Immunological and chemical properties of mouse alpha 1-protease inhibitors

We previously described the isolation and purification of two similar alpha 1-protease inhibitors from mouse plasma termed alpha 1-PI(E) and alpha 1-PI(T) because of their respective affinities for elastase and trypsin. Some of the biochemical and immunological properties of these proteins are reported. Both are acidic glycoproteins with pI's of 4.1-4.2. The plasma half-life of each inhibitor, determined after administration of the 125I-protein, is approximately 4 h both in normal mice and in mice after induction of the acute phase reaction. The two proteins have almost identical amino acid compositions and similar CNBr peptide maps. Tryptic maps, however, are considerably different. Reverse-phase chromatography separated alpha 1-PI(E) into three distinct isoforms, each eluting with approximately 60% acetonitrile. Under these conditions alpha 1-PI(T) shows a single peak, clearly different from those of alpha 1-PI(E). The three alpha 1-PI(E) isoforms have the same molecular weights on sodium dodecyl sulfate-gel electrophoresis and the same tripeptide sequence at their N-terminus, and appear to be immunologically identical. Polyclonal, monospecific antibodies to each native inhibitor, prepared in rabbits, showed no cross-reactivity when tested by functional assay or crossed immunoelectrophoresis. Interestingly, each antibody recognized epitopes on the C-terminal portion of its respective antigen. These studies confirm that alpha 1-PI(E) and alpha 1-PI(T), although highly similar, are products of different genes. Like human alpha 1-PI, the two mouse inhibitors are partially inactivated by mild oxidation with chloramine-T, losing all elastase inhibitor and lesser amounts of antichymotryptic and antitryptic activity. However, unlike the human protein, neither alpha 1-PI(E) nor alpha 1-PI(T) was found to have a methionine residue at its P1 site.     

Recombinant alpha 1-proteinase inhibitor blocks antigen- and mediator-induced airway responses in sheep.

Alpha(1)-proteinase inhibitor (alpha(1)-PI) is a natural serine protease inhibitor. Although mainly thought to protect the airways from neutrophil elastase, alpha(1)-PI may also regulate the development of airway hyperresponsiveness (AHR), as indicated by our previous findings of an inverse relationship between lung alpha(1)-PI activity and the severity of antigen-induced AHR. Because allergic stimulation of the airways causes release of elastase, tissue kallikrein, and reactive oxygen species (ROS), all of which can reduce alpha(1)-PI activity and contribute to AHR, we hypothesized that administration of exogenous alpha(1)-PI should protect against pathophysiological airway responses caused by these agents. In untreated allergic sheep, airway challenge with elastase, xanthine/xanthine oxidase (which generates ROS), high-molecular-weight kininogen, the substrate for tissue kallikrein, and antigen resulted in bronchoconstriction. ROS and antigen also induced AHR to inhaled carbachol. Treatment with 10 mg of recombinant alpha(1)-PI (ralpha(1)-PI) blocked the bronchoconstriction caused by elastase, high-molecular-weight kininogen, and ROS, and the AHR induced by ROS and antigen. One milligram of ralpha(1)-PI was ineffective. These are the first in vivo data demonstrating the effects of ralpha(1)-PI. Our results are consistent with and extend findings obtained with human plasma-derived alpha(1)-PI and suggest that alpha(1)-PI may be important in the regulation of airway responsiveness.     

Inactivation of serine proteinase inhibitors (serpins) in human plasma by reactive oxidants

Activated polymorphonuclear neutrophils (PMN) and macrophages generate oxidizing agents similar to or identical with N-chloroamines. Mimicking this oxidation in normal human plasma by usage of chloramine T (CT), we observed an oxidant concentration-dependent inactivating effect on plasma alpha 2-plasmin inhibitor (alpha 2-PI), antithrombin III (AT III), and alpha 1-proteinase inhibitor (alpha 1-PI). 20-50 mumol CT/ml plasma are necessary for almost complete inactivation of alpha 2-PI and AT III-activity, i.e. about 2-5 times the dose necessary for inactivation of alpha 1-PI which has already been classified as "oxidant sensitive". The inactivation of alpha 1-PI, alpha 2-PI and AT III in plasma by oxidants is the result of a specific oxidative damage since C1-inhibitor, serine proteinases and complexes of plasmin and alpha 2-PI were chloramine resistant under the conditions used. According to our results, the amount of chloramines released by 1 x 10(6) activated PMN, namely ca. 10 nmol (see Weiss et al. Science 222 625-628, 1983) would be sufficient to destroy alpha 1-PI and alpha 2-PI activity of 1.5 and 0.4 microliter of human plasma, respectively. Consequently, activated leukocytes may be able to create a microenvironment in which elastase as well as plasmin and thrombin can display their proteolytic activity unchecked by their regulator proteins. Oxidation may provide a general basis for altering enzyme/inhibitor balances.     

The in vitro interactions of rat pancreatic elastase and normal and inflammatory ray serum.

The partition of labelled rat pancreatic elastase (EC 3.4.21.11) between the different protease inhibitors of rat plasma was studied at different levels of saturation of the inhibitors of rat plasma was studied at different levels of saturation of the inhibitor capacity of plasma with the enzyme. The reaction mixtures were analysed by immunoelectrophoretic methods utilizing specific antisera against the different inhibitors and by gel filtration on Sephadex G-200. Rat serum was shown to contain four elastase binding proteins. alpha 1-antitrypsin, alpha 1-macroglobulin and alpha 2-acute phase protein and alpha 1-inhibitor 3 which exhibits immunologic cross-reaction with human inter-alpha-trypsin inhibitor and is of similar molecular weight. With minute amounts of labelled elastase the partition among the binding protein was alpha 1-macroglobulin 60%, alpha 1-antitrypsin 24% and alpha 1-I3 16%. The 60% value of alpha 1-M bound radioactivity in normal serum corresponds to the sum of alpha 1-M and alpha 2-AP labelling in inflammatory serum.     

Aprotinin turn-over studies in dog and in man with severe acute pancreatitis

The elimination of aprotinin after intravenous infusion was exponential until 95% of the dose was cleared from the plasma after 1 h in dogs with bile-induced pancreatitis. The half-life of this part of the elimination was 10 min. The concentration of aprotinin in the peritoneal fluid reached a maximum plateau after 1 h. Direct intra-abdominal infusion of aprotinin was followed by a relatively slow elimination of the inhibitor from the cavity. One hour after the infusion the concentration of aprotinin in the peritoneal exudate was about 50% of the initial value. Four hours later the concentration of inhibitor with unchanged immunoreactivity and inhibiting capacity was still about 25% of the initial value. Based on the results of this experimental study an intraperitoneal dosage schedule for aprotinin was tested in three patients with haemorrhagic pancreatitis. A total amount of 14 X 10(6) KIU was given in repeated dosages during 18 h. This resulted in a minimum level of aprotinin in the peritoneal exudate of about 10 mumol/l. According to our earlier published data this level should largely block trypsin-induced effects relevant in pancreatitis. In conclusion; due to the rapid elimination of aprotinin from plasma, after i.v. application a therapeutically useful concentration is never reached in the peritoneum, while the elimination from the peritoneum is relatively slow, thus providing therapeutically useful concentrations which can be maintained for some time after i.p. application.     

Quantification of kininogen in human renal medulla

Renal kininogen was detected in human medullary tissue as well as human medullary tubule suspensions. After treatment with pig pancreatic kallikrein or human renal cortical homogenate liberated kinin was measured by bradykinin radioimmunoassay. In the absence of inhibitors kinins were degraded by kininases located in the same part of the kidney. Several known inhibitors of kininase I and II did not inhibit this activity. Endogenous medullary kininase was inhibited by preincubation of homogenates at 56 degrees C for one hour or by addition of 0.25 mmol/l HgCl2. Under these conditions endogenous medullary kinin release amounted to 9-26 nmol/g protein. The action of renal cortical kininogenase on kinin formation from papillary kininogen was completely inhibited by addition of 1 mumol/l aprotinin. Kininogen examined in renal tubule suspensions revealed an increase in amount per g protein compared to homogenates, confirming the tubular localization of renal kininogen.     

Mouse alpha 1-protease inhibitor is not an acute phase reactant

Mouse plasma contains two major protease inhibitors, alpha 1-protease inhibitor (alpha 1-PI) and contrapsin, which have high affinity for bovine trypsin. Systemic injury, such as turpentine-induced inflammation, did not change the plasma concentration of alpha 1-PI, but increased that of contrapsin by 50%. The concentration of hepatic alpha 1-PI mRNA was determined by Northern blot hybridization and was not significantly affected by the acute phase reaction. J.M. Frazer, S.A. Nathoo, J. Katz, T.L. Genetta, and T.H. Finley [1985) Arch. Biochem. Biophys. 239, 112-119) have reported a threefold increase of mRNA for the elastase specific alpha 1-PI but this increase was not demonstrated by the present study. The mRNAs for known mouse acute phase plasma proteins were, however, stimulated severalfold by the same treatment. These results indicate that in the mouse, as opposed to human, alpha 1-PI is not an acute phase reactant.     

Microheterogeneity of human kininogen by isoelectric focusing and crossed immunoelectrophoresis.

LMW kininogen was isolated from whole human plasma by gel filtration on Sephadex G-200 (Kav 0.34) followed by DEAE-chromatography according to earlier established methods. Further purification was performed with specific Sepharose-antibody columns to remove protein contaminants, avoiding procedures which may denature kininogen. The microheterogeneity was investigated by isoelectric focusing in column in the pH-gradients 3.5-10, 4-6 and 3.5-5. Kininogen components were determined by single radial immunodiffusion against monospecific anti-human kininogen serum, in comparison with focusing of whole plasma. 40% of isolated as well as whole plasma kininogen focused at pI 4.5; the respective focusing ranges were pI 4.4-4.7 (60--80%) and pI 4.3-4.6 (92%). The results were verified by crossed immunoelectrophoresis. The pI 4.5 component is apparently the main native form of human kininogen as shown by focusing of whole human blood bank plasma. Earlier described difficulty of separating kininogen and alpha2HS-glycoprotein was verified by crossed immunoelectrophoresis which showed approximately seven kininogen components after focusing in polyacrylamide gel electrophoresis at pI 4.5-5.0 and four alpha 2HS components at pI 4.2-4.6.     

Plasma protein and liver mRNA levels of two closely related murine alpha 1-protease inhibitors during the acute phase reaction

Plasma levels of alpha 1-PI(T) and alpha 1-PI(E), two closely related murine alpha 1-protease inhibitors, having affinities for trypsin and elastase, respectively, were compared to changes in specific liver mRNA levels after induction of the acute-phase reaction by subcutaneous injection of turpentine. In earlier, qualitative experiments an increase in plasma levels of alpha 1-PI(E), but not alpha 1-PI(T), during the acute-phase reaction had been shown. It is now shown that stimulation of plasma alpha 1-PI(E) levels reaches a maximum of 35-50% above baseline 12 h after induction of the acute-phase response using either a functional or immunological assay to measure protease inhibitor activity. Consistent with earlier observations, little or no change in plasma levels of alpha 1-PI(T) is seen. Determination of mRNA levels in the mouse liver specific for alpha 1-PI(E) and alpha 1-PI(T) was accomplished using a cell-free translation system followed by immunoprecipitation of the 35S-labeled protease inhibitors. The apparent Mr's of alpha 1-PI(E) and alpha 1-PI(T) synthesized in vitro are 42K and 46K, respectively. Apparent Mr's of the native proteins in plasma are 55K and 65K. Unexpectedly, mRNA levels for both alpha 1-PI(E) and alpha 1-PI(T) were found to increase after induction of the acute-phase reaction. Maximal stimulation for both mRNAs was approximately 300% and occurred 9 h after turpentine administration. Under these conditions, levels of translatable albumin mRNA in the mouse liver decreased to 40% of baseline in 6-9 h.     

Isolation and characterization of alpha1-proteinase inhibitor from common carp (Cyprinus carpio) seminal plasma

Using a three-step procedure, we purified (79 and 51.6-fold to homogeneity) and characterized the two isoforms (a and b) of alpha1-proteinase inhibitor-like protein from carp seminal plasma. The isoforms have molecular masses of 55.5 and 54.0 kDa, respectively. These inhibitors formed SDS-stable complexes with cod and bovine trypsin, chymotrypsin and elastase. The thirty-three amino acids within the reactive loop SLPDTVILNRPFLVLIVEDTTKSILFMGKITNP were identified for isoform b. The same first ten amino acids were obtained for isoform a, and this sequence revealed 100% homology to carp alpha1-proteinase inhibitor (alpha1-PI) from perimeningeal fluid. Both isoforms of alpha1-PI are glycoproteins and their carbohydrate content was determined to be 12.6 and 12.1% for a and b, respectively. Our results indicated that alpha1-PI is one of the main proteins of carp seminal plasma. Using polyclonal anti-alpha1-PI antibodies, alpha1-PI was for the first time localized to the carp testis. The presence of alpha1-PI in testis lobules and in the area surrounding spermatides suggests that this inhibitor may be involved in the maintenance of testis connective tissue integrity, control of spermatogenesis or protection of tissue and spermatozoa against unwanted proteolysis. Since similar alpha1-PI has been identified in rainbow trout semen it can be suggested that the presence of alpha1-PI in seminal plasma is a common feature of cyprinid and salmonid fish.     

Purification and biochemical characterization of ovine alpha-1-proteinase inhibitor: mechanistic adaptations and role of Phe350 and Met356

The glycoprotein alpha-1-proteinase inhibitor (alpha-1-PI) is a member of the serpin super family that causes rapid and irreversible inhibition of redundant serine protease activity. A homogenous preparation of ovine alpha-1-PI, a 60 kDa protein was obtained by serially subjecting ovine serum to 40-70% (NH(4))(2)SO(4) precipitation, Blue Sepharose, size-exclusion, and concanavalin-A chromatography. Extensive insights into the trypsin, chymotrypsin, and elastase interaction with ovine alpha-1-PI, point towards the involvement of Phe(350) besides the largely conserved Met(356) in serine protease recognition and consequent inhibition. The N-terminal of C-terminal peptides cleaved on interaction with elastase, trypsin, and chymotrypsin prove the presence of diffused sub-sites in the vicinity of Met(356) and the strategically positioned Pro anchored peptide stretch. Further, human alpha-1-PI is more thermolabile compared to ovine alpha-1-PI, higher thermolability is mainly attributed to poorer glycosylation. The enzymatic deglycosylation of human and ovine alpha-1-PI results in diminished thermostability of the inhibitors, with sharp decrease in thermal transition temperatures but retaining their inhibitory potency. Homology modeling of the deduced amino acid sequence of ovine alpha-1-PI using the human alpha-1-PI template has been used to explain the observed inhibitor-protease interactions.     

Acute phase plasma proteins in kininogen-deficient Brown Norway rats

We examined whether Brown Norway rat plasma (BN/May Pfd f) contains alpha 1-cysteine proteinase inhibitor (alpha 1-CPI), also called major acute phase alpha 1-protein or T-kininogen. T-kininogen is a low molecular weight kininogen from which kinin can be released by trypsin but not by kallikreins. The BN plasma reacted with rabbit anti-alpha 1-CPI gamma globulins. Purified alpha 1-CPI released a kinin-like activity with trypsin and with homogenate of salivary glands, as Brown Norway rat plasma did. High concentration of added rat urine induced a small release (10%) of kinin from alpha 1-CPI. Preincubation of Brown Norway rat plasma with rabbit anti-rat alpha 1-CPI gamma-globulins nearly suppressed the kinin-forming substrate of trypsin in this plasma. These results indicated that plasma of our Brown Norway rats contains only alpha 1-CPI as kinin-forming substrate. This plasma contains low amount of alpha 2-macroglobulin, while its content in orosomucoid and haptoglobin was a little larger than that of Wistar rat plasma.     

Development and evaluation of an ELISA for quantification of human alpha-1-proteinase inhibitor in complex biological mixtures

Human alpha-1-proteinase inhibitor(1) (alpha(1)-PI) is the most abundant serine protease inhibitor in plasma. Its major function is inhibition of neutrophil elastase in lungs. alpha(1)-PI deficiency may result in severe, ultimately fatal emphysema. Three plasma-derived (pd-) alpha(1)-PI products are licensed in the US for replacement therapy of deficient patients. The recombinant versions (r-alpha(1)-PI), proposed as alternatives to pd-alpha(1)-PI products, have been under intensive investigation. For accurate determination of alpha(1)-PI from different sources and in various forms, there is an obvious need for reliable standardized assays for alpha(1)-PI quantification and potency measurements. As a part of our multi-step research focused on alpha(1)-PI structure-function investigation, we have established a simple and reproducible double-sandwich ELISA based on commercially available polyclonal antibodies. The developed ELISA allows the quantification of both pd-alpha(1)-PI and r-alpha(1)-PI in various complex matrices. A validation of the ELISA was performed with the working range of the assay (3.1-50 ng/ml) established on the bases of the following parameters: linearity (3-100 ng/ml, r(2)=0.995); accuracy (87.3-114.6% recovery); intra-assay precision (%CV, 2.8%); inter-assay plate-to-plate precision (3.9% per day and 4.1% day-to-day); detection limit (1.10 ng/ml); and quantification limit (3.34 ng/ml). The analytical performance of the alpha(1)-PI ELISA indicates that this assay can be used for monitoring concentration levels of alpha(1)-PI in multi-component biological matrices, based on the following: (a) quantification of r-alpha(1)-PI in various fermentation mixtures (E. coli and A. niger); (b) investigation of alpha(1)-PI enzymatically digested in the conditions of harsh fungal proteolysis; (c) evaluation of thermally polymerized alpha(1)-PI; (d) quantification of alpha(1)-PI in human serum; and (e) comparative quantification of alpha(1)-PI in commercially available products.     

Increased plasma concentrations of C9, C1-inhibitor and alpha 1-protease inhibitor associated with cigarette smoking

The association between various parameters of acute and chronic smoking status and plasma levels of three proteins, C9, C1-inhibitor (C1-INH) and alpha 1-protease inhibitor (alpha 1-PI) were determined for 49 male cigarette smokers and 49 age-matched nonsmokers (mean age = 32.2 years). The mean number of cigarettes smoked was 28.7 per day while the cumulative consumption was only 18.1 pack-years. Plasma levels of all three proteins were significantly higher in the smokers than nonsmokers. Plasma C9 and alpha 1-PI concentrations correlated with cumulative cigarette consumption and plasma nicotine concentrations. While C1-INH concentration did not correlate with either cumulative cigarette consumption or plasma nicotine concentration, it correlated significantly with serum thiocyanate concentration. No consistent correlation was found between plasma concentration of these proteins and parameters of pulmonary function.     

Differential inhibitory properties of dog and human alpha 1-proteinase inhibitors

Dog alpha 1-proteinase inhibitor (alpha 1-PI) was found to be an effective inhibitor of bovine chymotrypsin and also of porcine pancreatic elastase as in the case of human inhibitor. The dog inhibitor inactivated both proteinases at a molar ratio of 1:1. However, compared to the human inhibitor, dog alpha 1-PI was a relatively poor inhibitor of bovine trypsin. The association rate constants (kass) of the interactions of dog alpha 1-PI with bovine chymotrypsin and with porcine elastase were determined to be 6.9 +/- 0.3 X 10(6) M-1 s-1 and 6.4 +/- 0.1 X 10(5) M-1 s-1, respectively. These values are 1.3- and 2.7-fold higher than the corresponding values for the human inhibitor. On the other hand, kass for the dog inhibitor with bovine trypsin (2.6 +/- 0.3 X 10(4)M-1 s-1) was found to be about 5 times smaller than that of the human inhibitor.     

Structure of human alpha 2-plasmin inhibitor deduced from the cDNA sequence

We have isolated three cDNA clones for human alpha 2-plasmin inhibitor (alpha 2-PI). Two clones are from human hepatoma cell line, Hep G2, and cover the entire protein coding region plus the 3'-flanking region up to the poly(A) sequence, and the other clone is from human liver and contains the carboxyl-terminal half. The total length of the cDNAs is 2.29 kb, corresponding to more than 95% of the full-length mRNA. alpha 2-PI seems to consist of 452 amino acid residues plus 39 amino acid residues for the signal peptide. The amino acid sequence shows 23 to 28% homology to those of five other protease inhibitors, plasminogen activator inhibitor (PAI), protein C inhibitor (PCI), alpha 1-antitrypsin (alpha 1-AT), antithrombin III (AT III), and alpha 1-antichymotrypsin (alpha 1-AC). alpha 2-PI seems to be the most distantly related among these inhibitors. Comparison of the phylogenetic trees of proteases and their inhibitors indicates that four proteases, namely elastase (or trypsin), chymotrypsin, plasminogen activator, and thrombin, may have evolved concurrently with the corresponding inhibitors. However, alpha 2-PI and PCI seem to have evolved asynchronously from their substrates. The data suggest that alpha 2-PI may originally have inhibited some protease other than plasmin, and protein C may have had an inhibitor different from the present one early in its evolutionary history.     

Isolation of an enzymatically active tissue kallikrein from human seminal plasma by immunoaffinity chromatography

A tissue kallikrein from human seminal plasma was isolated by immunoaffinity chromatography and characterized. Its molecular mass was determined by gel filtration to be approximately 40000 Da. The enzyme preparation liberates kinin from human HMW kininogen (specific activity: 0.594 HMW kininogen-U/mg), lowers the blood pressure of dogs after intravenous injection (specific activity: 1740 biol. kallikrein unit/mg) and is strongly inhibited by aprotinin but not by soybean trypsin inhibitor. N alpha-Acetyl-L-phenylalanyl-L-arginine ethyl ester, D-valyl-L-leucyl-L-agrine ethyl ester and N-benzyloxycarbonyl-L-tyrosine p-nitrophenyl ester are cleaved with identical rates by the enzyme from human seminal plasma and human urinary kallikrein.     

Myeloperoxidase-catalyzed inactivation of alpha 1-protease inhibitor by human neutrophils

We have examined the effect of the myeloperoxidase-hydrogen peroxide-halide system and of activated human neutrophils on the ability of serum alpha 1-protease inhibitor (alpha 1-PI) to bind and inhibit porcine pancreatic elastase. Exposure to the isolated myeloperoxidase system resulted in nearly complete inactivation of alpha 1-PI. Inactivation was rapid (10 to 20 s); required active myeloperoxidase, micromolar concentrations of H2O2 (or glucose oxidase as a peroxide generator), and a halide cofactor (Cl- or I-); and was blocked by azide, cyanide, and catalase. Intact neutrophils similarly inactivated alpha 1-PI over the course of 5 to 10 min. Inactivation required the neutrophils, a halide (Cl-), and a phorbol ester to activate secretory and metabolic activity. It was inhibited by azide, cyanide, and catalase, but not by superoxide dismutase. Neutrophils with absent myeloperoxidase or impaired oxidative metabolism (chronic granulomatous disease) failed to inactivate alpha 1-PI, and these defects were specifically corrected by the addition of myeloperoxidase or H2O2, respectively. Thus, stimulated neutrophils secrete myeloperoxidase and H2O2 which combine with a halide to inactivate alpha 1-PI. We suggest that leukocyte-derived oxidants, especially the myeloperoxidase system, may contribute to proteolytic tissue injury, for example in elastase-induced pulmonary emphysema, by oxidative inactivation of protective antiproteases.     

Ig-binding bacterial proteins also bind proteinase inhibitors

Protein G is a streptococcal cell wall protein with separate binding sites for IgG and human serum albumin (HSA). In the present work it was demonstrated that alpha 2-macroglobulin (alpha 2M) and kininogen, two proteinase inhibitors of human plasma, bound to protein G, whereas 23 other human proteins showed no affinity. alpha 2M was found to interact with the IgG-binding domains of protein G, and in excess alpha 2M inhibited IgG binding and vice versa. A synthetic peptide, corresponding to one of the homologous IgG-binding domains of protein G, blocked binding of protein G to alpha 2M. Protein G showed affinity for both native and proteinase complexed alpha 2M but did not bind to the reduced form of alpha 2M, or to the C-terminal domain of the protein known to interact with alpha 2M receptors on macrophages. Binding of protein G to alpha 2M and kininogen did not interfere with their inhibitory activity on proteinases, and the interaction between protein G and the two proteinase inhibitors was not due to proteolytic activity of protein G. The finding that protein G has affinity for proteinase inhibitors was generalized to comprise also other Ig binding bacterial proteins. Thus, alpha 2M and kininogen, were shown to bind both protein A of Staphylococcus aureus and protein L of Peptococcus magnus. The results described above suggest that Ig-binding proteins are involved in proteolytic events, which adds a new and perhaps functional aspect to these molecules.     

Purification and partial characterization of feline alpha1-proteinase inhibitor (falpha1-PI) and the development and validation of a radioimmunoassay for the measurement of falpha1-PI in serum

Alpha(1)-proteinase inhibitor (alpha(1)-PI) of the domestic cat (Felis catus) was purified from serum and a radioimmunoassay (RIA) for the measurement of feline alpha(1)-PI concentration in serum was developed and validated. Feline alpha(1)-PI (falpha(1)-PI) was isolated using ammonium sulfate precipitation, anion-exchange, size-exclusion, ceramic hydroxyapatite, and hydrophobic interaction chromatography. The molecular weight of falpha(1)-PI was estimated at 57,000 and the relative molecular mass (M(r)) was determined to be approximately 54.5 kDa. Isoelectric focusing revealed four bands with isoelectric points (pI) between 4.3 and 4.5. The N-terminal amino acid sequence of the first 19 residues was Glu-Gly-Leu-Gln-Gly-Ala-Ala-Val-Gln-Glu-Thr-Val-Ala-Ser-Gln-His-Asp-Gln-Glu. Antiserum against feline alpha(1)-PI was raised in rabbits. Tracer was produced by iodination ((125)I) of feline alpha(1)-PI using the chloramine T method. A radioimmunoassay was established and validated by determination of sensitivity, dilutional parallelism, spiking recovery, intra-assay variability, and inter-assay variability. A control range for serum feline alpha(1)-PI concentration was established from 50 healthy cats using the central 95th percentile. The sensitivity of the assay was 0.042 mg/ml. Observed to expected ratios for serial dilutions ranged from 105% to 141.18% for four different serum samples at dilutions of 1 in 35,000, 1 in 70,000, 1 in 140,000 and 1 in 280,000. Observed to expected ratios for spiking recovery ranged from 88.14% to 152.17% for four different serum samples and five different spiking concentrations. Coefficients of variation for four different serum samples were 4.57%, 6.45%, 8.52%, and 4.27% for intra-assay variability and 6.88%, 9.57%, 7.44%, and 9.94% for inter-assay variability. The reference range was established as 0.25-0.6 mg/ml. In summary, feline alpha(1)-PI was successfully purified from serum using a rapid and efficient method. The radioimmunoassay described here is sensitive, linear, accurate, precise, and reproducible and will facilitate further studies of the physiological or potential pathological role of alpha(1)-PI in cats.