molecular weight of the AI was calculated from the starting buffer, was connected by siphon to a second 3 liter sedimentation (s) and diffusion coefficient (D), assuming beaker filled with 2500 ml of starting buffer containing a partial specific volume of 0.73. s was estimated by ultra- 0.3 M NaCl. The flow rate was adjusted to 100 ml/hr and centrifugation in a 6.9-31.0% linear sucrose density gradient 20-ml fractions were collected. The fractions containing the using an L2 Spinco machine and an SW 50 rotor. D was AI were pooled and concentrated to approximately 50 ml estimated according to Andrews (5) employing a 3.5 X 170 in an Amicon concentration device using an XM 50 ultra- cm Sephadex column equilibrated with 0.05 M phosphate filter. The AI containing material from four separate TEAE buffer, pH 7.0. Reference substances used were: equine cellulose columns was combined and then dialyzed against cytochrome c (13 X 10- cm'/sec), hemoglobin (6.8 X 10V 0.02 M phosphate buffer, pH 7.0, containing 0.07 M NaCl cm/sec), transferrin (5.0 X 104 cm¶/sec), 'yG globulin-WI (starting buffer II). It was then applied to a second TEAE (3.8 X 10- cm2/sec), and thyroglobulin--I (2.5 X 10' cm'/ column X 50 cm) equilibrated with starting buffer II sec). (3.5 The molecular weight was also determined employing the and the column was washed with 2 liters of this buffer polyacrylamide gel electrophoresis method according to before a NaCl concentration gradient was started. A 2 liter from beaker containing 1500 ml of starting buffer II served as Hedrick and Smith (6). The gel concentrations ranged 6 to 10%. As reference substances with known molecular mixing chamber and was connected by siphon with another and 2 liter beaker filled with 1500 ml 0.02 M phosphate buffer, weights were used: albumin in its monomeric, dimeric, M sodium chloride. The flow rate trimeric form (mol wt 65,000, 130,000, and 195,000, respec- pH 7.0, containing 0.25 tively) and urease (mol wt 485,000), Code: URC, Worth- was adjusted to approximately 70 ml/hr and fractions of 20 blue ml were collected. The fractions containing the AI were ington Biochemical Corp., Freehold, N. J. Bromphenol served as reference for determination of the Rm of the pooled and concentrated to 10 ml as described above. of was dialyzed various proteins. Its position at the end electrophoresis Pevikon block electrophoresis. The material was marked by the tip of a copper wire inserted prior to overnight against barbital buffer, pH 8.6, ionic strength staining of the gels.