Fink, Edwin

Haberland, Gert L.

Rohen, J. W.

Suzuki, T.

English

Open Access LMU

R e p r i n t f r o m KININOGENASES KALLIKREIN 4 t h Symposium on Physiological Properties and Pharmacological Rationale International Symposium held at the Academy of Sciences and Literature at Mainz, April 25 t h and 26 t h, 1977 Edited by G. L . Haberland, J. W. Rohen and T. Suzuki F. K. S C H A T T A U E R V E R L A G S T U T T G A R T - N E W Y O R K Development of a Radioimmunoassay for Hog Pancreatic Kallikrein and its Application in Pharmacological Studies E. F I N K The estimation of hog pancreatic Kallikrein is conventionally performed by pro-teolytic assays using either synthetic substrates or the natural substrate kininogen. However, these assays are not specific for Kallikrein in that they detect other pro-teases, too. Since we needed an assay suitable for studies in which the pharmacologi-cal effects after administration of Kallikrein are to be associated with the concentra-tion of the enzyme in blood and tissues where other proteases might be present we developed a highly sensitive and specific radioimmunoassay for hog pancreatic Kal-likrein. The radioimmunoassay method is based on the competitive inhibition of the bind-ing of labelled antigeii to its specific antibody by the unknown or by the unlabelled antigen used as a standard. The sensitivity of the method results from the use of radioisotopes and its specificity from the immunological reaction between the anti-gen and its antibody. Materials and methods The Kallikrein used for radioiodination and as standard in the R I A was a highly purified neuraminidase treated preparation (1). Rabbit anti-Kallikrein serum was kindly provided by Bayer A G . As second antiserum donkey anti-rabbit IgG serum, Welcome Company, was used. Na 1 2 5 I was purchased from Farbwerke Hoechst A G , Frankfurt/Main. Buffers: buffer A : 0.015M N a H 2 P 0 4 , 0.15M NaCl, 0.02% Merthiolat, 0.2% bovine serum albumin, 0 .01MEDTA, pH 7.4. Buffer B: 0.015MNaH 2 PO 4 ,0.15 M NaCl, 0.02% Merthiolat, 4% bovine serum albumin, pH 7.4. Buffer C: 0.015M N a 2 H P 0 4 , 0.15M NaCl, pH 7.4. Radioimmunoassay conditions Anti-Kallikrein serum was diluted with buffer B, dilutions of Kallikrein standard samples, unknowns, anti-rabbit IgG serum and 1 2 5I-Kallikrein were prepared with 48 E. Fink buffer A , the 1 2 5I-Kallikrein dilution (250000 cpm/ml) contained 70 ug/ml rabbit IgG. The incubation mixtures were set up according to the following Table 1: Table 1. Incubation mixtures. Buffer A serum Ml Buffer B serum Ml Sample serum Ml Anti-Kallikrein serum Hi 125I-Kallikrein serum Ml N 200 100 — — 100 Bo 200 - - 100 100 Samples 100 - 100 100 100 After an incubation period of 24-48 h 100 ul of the dilution of the second an-tiserum were added followed by 0.5 ml buffer A after 30 min. After 10 min centrifu-gation at 6000g the supernatant was removed by aspiration and the precipitates were counted in a well type gamma counter. P r e p a r a t i o n of 1 2 5 I - K a l l i k r e i n For the labelling of Kallikrein with Na 1 2 5 I a modification (2) of the chloramine-T method of GREENWOOD and H U N T E R (3) was employed. The labelled Kallikrein was separated from free iodine by gel filtration of the reaction mixture through a Sephadex G-75 column (1.3 x 15cm) equilibrated with buffer C. Fractions of 0.8ml were collected. The 5 fractions of the Kallikrein peak with the highest radioactivity were combined and diluted with buffer A to a concentration of 250000 cpm/ml. A specific activity between 40 and 130 uCi/ug was achieved. The preparations were used in the RIA for up to 4 weeks. Results and discussion Radioimmunoassay Fig. 1 shows the binding of 1 2 5I-Kallikrein (28000 cpm) to antibody after incuba-tion at 21°C for 24 hours using increasing dilutions of the antiserum. More than 80% of the 1 2 5I-Kallikrein were bound indicating the high quality of the antiserum. In order to find the optimal incubation conditions the kinetics of the binding of 1 2 5I-Kallikrein to the antibody was studied at 8, 22 and 37°C. The results are sum-marized in Fig. 2. The lowest reaction rate is observed at 8°C. At 37°C the initial binding rate is insignificantly higher than at 22°C and slows down after 8 h. At Radioimmunoassay for Hog Pancreatic Kallikrein 49 k-134 1:200000 1:800000 1:3200000 1:12800000 1:400000 1:1600000 1:6400000 Dilution of ant iserum (Kallikrein) Fig. 1. Antiserum titration curve for 125I-Kallikrein (hog pancreas). For incubation conditions see text. equilibrium, which is reached after different incubation periods for the different temperatures, the amount of bound radioactivity is highest at 22°C. Incubation periods longer than 72h result in a significant decrease of binding. We interpret this phenomenon by loss of immunologic reactivity of the 1 2 5I-Kallikrein and/or the anti-body. Based on these studies incubation at 22°C for 24—48h was chosen for the routine RIA. For the separation of Kallikrein-antibody complex from free Kallikrein by the second antibody method similar studies showed that maximum precipitation was achieved already after 15-min incubation at 22°C. The evaluation of the R I A was done either manually using logit-log graph paper (4; Fig. 3 shows a typical standard curve) or by means of a computer employing the spline approximation method (5). The lowest level of detection for hog pancreatic Kallikrein in the R I A was between 40 and 300 pg per 100 ul sample volume, depend-ing on the quality of the labelled Kallikrein and the incubation conditions. The coef-ficient of variation for triplicates was usually between 6 and 14%. Measurement of K a l l i k r e i n i n serum Since in the pharmacological studies it would be necessary to measure the Kalli-krein concentration in the serum of dogs we investigated the influence of dog serum 50 E. Fink 99-1 2 -, k-70 1 I I 1 1 1 1 1 1 0.04 0.08 0.16 0.31 0.63 1.25 2.5 5 Porcine pancreatic Kallikrein (ng) Fig. 3. Standard curve for the radioimmunoassay of hog pancreatic Kallikrein. For incubation conditions see text. The data are presented in the form of the logit-log plot (4). Radioimmunoassay for Hog Pancreatic Kallikrein 51 99 - . 9 8 -2 -1 0.16 T —r~ 2.5 0.31 0.63 1.25 Porcine pancreatic Kallikrein (ng) k - 7 0 1 20 Fig. 4. Standard curve for the radioimmunoassay of hog pancreatic Kallikrein in the presence of dog serum. • — • without serum, O O and • • in the presence of 20 ul and 100 ul serum. For incuba-tion conditions see text. on the RIA. Fig. 4 demonstrates that the presence of serum has indeed an influence, the slope of the standard curve depends on the amount of serum added to the incuba-tion mixture. Therefore, if serum samples are to be assayed, the standard curve has to be set up under addition of the same volume of serum to the standard incubation mix-tures as applied in the unknowns. The influence of serum on the immunoreactivity of Kallikrein in the RIA may be caused by complex formation with serum inhibitors and/or by unspecific interactions of serum components. The formation of a complex of Kallikrein with (a) serum component(s) was de-monstrated by gel filtration experiments. A mixture of Kallikrein and dog serum was applied to a Sephacryl S-200 column and the eluate was tested by R I A . Two peaks were detected, one in the normal elution position of Kallikrein, the other approxi-mately in the position of the albumin peak. This second peak might represent the complex of Kallikrein with a rantitrypsin. Some proteases were checked for cross-reactivity by the RIA; hog-trypsin and bovine trypsin and chymotrypsin showed no cross-reactivity at all whereas hog sub-mandibular and urinary Kallikreins had immunoreactivities comparable to the pan-creatic Kallikrein. We have not yet investigated Kallikreins of other species. 52 E. Fink Pharmacological studies The R I A for hog pancreatic Kallikrein offers the possibility to study the long known pharmacological effects of Kallikrein in relation to the Kallikrein concentra-tion in the blood. As already described by FREYand K R A U T (6) and FREY, W E R L E and SACKERS (7) intravenous injection of Kallikrein causes an immediate drop of the mean blood-pressure which is due to the release of kinin from kininogen. If Kalli-krein is applied intramuscularly the response of the blood-pressure is delayed and is less dramatical. Fig. 5 summarizes the changes of blood pressure and kininogen concentration in plasma in relation to the Kallikrein concentration in serum after intravenous injec-250-1 2*150 100 -5 0 -100-80--g 6 0 -40 20-1 100 80 H SL 40H 20-Kininogen (ng/mg plasma-prote in) ~i 1 1 1 1 1 J0.5mg J0.5mg J0.5mg Kallikrein (ng/ml serum) T 1 1 1 -Blood-pressure (%) —J— 50 l o ö " ~~I— 150 200 250 300 Time (min) Fig. 5. Changes of mean blood-pressure and plasma kininogen concentration in relation to the Kallikrein concentration after intravenous injection. For details see text. Radioimmunoassay for Hog Pancreatic Kallikrein 53 Time (min) Fig. 6. Response of the mean blood-pressure in relation to the Kallikrein concentration after intramuscu-lar injection. For details see text. tion. After injection of 0.5mg Kallikrein into the left femoral vein of a dog (15 kg body weight, pentobarbital anesthesia) samples of blood were taken from the right femoral vein. The highest concentration of Kallikrein was found in the sample ob-tained within 1 min after injection. The concentration dropped rapidly within 6 min from 530 ng/ml to 300 ng/ml. After this initial phase the half-life of Kallikrein was approximately 1 hour. After repeated injections of Kallikrein basically identical elimination kinetics were observed. The kininogen concentration on the upper pan-nel is expressed as ng bradykinin equivalents releasable per mg plasma protein by in-cubation of plasma samples with trypsin. It was measured in the bioassay with the iso-lated rat uterus (8) using synthetic bradykinin as reference. Immediately after the Kallikrein injection the kininogen concentration dropped from 600 ng bradykinin equivalents per mg plasma protein to 350 ng. This level was relatively constant for 2h in spite of the presence of relatively high concentrations of immunologically active Kallikrein, an indication that the Kallikrein circulating in the blood must be present mainly as an enzymatically inactive form. A second injection of Kallikrein caused a drop of the kininogen concentration below the level of detection. The response of the mean blood-pressure, recorded from the carotid artery, indicates that at the time of the third injection there was still some kininogen present in the blood. After intramuscular injection of Kallikrein the maximum of the Kallikrein con-centration in the blood is much lower and the time period necessary to reach it is longer than after intravenous injection (Fig. 6). The rate of the decrease of the Kal-likrein concentration in the blood is comparable to the slow phase of elimination aft-er intravenous injection. The blood-pressure reached a minimum within 5 min after 54 E. Fink injection when the maximum of the Kallikrein concentration in the blood was not yet reached. After the initial phase the blood-pressure remained constant at a level somewhat lower than before injection. Concluding from the results presented here the radioimmunoassay for porcine pancreatic Kallikrein is a highly suitable tool for the study of the pharmacological ef-fects of this enzyme. It is expected that it will be possible to get new insights into the action and fate of pancreatic Kallikrein in the organism by using this tool. Acknowledgements This work was generously supported by a grant of the Deutsche Forschungsgemeinschaft. The skilful technical assistance of Mrs. C. GÜTTEL is gratefully acknowledged. Anti-Kallikrein serum and hog pancreatic Kallikrein for pharmacological studies were kindly provided by Bayer AG, the highly purified Kallikrein used for iodination and as reference was a gift of Dr. F. FIEDLER. I am very grateful to Prof. P. C. SCRIBA for the kind permission to use the facilities of his laboratory for radioactive work. I am indebted to Dr. F. ERHARDT for his helpful discussions in developing the radioimmunoassay. I wish to thank very much Prof. H. FRITZ for his stimulating discussions and comments and for the in-spiring atmosphere he creates in our department. References (1) FIEDLER, F.: Pig pancreatic kallikreins A and B. In: LORAND, E. (ed.): Methods in enzymology, Vol. 45, 289-303, Academic Press, New York 1976. (2) ERHARDT, F . , I. MARSCHNER, R. C. PICKART, P. C. SCRIBA: Verbesserung und Qualitätskontrolle der radioimmunologischen Thyreotropin-Bestimmung. Z. klin. Chem. klin. Biochem. 1 1 : 381-387 (1973). (3) GREENWOOD, F. C , W. M. HUNTER, J. GLOVER: The preparation of 131I-labeled human growth hor-mon of high specific radioactivity. Biochem. J. 8 9 : 114-123 (1963). (4) RODBARD, D. , W. BRIDSON, P. L. RAYFORD: Rapid calculation of radioimmunoassay results. J . Lab. clin. Med. 74: 770-781 (1969). (5) MARSCHNER, I., F . ERHARDT, P. C. SCRIBA: Calculation of the radioimmunoassay standard curve by "spline function". In: Radioimmunoassay and related procedures in medicine, Vol. I, 111-122, IAEA, Vienna 1974. (6) FREY, E. K., H . KRAUT: Ein neues Kreislaufhormon und seine Wirkung. Naunyn-Schmiedeberg's Arch. exp. Path. Pharmak. 1 3 3 : 1-56 (1928). (7) FREY, E. K., E. WERLE, E. SACKERS: Über die Wirkung intravenöser und intramuskulärer Kallikrein-injektionen beim Menschen. Z. ges. exp. Med. 9 6 : 404—413 (1935). (8) MANN, K., R. GEIGER, E. WERLE: A sensitive kinin liberating assay for kininogenase in rat urine, iso-lated glomeruli and tubules of rat kidney. In: Adv. exp. Med. Biol. (BACK, N., F . SICUTERL G . L. HABERLAND, [eds.]) 70: 65-73, Plenum Press, New York 1976. 

Ein neues Kreislaufhormon und seine Wirkung.

Pig pancreatic kallikreins A

Rapid calculation of radioimmunoassay results.

SACKERS: Über die Wirkung intravenöser und intramuskulärer Kallikreininjektionen beim Menschen.

SCRIBA: Calculation of the radioimmunoassay standard curve by &quot;spline function&quot;. In: Radioimmunoassay and related procedures in medicine, Vol. I, 111-122, IAEA,

SCRIBA: Verbesserung und Qualitätskontrolle der radioimmunologischen Thyreotropin-Bestimmung.

The preparation of 131I-labeled human growth hormon of high specific radioactivity.

WERLE: A sensitive kinin liberating assay for kininogenase in rat urine, isolated glomeruli and tubules of rat kidney.

Development of a Radioimmunoassay for Hog Pancreatic Kallikrein and its Apllication in Pharmacological Studies

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Development of a Radioimmunoassay for Hog Pancreatic Kallikrein and its Apllication in Pharmacological Studies

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