A method to evaluate the antioxidant system for radicals in erythrocyte membranes

The erythrocyte defense system against cellular oxidants is complex and efficient. Free radicals generated in cell membranes, however, are relatively sequestered from the cell's antioxidant mechanisms. When an oxidant challenge exceeds the capacity of the erythrocyte's antioxidant system, membrane damage may occur, causing red cell destruction and hemolytic anemia. In this study, we present a method for monitoring radical reduction in erythrocyte membranes, using fatty acid spin labels with nitroxide radicals on the hydrocarbon chains. About 50 microL of packed (about 5-6 x 10(8)), carbon monoxide (CO)-gassed red blood cells are used. The electron paramagnetic resonance signals of the 5-doxylstearic acid spin labels in the intact cells are obtained as a function of time, at 37 degrees C over a period of 2 h. The pseudo first-order rate constant for reduction of the spin label in normal adult intact cells under our experimental conditions is 4.3 +/- 1.8 x 10(-3)/min. The reproducibility and variability of the measurements are discussed. Since the measurements we describe reflect the extent of radical reductions occurring in cell membranes, we suggest that this method can be used to measure the ability to defend oxidants in membranes of erythrocytes with defective antioxidant systems. This method is particularly useful for measuring the modification of the antioxidant system toward radicals in membranes by drugs, chemicals, or environmental toxins.     

Nitric oxide inhibition of free radical-mediated cholesterol peroxidation in liposomal membranes

The ability of nitric oxide ((*)NO) to inhibit propagative lipid peroxidation was investigated using unilamellar liposomes (LUVs) constituted with egg phosphatidylcholine (PC) or 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), [(14)C]cholesterol (Ch), and a nonregenerable singlet oxygen-derived primer, 5alpha-hydroperoxycholesterol (5alpha-OOH). Exposing LUVs to ascorbate and a lipophilic iron chelate at 37 degrees C resulted in an exponential decay of 5alpha-OOH and accumulation of free radical-derived 7alpha- and 7beta-hydroperoxycholesterol (7alphabeta-OOH), as detected by high-performance liquid chromatography with electrochemical detection. Thiobarbituric acid-reactive species (TBARS) were generated concurrently in egg PC-containing LUVs. Including the (*)NO donor spermine NONOate (SPNO, 5-50 microM) or S-nitroso-N-acetyl-D,L-penicillamine (SNAP, 50-100 microM) in the reaction mixture had no effect on 5alpha-OOH decay (suggesting that iron was not redox-inhibited) but slowed TBARS and 7alphabeta-OOH accumulation in a strongly dose-dependent fashion. Decomposed SPNO or SNAP had no such effects, implying that (*)NO was the responsible agent. Accumulation of several [(14)C]Ch oxidation products, detected by high-performance thin-layer chromatography with phosphorimaging, was similarly diminished by active SPNO or SNAP. Concomitantly, a new band referred to as RCh.4 appeared, the radioactivity of which increased as a function of incubation time and (*)NO donor concentration. RCh.4 material was also generated via direct iron/ascorbate reduction of 7alpha-OOH in the presence of (*)NO, consistent with 7alpha-nitrite (7alpha-ONO) identity. However, various other lines of evidence suggest that RCh.4 is not 7alpha-ONO, but rather 5alpha-hydroxycholesterol (5alpha-OH) generated by reduction of 5alpha-ONO arising from 7alpha-ONO rearrangement. 5alpha-OH was only detected when (*)NO was present in the reaction system, thus providing indirect evidence for the existence of nitrosated Ch intermediates arising from (*)NO chain-breaking activity.     

Effect of diamide on protein oxidation and physico-chemical properties of lipids in erythrocyte membranes

The effect of diamide on the physicochemical state of proteins and lipids of human erythrocyte membrane was studied. It was found that diamide at a concentration of 1 mM decreases the content of the SH-groups of membrane proteins by approximately 50%, resulting in enhanced vesiculation of erythrocytes upon metabolic exhaustion of cells. It was shown using fluorescein isothiocyanate-labeled concanavalin A and 4,4'-diisothiocyano-2,2'-stilbene disulfonate that diamide changes the structural state of the main integral protein of erythrocyte membranes, the band 3 protein. Changes in the microviscosity of the membrane lipid bilayer depending on diamide concentration were determined from the changes in the fluorescence parameters of the lipophilic probes (pyrene and 1,6-diphenyl-3,5-hexatriene). The level of lipid peroxidation products in membranes remained unchanged. It follows from these data that the SH-oxidizing agent diamide does not directly interact with the lipid bilayer of membrane and produces changes in the physicochemical state of lipids presumably by disrupting protein-lipid interactions that take place upon oxidation of the SH-groups and cross-linking of membrane proteins.     

Free radical-mediated oxidation of free amino acids and amino acid residues in proteins

Summary. We summarize here results of studies designed to elucidate basic mechanisms of reactive oxygen (ROS)-mediated oxidation of proteins and free amino acids. These studies have shown that oxidation of proteins can lead to hydroxylation of aromatic groups and aliphatic amino acid side chains, nitration of aromatic amino acid residues, nitrosylation of sulfhydryl groups, sulfoxidation of methionine residues, chlorination of aromatic groups and primary amino groups, and to conversion of some amino acid residues to carbonyl derivatives. Oxidation can lead also to cleavage of the polypeptide chain and to formation of cross-linked protein aggregates. Furthermore, functional groups of proteins can react with oxidation products of polyunsaturated fatty acids and with carbohydrate derivatives (glycation/glycoxidation) to produce inactive derivatives. Highly specific methods have been developed for the detection and assay of the various kinds of protein modifications. Because the generation of carbonyl derivatives occurs by many different mechanisms, the level of carbonyl groups in proteins is widely used as a marker of oxidative protein damage. The level of oxidized proteins increases with aging and in a number of age-related diseases. However, the accumulation of oxidized protein is a complex function of the rates of ROS formation, antioxidant levels, and the ability to proteolytically eliminate oxidized forms of proteins. Thus, the accumulation of oxidized proteins is also dependent upon genetic factors and individual life styles. It is noteworthy that surface-exposed methionine and cysteine residues of proteins are particularly sensitive to oxidation by almost all forms of ROS; however, unlike other kinds of oxidation the oxidation of these sulfur-containing amino acid residues is reversible. It is thus evident that the cyclic oxidation and reduction of the sulfur-containing amino acids may serve as an important antioxidant mechanism, and also that these reversible oxidations may provide an important mechanism for the regulation of some enzyme functions.     

Iron stimulation of free radical-mediated porphyrinogen oxidation by hepatic and renal mitochondria

Mitochondria from rat liver and kidney catalyze oxidation of uroporphyrinogen in the presence of NADH or succinate and the respiratory chain inhibitor, NaN3. The rate of porphyrinogen oxidation was substantially accelerated when iron as Fe+3-EDTA was added to reaction mixtures. This effect was partially attenuated by catalase, reduced glutathione (GSH) and other free radical scavengers. These results suggest that iron stimulates free radical-mediated porphyrinogen oxidation by tissue mitochondria under conditions of perturbed mitochondrial respiratory function. These observations suggest a mechanism by which iron could contribute to excess porphyrin excretion in various inherited or chemically-induced porphyrias.     

Nitric oxide inhibition of free radical-mediated lipid peroxidation in photodynamically treated membranes and cells

Of the numerous biological activities attributed to nitric oxide ((*)NO), relatively little is known about its ability to intercept lipid-derived free radicals and thus protect cells against the damaging effects of lipid peroxidation, particularly in photodynamic settings. To address this, we asked how the (*)NO donor spermine-NONOate (SPER/NO) would affect porphyrin (PpIX)-photosensitized, iron/ascorbate-amplified chain peroxidation in cholesterol (Ch)/phospholipid (0.8:1.0, mol/mol) liposomes. Several Ch oxidation products (ChOX) were monitored by high performance chromatographic techniques. When added immediately before irradiation, SPER/NO (0.4 mM) had no effect on accumulation of 5alpha-hydroperoxide, a primary singlet oxygen-derived ChOX, but strongly suppressed the secondary species arising from postphotooxidation chain reactions, including 7alpha/7beta-hydroperoxides, 7alpha/7beta-hydroxides, and 5,6-epoxides. Metabolism of exogenous 5-aminolevulinate to PpIX in COH-BR1 tumor cells sensitized them to ChOX photogeneration and necrotic photokilling. When present during irradiation, active (but not decomposed) SPER/NO strongly inhibited both effects. These findings support the hypothesis that suitably presented NO, by intercepting lipid-derived radicals, can antagonize the antitumor effects of photodynamic therapy and other oxidative therapies.     

Kinetics and mechanism of the chemiluminescence associated with the free radical-mediated oxidation of amino acids

When amino acids are incubated in the presence of a free radical source [2,2′-azobis(2-amidinopropane) dihydrocloride], only tyrosine (Tyr) and tryptophan (Trp) produce significant chemiluminescence. The relationship between the observed light intensity, the rate of the oxidation process and the substrate concentration is complex and can not be explained in terms of the formation of excited states in termination processes involving two peroxyl radicals (Russell's mechanism). The observed increase in light emission with the incubation time, for both Trp and Tyr, would indicate the participation of more than one reaction product as intermediates in the pathways leading to the production of excited molecules. However, the fact that after product accumulation a high proportion of the observed luminescence is quenched by Trolox addition, implies that the main chemiluminescent process must involve the interaction of product(s) and free radicals. From the effect of added Ebselen, it is proposed that hydroperoxides and peroxides, formed along the reaction path, are the intermediates whose accumulation leads to the observed increase in chemiluminescence with elapsed time. The observed time profiles and the proposed mechanism strongly resemble those associated with the oxidation of complex biological systems, suggesting that protein oxidation could be one of the main sources of chemiluminescence in biological oxidations. Copyright © 2000 John Wiley & Sons, Ltd.     

2'-Deoxycytidine glycols, a missing link in the free radical-mediated oxidation of DNA.

2'-Deoxycytidine glycols (5,6-dihydroxy-5, 6-dihydro-2'-deoxycytidine) are major products of the hydroxyl radical-induced oxidation of 2'-deoxycytidine resulting from either a Fenton reaction or exposure to ionizing radiation. Because of their instability, however, the glycols have not previously been characterized. Instead, the impetus has been placed on the primary decomposition products of 2'-deoxycytidine glycols, which includes 5-hydroxy-2'-deoxycytidine, 5-hydroxy-2'-deoxyuridine, and 2'-deoxyuridine glycols. Here, we have identified one of the four possible diastereomers of 2'-deoxycytidine glycols by product analyses of decomposition products, (1)H NMR, and mass spectrometry. This glycol was observed to decompose with a half-life of 50 min at 37 degrees C in buffered neutral solutions and preferentially undergo dehydration to 5-hydroxy-2'-deoxycytidine. The rate of decomposition was strongly dependent on pH (2-10) and the concentration of phosphate ion (10-300 mM). Next, we report on the deamination of cytosine glycols to uracil glycols in oxidized DNA using acid hydrolysis and high performance liquid chromatography analysis with electrochemical detection to monitor 5-hydroxycytosine and 5-hydroxyuracil. The results showed that the lifetime of cytosine glycols is greatly enhanced in DNA (34-fold; half-life, 28 h), and that deamination accounts for at least one-third of the total decomposition. The relatively long lifetime of cytosine glycols in DNA suggests that this important class of DNA oxidation products will be significantly involved in repair and mutagenesis processes.     

Serum thyroid hormones and performance of offspring in ewes receiving propylthiouracil with or without melatonin

Two experiments were conducted during mid-gestation to examine effects in ewes of propylthiouracil (PTU) treatment alone or with melatonin on serum thyroid hormones, postpartum reproduction, and lamb performance. In the first experiment, beginning on day 0 (first day of treatment when all animals were 72.2+/-0.9 days of gestation), ewes received daily treatments (gavage) consisting of either 0mg (n=6) or 40 mg (n=6) PTU/kg body weight/day for 15 days. After 15 days, the 40 mg dosage was decreased to 20mg/kg body weight for an additional 20 days (35 days of PTU). Serum thyroxine (T4) did not differ (P>0.10) between groups through day 4; but on day 5, control females had a serum value of 67 ng/ml compared with 46 (+/-5)ng/ml for PTU-treated ewes (P=0.02). On the last day that 40 mg of PTU was administered, serum T4 averaged 67 and 7 (+/-5)ng/ml (P<0.001) in the two respective groups. Serum T4 remained low and was 80 and 1 ng/ml (P<0.001) in control and treated ewes on day 34. Serum T4 rose gradually after PTU but remained different from that observed in control ewes through day 48. Lambs from control and treated ewes had similar (P=0.46) T4 values at birth but lambs from PTU-treated ewes had lower (P=0.03) birth weights than did those from control ewes. Serum progesterone (P4) after parturition indicated a lack of cyclicity in all ewes. In the second experiment, beginning on day 0 (76.8+/-4.7 days of gestation), ewes received PTU as in Experiment 1. In addition, after 15 days of PTU, melatonin was given (i.m. injections at 5mg/day) for 30 days. Propylthiouracil decreased (P0.60) for lambs born to control and treated ewes. Female offspring of PTU+melatonin-treated dams reached puberty, became anestrus, and returned to cyclicity at similar (P>0.10) times to contemporary ewe lambs. Results indicate that 40/20mg PTU alone or with melatonin does not induce cyclicity after lambing in spring lambing ewes and has little effect on offspring performance.     

Serum thyroid hormones and reproductive characteristics of Rambouillet ewe lambs treated with propylthiouracil before puberty

Twenty-four Rambouillet ewe lambs (average weight=43.7+/-1.2 kg, approximately 6 months of age) were used to examine the effect of thyroid suppression before the onset of puberty on serum thyroid hormones, body weights (BW), and reproductive performance. Beginning in early September, ewe lambs were randomly assigned to three treatments (n=8 lambs/treatment). All animals remained in a single pen (4 x 12 m) with access to salt, water, shade and alfalfa hay (2.5 kg per animal per day) throughout the experiment. Beginning on Day 0 (first day of treatment), all ewe lambs received daily treatments (gavage) for 15 days consisting of 0, 20, or 40 mg 6-N-propyl-2-thiouracil(PTU)/kg BW per day. Beginning on Day 15, the 20 and 40 mg treatments were lowered to 10 and 20 mg PTU/kg BW, respectively. All animals were treated for 28 days. Ovarian cyclicity was determined by twice weekly progesterone (P(4)) analysis. Thyroxine (T(4)) concentrations were similar on Day 0 (61.6, 54.8 and 56.9+/-2.5 ng/ml, P=0.17) in ewe lambs receiving 0, 20 and 40 mg PTU/kg BW, respectively. By Day 7, both PTU-treated groups had T(4) values less than 20 ng/ml (9.0 and 15.4+/-2.5 ng/ml) compared with 78.5 ng/ml in controls (P<0.01). By 7 days after termination of PTU treatment, serum T(4) had risen to 29.1 and 26.9 (+/-2.9)ng/ml in the 20/10 and 40/20 PTU groups, respectively. On Day 66, control ewes had 55.0 ng T(4)/ml compared with 43.1 and 39.0 (+/-2.6 ng/ml) for ewes in the 20/10 and 40/20 groups, respectively (linear, P<0.01). Serum triiodothyronine (T(3)) followed a similar pattern to that observed for T(4). Ewe lamb BW were similar (P>0.50) among groups throughout the treatment period. However, following the treatment, PTU-treated ewes tended (P<0.10) to weigh less than controls. Average Julian day of puberty was also similar (P>0.50) among treatments (286, 288 and 288+/-5 days; control, 20/10 and 40/20, respectively). Control ewes had a pregnancy rate of 75%, while both PTU-treated groups had pregnancy rates of 88% (P>0.20). The administration of PTU resulted in a rapid decline in serum T(4) and T(3) but neither time of puberty nor pregnancy rates were affected by lowered thyroid hormones.     

Chronobiological study of free thyroid hormones in hypothyroid and hyperthyroid subjects

Previous reports demonstrate a circadian rhythm of the free thyroid hormones in healthy subjects. In this study we evaluated circadian variation of FT3 and FT4 in hyperthyroid and hypothyroid states. We considered six hyperthyroid patients, six hypothyroid patients and six control subjects. Blood samples were taken two hours apart from a catherterized arm vein. Data were evaluated by Halberg's cosinor analysis. The results show that FT3 and FT4 exhibit a circadian rhythm in healthy subjects, not evident in hyperthyroid and hypothyroid patients.     

Interaction between thyroid hormones and erythrocyte membranes: competitive inhibition of binding 131 I-L-triiodothyronine and 131 I-L-thyroxine by their analogs.

Molecular structural characteristics of thyroid hormones which influence binding to the erythrocyte membranes were investigated by competitive binding experiments. The ability of thyroid hormone analogs to displace 131 I-L-thyroxine and 131 I-L-triiodothyronine from the membranes was considered evidence of their competitive binding. The diphenyl ether linkage (thyronine) was essential as compounds with a single aromatic ring were weakly competitive. The presence of three iodine atoms at 3, 5 and 3' positions on thyronine was optimal for maximal competitive binding. There was weak competitive binding of analogs if chlorine or bromine was substituted for iodine. The alanine side chain was required for optimal binding as N-acetyl-l-thyroxine and various deaminated analogs were poor competitors compared to T4 and T3. L-isomers of T4 and T3 showed greater competitive binding to erythrocyte membranes than the corresponding d-isomers.     

Recycling of the ascorbate free radical by human erythrocyte membranes.

Reduction of the ascorbate free radical (AFR) at the plasma membrane provides an efficient mechanism to preserve the vitamin in a location where it can recycle alpha-tocopherol and thus prevent lipid peroxidation. Erythrocyte ghost membranes have been shown to oxidize NADH in the presence of the AFR. We report that this activity derives from an AFR reductase because it spares ascorbate from oxidation by ascorbate oxidase, and because ghost membranes decrease steady-state concentrations of the AFR in a protein- and NADH-dependent manner. The AFR reductase has a high apparent affinity for both NADH and the AFR (< 2 microM). When measured in open ghosts, the reductase is comprised of an inner membrane activity (both substrate sites on the cytosolic membrane face) and a trans-membrane activity that mediates extracellular AFR reduction using intracellular NADH. However, the trans-membrane activity constitutes only about 12% of the total measured in ghosts. Ghost AFR reductase activity can also be differentiated from NADH-dependent ferricyanide reductase(s) by its sensitivity to the detergent Triton X-100 and insensitivity to enzymatic digestion with cathepsin D. This NADH-dependent AFR reductase could serve to recycle ascorbic acid at a crucial site on the inner face of the plasma membrane.     

Effects of propylthiouracil and methylmercaptoimidazol on metabolism of thyroid hormones by cultured monkey hepatocarcinoma cells.

Monkey hepatocarcinoma cell monolayer cultures (NCLP-6E) metabolized thyroxine, 3,5,3'-triiodothyronine, 3,3',5'-triiodothyronine and 3,3'-diiodothyronine by phenolic and nonphenolic ring deiodinations and sulfation of the deiodinated products, as shown in previous work with this system. The effects of the antithyroid drugs, propylthiouracil (PTU) and methylmercaptoimidazole (MMI), on these processes was investigated. PTU, at 0.1 and 1 mM, inhibited only phenolic ring deiodination. MMI at 1 mM had no effect, but 32 mM inhibited deiodination of both rings as well as sulfation. The findings suggest that the increased serum rT3 level caused by PTU in vivo is the result of decreased rT3 deiodination, in contrast to the increased rT3 production which is caused by starvation.     

Preparation and properties of thyroid cell membranes

Summary Calf and human thyroids have been disrupted by nitrogen microcavitation, and the thyroid membranes prepared by repeated centrifugation in low ionic strength buffers. Two classes of membranes were prepared by centrifugation on a discontinuous gradient of ficoll. A lighter fraction was comprised of somewhat larger vesicles; they were higher in Na⁺–K⁺-activated ATPase, phosphodiesterase, and 5-nucleotidase than was the heavier fraction. The heavier fraction had a higher nicotinamide adenine nucleotide dehydrogenase-diaphorase activity. Thus the lighter fraction appears to have been enriched in fragments derived from the plasma membrane.     

Lipid oxidation in biological membranes. Electron transfer proteins as initiators of lipid autoxidation.

Biological lipid autoxidation has been studied in a model system composed of sonicated phospholipids as substrate and electron transfer proteins found in membranes as possible catalysts. Heme compounds, flavoproteins, and iron-sulfur proteins were examined for their ability to initiate lipid autoxidation. Among many heme compounds tested, the most active were hematin ?microperoxidase ? methemoglobin > cytochrome c. With fresh preparations of phospholipids, reaction rates (nanomoles of oxygen/minute nanomoles of heme) ranged from 5 (cytochrome c) to 350 (hematin). Only the oxidized heme compounds were active as catalysts. Reduced heme compounds, flavoproteins and riboflavin were inactive. In the presence of heme compounds, aged preparations of sonicated phospholipids were much more rapidly oxidized than fresh preparations. They also had a higher content of fatty acid hydroperoxides as judged from their characteristic diene absorption peak at 234 nm. This observation agrees with the postulated mechanism of lipid autoxidation by heme compounds, namely, homolytic scission of preformed fatty acid hydroperoxides. Iron-sulfur proteins were also active as initiators of lipid autoxidation when destabilized in the presence of an appropriate iron chelator (o-phenanthroline or 2,2′-bipyridine) or a chaotropic ion. Oxygen uptake rates (nanomoles of oxygen/minute × milligrams of protein) varied from about 200 for an iron-sulfur protein isolated from complex I to about 5500 for Clostridium pasteurianum ferredoxin. However, per nanomole of labile sulfide, the rates for all active iron-sulfur proteins were 4–7 nmol of oxygen/min × nmol of labile sulfide.Superoxide-generating systems did not initiate lipid autoxidation, nor did erythrocuprein inhibit the autoxidations induced by heme compounds or ferredoxin. However, lipid oxidations induced by two other iron-sulfur proteins were partially inhibited by erythrocuprein. It is concluded that in the above system Superoxide anion is neither an initiator nor an obligatory intermediate of lipid autoxidation.     

Evaluation of radiation damage to rat and dog erythrocyte membranes based on changes in their sedimentation properties

It was shown that injury to plasma membranes leads to a change in the sedimentation behaviour of erythrocytes: the maximum effect is produced when a protein component of the membrane is affected. The same dose-dependent character of the change in erythrocyte sedimentation in urografin is observed during the first 24 h after gamma-irradiation of rats and dogs.     

Fluidity properties and liquid composition of erythrocyte membranes in Chediak-Higashi syndrome

We have earlier shown through electron spin resonance (ESR) studies of leukocytes that membranes of cells from both Chediak-Higashi syndrome (CHS) mice and humans have abnormally high fluidity. We have extended our studied to erythrocytes. Erythrocytes were labeled with the nitroxide-substituted analogue of stearic acid, 2-(3-carboxypropyl)-4,4- dimethyl-2-tridecyl-3-oxazolidinyloxyl, and ESR spectra were obtained. Order parameter, S, at 23 degrees C, was 0.661 and 0.653 for erythrocytes of normal and CHS mice (P less than 0.001). S was 0.684 for normal human erythrocytes and 0.675 (P less than 0.001) for CHS erythrocytes at 25 degrees C. Because S varies inversely to fluidity, these results indicate that CHS erythrocytes tend to have higher fluidity than normal. In vitro treatment of both mice and human CHS erythrocytes with 10 mM ascorbate returned their membrane fluidity to normal. We prepared erythrocyte ghosts and extracted them with CHCl3:CH3OH (2:1). Gas-liquid chromatography analysis showed a greater number of unsaturated fatty acids for CHS. The average number of double bonds detected in fatty acids for mice on a standard diet was 1.77 for normal and 2.02 for CHS (P less than 0.04); comparison of human erythrocytes from one normal control and one CHS patient showed a similar trend. Our results suggest that an increased proportion of unsaturated fatty acids may contribute to increased fluidity of CHS erythrocytes. Our observation that both leukocytes and erythrocytes of CHS have abnormal fluidity indicates that CHS pathophysiology may relate to a general membrane disorder.     

Isolation and properties of a T-kininogenase from bovine erythrocyte membranes

A kininogenase from bovine erythrocyte membranes has been purified 140-fold by affinity chromatography on pepstatin A-Agarose followed by ion exchange chromatography on CM Cellulose. The purified enzyme showed an apparent molecular weight of 31,000 daltons as measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. ItspH optimum is 7.5, and it was totally inhibited by soybean trypsin inhibitor, phenylmethylsulfonylfluoride, aprotinin, pepstatin, and dithiotreitol, suggesting the presence of a disulfide bond(s) whose integrity is(are) essential for maintaining the native three-dimensional structure. The referred enzyme was able to release kinin from a substrate partially purified from rat plasma. The kininogenase was activated by Zn²⁺, Ca²⁺, and cysteine-HCl.