Activation of mammalian endplate receptors by iontophoretic application of reducing and oxidizing sulphydryl reagents

The effects of microelectrophoretic application of sulphydryl reagents at both the endplate region and extrasynaptic sites have been studied in the mouse diaphragm neuromuscular preparation. Two groups of sulphydryl reagents have been tested: oxidizing agents and reducing agents. Reagents were applied iontophoretically by means of a microelectrophoretic programmer with constant current source. Both groups of sulphydryl reagents elicit depolarizations only when applied at the endplate region. Presynaptic components were ruled out by addition of magnesium chloride to the bathing medium. After blockade of neuromuscular transmission, the depolarizing effect of both groups of sulphydryl reagents persisted. The present results suggest the presence of reactive disulphide bonds and SH groups in the receptor complex of the mammalian neuromuscular junction. These groups could be located at the alpha subunit of the acetylcholine receptor.     

Oxidation and reduction of pig skeletal muscle ryanodine receptors

Time-dependent effects of cysteine modification were compared in skeletal ryanodine receptors (RyRs) from normal pigs and RyR(MH) (Arg(615) to Cys(615)) from pigs susceptible to malignant hyperthermia, using the oxidizing reagents 4,4'-dithiodipyridine (4, 4'-DTDP) and 5,5'-dithio-bis(2-nitrobenzoic acid) (DTNB) or the reducing agent dithiothreitol (DTT). Normal and RyR(MH) channels responded similarly to all reagents. DTNB (1 mM), either cytoplasmic (cis) or luminal (trans), or 1 mM 4,4'-DTDP (cis) activated RyRs, introducing an additional long open time constant. 4,4'-DTDP (cis), but not DTNB, inhibited channels after >5 min. Activation and inhibition were relieved by DTT (1-10 mM). DTT (10 mM, cytoplasmic or luminal), without oxidants, activated RyRs, and activation reversed with 1 mM DTNB. Control RyR activity was maintained with 1 mM DTNB and 10 mM DTT present on the same or opposite sides of the bilayer. We suggest that 1) 4,4'-DTDP and DTNB covalently modify RyRs by oxidizing activating or inhibiting thiol groups; 2) a modified thiol depresses mammalian skeletal RyR activity under control conditions; 3) both the activating thiols and the modified thiols, accessible from either cytoplasm or lumen, reside in the transmembrane region; 4) some cardiac sulfhydryls are unavailable in skeletal RyRs; and 5) Cys(615) in RyR(MH) is functionally unimportant in redox cycling.     

Oxidative inactivation of rhodanese by hydrogen peroxide produces states that show differential reactivation

Controlled conditions have been found that give complete reactivation and long term stabilization of rhodanese (EC 2.8.1.1) after oxidative inactivation by hydrogen peroxide. Inactivated rhodanese was completely reactivated by reductants such as thioglycolic acid (TGA) (100 mM) and dithiothreitol (DTT) (100 mM) or the substrate thiosulfate (100 mM) if these reagents were added soon after inactivation. Reactivability fell in a biphasic first order process. At pH 7.5, in the presence of DTT inactive rhodanese lost 40% of its reactivability in less than 5 min, and the remaining 60% was lost more gradually (t 1/2 = 3.5 h). TGA reactivated better than DTT, and the rapid phase was much less prominent. If excess reagents were removed by gel filtration immediately after inactivation, there was time-independent and complete reactivability with TGA for at least 24 h, and the resulting samples were stable. Reactivable enzyme was resistant to proteolysis and had a fluorescence maximum at 335 nm, just as the native protein. Oxidized rhodanese, Partially reactivated by DTT, was unstable and lost activity upon further incubation. This inactive enzyme was fully reactivated by 200 mM TGA. Also, the enzyme could be reactivated by arsenite and high concentrations of cyanide. Addition of hydrogen peroxide (40-fold molar excess) to inactive rhodanese after column chromatography initiated a time-dependent loss of reactivability. This inactivation was a single first order process (t 1/2 = 25 min). Sulfhydryl titers showed that enzyme could be fully reactivated after the loss of either one or two sulfhydryl groups. Irreversibly inactivated enzyme showed the loss of one sulfhydryl group even after extensive reduction with TGA. The results are consistent with a two-stage oxidation of rhodanese. In the first stage there can form sulfenyl and/or disulfide derivative(s) at the active site sulfhydryl that are reducible by thioglycolate. A second stage could give alternate or additional oxidation states that are not easily reducible by reagents tried to date.     

Degradation of somatomedin A by various organ homogenates from rats

Degradative activities of somatomedin A (SMA) have been examined in various tissue homogenates of rat using trichloracetic acid precipitable radioactivity of 125I-SMA. Kidney and testis showed higher specific activities and liver and brain lower activities. They were dependent on SH reagents; 0.5 mM HgCl2 inhibited the degradative activity of liver completely and 1 mM dithiothreitol (DTT) augmented the activity slightly. The activities in liver were separated by differential centrifugation; about 90 per cent of the total activity in the whole homogenate was recovered in the supernatant fraction at 100,00 x g for 60 min, and 10 per cent in the precipitate. The pH profile of each fraction was different; that of the supernatant showed a single peak at pH 7.4 and that of the pellet revealed two peaks at pH 5.9 and 7.4. However, both fractions showed similar SH-dependency.     

Quantification of protein thiols and dithiols in the picomolar range using sodium borohydride and 4,4'-dithiodipyridine

Experimental determination of the number of thiols in a protein requires methodology that combines high sensitivity and reproducibility with low intrinsic thiol oxidation disposition. In detection of disulfide bonds, it is also necessary to efficiently reduce disulfides and to quantify the liberated thiols. Ellman's reagent (5,5'-dithiobis-[2-nitrobenzoic acid], DTNB) is the most widely used reagent for quantification of protein thiols, whereas dithiothreitol (DTT) is commonly used for disulfide reduction. DTNB suffers from a relatively low sensitivity, whereas DTT reduction is inconvenient because the reagent must be removed before thiol quantification. Furthermore, both reagents require a reaction pH > 7.0 where oxidation by ambient molecular oxygen is significant. Here we describe a quick and highly sensitive assay for protein thiol and dithiol quantification using the reducing agent sodium borohydride and the thiol reagent 4,4'-dithiodipyridine (4-DPS). Because borohydride is efficiently destroyed by the addition of acid, the complete reduction and quantification can be performed conveniently in one tube without desalting steps. Furthermore, the use of reverse-phase high-performance liquid chromatography for the thiol quantification by 4-DPS reduces the detection limit to the picomolar range (equivalent to 1 microg of a 50-kDa protein containing 1 thiol) while at the same time maintaining low pH throughout the procedure.     

Disulfide bond formation is not required for human chorionic gonadotropin subunit association. Studies with dithiothreitol in JEG-3 cells

To study the influence of disulfide bridge formation on the assembly of the subunits of human chorionic gonadotropin in JEG-3 choriocarcinoma cells, dithiothreitol (DTT) was used to create a reducing milieu in the endoplasmic reticulum (ER) in vivo. In the presence of 5 mM DTT during pulse-chase experiments all of the beta-subunit precursors observed in unperturbed cells (pbeta(0), pbeta(1), pbeta(2), and beta(*)) collapsed into the pbeta(0) form. The reducing milieu of the ER was reoxidized in less than 5 min after removal of DTT from the medium. DTT markedly increased the half-life of the pbeta(0) precursor from 8.8 to 65.2 min. Under reoxidation conditions, the beta-subunit precursors folded back from pbeta(0) in less than 5 min. In unperturbed JEG-3 cells, the alpha-subunit was present in both fully glycosylated and monoglycosylated precursor (pre-alpha) forms. The attachment of the second N-linked glycan residue of the alpha-subunit was accelerated in the presence of DTT, and consequently pre-alpha-subunit was missing from the DTT-treated cultures. The formation of alphabeta-dimers appeared to be at least partially independent of the oxidation state in the ER. The alphabeta-dimer was present under conditions in which disulfide bridge formation was prevented by exposure to 5 mM DTT before and during the pulse period. This clearly suggests that the human chorionic gonadotropin subunits may acquire association-competent conformations even when no disulfide bridge formation has taken place.     

Influence of liposome charge and composition on their interaction with human blood serum proteins

The roles of sulfhydryl and disulfide groups in the specific binding of synthetic cannabinoid CP-55,940 to the cannabinoid receptor in membrane preparations from the rat cerebral cortex have been examined. Various sulfhydryl blocking reagents including p-chloromercuribenzoic acid (p-CMB), N-ethylmaleimide (NEM), o-iodosobenzoic acid (o-ISB), and methyl methanethiosulfonate (MMTS) inhibited the specific binding of [³H]CP-55,940 to the cannabinoid receptor in a dose-dependent manner. About 80–95% inhibition was obtained at a 0.1 mM concentration of these reagents. Scatchard analysis of saturation experiments indicates that most of these sulfhydryl modifying reagents reduce both the binding affinity (K_d) and capacity (B_max). On the other hand, DL-dithiothreitol (DTT), a disulfide reducing agent, also irreversibly inhibited the specific binding of [³H]CP-55,940 to the receptor and about 50% inhibition was obtained at a 5 mM concentration. Furthermore, 5mM DTT was abelt to dissociate 50% of the bound ligand from the ligand-receptor complex. The marked inhibition of [³H]CP-55,940 binding by sulfhydryl reagents suggests that at least one free sulfhydryl group is essential to the binding of the ligand to the receptor. In addition, the inhibition of the binding by DTT implies that besides free sulfhydryl group(s), the integrity of a disulfide bridge is also important for [³H]CP-55,940 binding to the cannabinoid receptor.     

Detection of reversible protein thiol modifications in tissues

Oxidation/reduction reactions of protein thiol groups (PSH) have been implicated in many physiological and pathological processes. Although many new techniques for separation and identification of modified cysteinyl residues in proteins have been developed, critical assessment of reagents and sample processing often are overlooked. We carefully compared the effectiveness of N-ethylmaleimide (NEM), iodoacetamide (IAM), and iodoacetic acid (IAA) in alkylating protein thiols and found that NEM required less reagent (125 vs. 1000 mol:mol excess), required less time (4 min vs. 4h), and was more effective at lower pHs (4.3 vs. 8.0) in comparison with IAM and IAA. The relative efficacy of dithiothreitol (DTT) and tris(2-carboxyethyl)phosphine (TCEP) for reducing protein disulfides suspended in NaPO(4) buffer or MeOH was assessed, and no differences in total normalized fluorescence were detected at the concentrations tested (10-100mM); however, individual band resolution appeared better in samples reduced with DTT in MeOH. In addition, we found that oxidation ex vivo was minimized in tissue samples that were homogenized in aqueous buffers containing excess molar quantities of NEM compared with samples homogenized in MeOH containing NEM. Using NEM for thiol alkylation, DTT for disulfide reduction, and mBBr for labeling the reduced disulfide and fluorimetric detection, we were able to generate an in-gel standard curve and quantitate total disulfide contents within biological samples as well as to identify changes in specific protein bands by scanning densitometry. We demonstrated that reagents and techniques we have identified for disulfide detection in complex samples are also applicable to two-dimensional electrophoresis separations.     

Essential disulfide and sulfhydryl groups for organic cation transport in renal brush-border membranes

The disulfide reducing agent, dithiothreitol (DTT) and the sulfhydryl-modifying reagents p-chloromercuribenzenesulfonic acid and N-ethylmaleimide (NEM) were employed to assess the role of disulfide and sulfhydryl groups in organic cation transport. The transport of N1-[3H]methylnicotinamide (NMN), a prototypic organic cation, was examined employing brush-border membrane vesicles isolated from the outer cortex of canine kidneys. DTT inhibited NMN transport reversibly with an IC50 of 250 microM/mg of protein. 5 mM NMN protected against DTT inactivation. The specificity of substrate protection was demonstrated by showing that D-glucose had no effect on the DTT inactivation of NMN transport and conversely that NMN had no effect on the DTT inactivation of D-glucose transport. Disulfide bonds reduced by DTT could be reoxidized by washing with excess buffer or by addition of 0.02% H2O2 thereby restoring NMN transport. p-Chloromercuribenzenesulfonic acid reversibly inactivated NMN transport with an IC50 of 25 microM/mg of protein. 5mM NMN protected against inactivation. NEM irreversibly inactivated transport with an IC50 of 250 microM/mg of protein. The rate of NMN inactivation by NEM followed pseudo-first order reaction kinetics. A replot of the data gave a linear relationship between the apparent rate constants and the NEM concentration with a slope of 1.3. The data are consistent with a simple bimolecular reaction mechanism and imply that one molecule of NEM inactivates 1 sulfhydryl group/active transport unit. The presence of 5 mM NMN affected the rate of NEM (2.5 mM) inactivation: the t1/2 values for inactivation in the presence and absence of substrate were 7.3 and 2.0 min, respectively. The results demonstrate an essential requirement for disulfide and sulfhydryl groups.     

Effects of sulfhydryl inhibitors on depolarizations-contraction coupling in frog skeletal muscle fibers

We have studied the effects of the sulfhydryl reagents on contractile responses, using either electrically stimulated single muscle fibers or short muscle fibers that were voltage-clamped with a two-microelectrode voltage-clamp technique that allows the fiber tension in response to membrane depolarization to be recorded. The sulfhydryl inhibitors para- chloromercuribenzoic acid (PCMB) and parahydroximercuriphenyl sulfonic acid (PHMPS), at concentrations from 0.5 to 2 mM, cause loss of the contractile ability; however, before this effect is completed, they change the fiber contractile behavior in a complex way. After relatively short exposure to the compounds, < 20 min, before the fibers lose their contractile capacity, secondary tension responses may appear after electrically elicited twitches or tetani. After losing their ability to contract in response to electrical stimulation, the fibers maintain their capacity to develop caffeine contractures, even after prolonged periods (120 min) of exposure to PHMPS. In fibers under voltage-clamp conditions, contractility is also lost; however, before this happens, long-lasting (i.e., minutes) episodes of spontaneous contractile activity may occur with the membrane polarized at -100 mV. After more prolonged exposure (> 30 min), the responses to membrane depolarization are reduced and eventually disappear. The agent DTT at a concentration of 2 mM appears to protect the fibers from the effects of PCMB and PHMPS. Furthermore, after loss of the contractile responses by the action of PCMB or PHMPS, addition of 2 mM DTT causes recovery of tension development capacity.     

Determination of the intravesicular pH of fragmented sarcoplasmic reticulum with 5,5-dimethyl-2,4-oxazolidinedione.

The intravesicular pH (pHi) of fragmented sarcoplasmic reticulum (SR) of the skeletal muscle was determined from the distribution of 5,5-dimethyl-2,4-oxazolidinedione (DMO), a weak organic acid, between the intra- and extravesicular spaces. The pHi's thus obtained were found to be slightly lower (0.02-0.17 pH unit) than the pH's of the external medium (pHe) at pH 6.5-8.5 in the presence of 105 mM KCl and 40 mM Tris-maleate buffer. The higher the pHe, the greater the pH gradient. When pHe was changed, pHi attained equilibrium within about 20 min, the time necessary for the separation of the SR by centrifugation. When 0.25 M sucrose and 5 mM Tris-maleate buffer were used instead of 105 mM KCl and 40 mM buffer, the pH gradient increased to 0.56. It was also demonstrated by direct measurements of pHe with a glass-electrode pH meter that K+ ions added to the external medium exchanged the intravesicular H+ ions. From these results it appears that the pH gradient across the SR membrane was at the Donnan equilibrium. In this state, the Donnan potentials corresponding to pH gradients of 0.17 and 0.56 were -9.3 and -30.6 mV, respectively.     

Effect of pH on stability of sarcoplasmic reticulum calcium pump in rabbit heart

The sarcoplasmic reticulum (SR) membranes isolated from rabbit heart were preincubated at pH 6.8 or 7.8 and their Ca²⁺ pump properties were compared at pH 6.8. The ATP-dependent azide insensitive oxalate-stimulated Ca²⁺ uptake was reduced more rapidly from the membranes preincubated at 37°C at pH 7.8 than from those preincubated at pH 6.8. The Ca²⁺–Mg²⁺-ATPase, and the Ca²⁺-dependent formation of 110 kDa acylphosphate were also inhibited by the preincubation at the higher pH. Including 1 mM DTT in the preincubation medium reduced the inactivation. The preincubation at 37°C in the presence or absence of DTT caused membranes to become more leaky as the loss of Ca²⁺ uptake was more rapid than that of ATPase or the acylphosphate formation. The loss of these activities was not accompanied by a breakdown of the protein as monitored in Western blots. It is hypothesized that the SR Ca²⁺ pump inactivation involves a key-SH group and that the lower pH provides a compensatory protective mechanism for the SR during acidosis.     

Redox-dependent modulation of the carrot SV channel by cytosolic pH

Currents mediated by a slow vacuolar (SV) channel were recorded and characterized in vacuoles from cultured carrot cells. The carrot channel shows the typical functional characteristics reported for channels of the SV category previously identified in other plants, i.e., slow voltage-dependent activation kinetics, current activation favoured by cytosolic calcium and permeability to different monovalent cations. The carrot channel is strongly activated by cytosolic reducing agents (such as dithiothreitol, DTT, and glutathione, GSH) and has a peculiar dependence on cytosolic pH, which, in turn, is affected by the concentration of cytosolic reducing agents. Specifically, in 1 mM DTT or GSH the channel displayed a maximum conductance at neutral pH. The normalized conductance did not depend significantly on DTT concentration at acidic pH, while at alkaline pH the attenuation of the normalized conductance declines with increasing DTT concentration. Our results suggest two pH-titratable groups within the carrot SV channel, one of these depending on cysteine residues exposed to the cytosolic side of the vacuole.     

Pharmacological intervention in oxidant-induced calcium pump dysfunction of dog heart

Micromolar concentrations of HOCl, an oxidant produced by activated neutrophils, inhibited Ca2+ uptake and Ca2+ATPase of isolated dog heart sarcoplasmic reticulum (SR). DTT antagonized completely the HOCl effect only when it was given within 5 min after the addition of HOCl. When the pharmacological intervention was delayed, the recovery with DTT was not complete, and administration of DTT 30 min after the start of HOCl's reaction with SR resulted in only a small improvement in SR Ca2+ uptake. Although H2O2 and Fe ion-chelate (a free radical-generating procedure) also inhibited Ca2+ uptake and ATPase, the concentrations required were very large. The response of cardiac sarcolemmal and skeletal muscle SR calcium pumps to oxidants was similar to that of the cardiac SR calcium pump.     

Novikoff hepatoma deoxyribonucleic acid polymerase. Sensitivity of the beta-polymerase to sulfhydryl blocking agents.

Unlike other beta-class eukaryotic DNA polymerases, the enzyme purified from the Novikoff hepatoma is inhibited by both sulfhydryl blocking agents N-ethylmaleimide (NEM) and p-hydroxymercuribenzoate (pHMB). The degree of sensitivity varies depending on the enzyme purity, pH of the reaction, and the presence of sulfhydryl reducing agents. Novikoff beta-polymerase activity is unaffected by the presence of 2-mercaptoethanol (2-Me) or dithiothreitol (DTT); however, the combination of 2-mercaptoethanol and NEM or pHMB acts to reverse the inhibition of the sulfhydryl blocking agent. The reversal of inhibition involves more than just a titration of NEM with 2-mercaptoethanol since a) the combination of these two reagents actually stimulates the DNA polymerase, and b) dithiothreitol did not reverse the inhibition. Binding of the polymerase to DNA did not affect the enzyme sensitivity to NEM.     

Carbachol contracture of stomach smooth muscle of the newt in calcium-free solution

A small muscle preparation of stomach circular muscle of the newt responded to carbachol (CCh) with a phasic contracture. At 20 degrees C, in Ca-free Ringer solution (+1 mM EGTA), the amplitude of CCh contracture was very rapidly inhibited to less than 10% of that in normal Ringer solution (1.8 mM Ca). The amplitude of this CCh contracture was markedly enhanced with increasing [K]0. CCh contracture in Ca-free Ringer solution was also enhanced after K contracture was induced once in the presence of 1.8 mM Ca, followed by soaking in normal Ringer solution. The amplitude of this enhanced CCh contracture persisted up to about 5 min, following rapid decrease to about 70%, and then gradually decreased to a steady level in Ca-free Ringer solution. This decrease in amplitude was prevented by increasing [K]0 during soaking in Ca-free solution; even when the temperature was elevated from 20 to 35 degrees C during the periods of soaking in Ca-free solution, CCh contracture was inhibited only by about 20% in Ca-free high K solution, whereas in Ca-free or Ca-free low Na (Tris) Ringer solution it was inhibited by more than 50%.     

Acyl cystamine: small-molecular foldase mimics accelerating oxidative refolding of disulfide-containing proteins

Based on the structural characteristic of Protein disulfide isomerases and DsbA that have hydrophobic regions around the active sites, hydrophobic alkyl tails are linked to cystamine to create new small molecular foldase mimics, acyl cystamine. Both the oxidizing power and oxidation specificity of cystamine are enhanced by n-octanoyl or n-hexanoyl tail. N-octanoyl and n-hexanoyl cystamine are very effective to facilitate oxidative protein refolding at strong reducing environments. In the presence of 0.42 mM DTT, the activity recovery of lysozyme is over 90% by 90-min refolding with 0.1 mM n-octanoyl cystamine and 0.1 mM cystamine as oxidant, while almost no activity is recovered with 0.2 mM GSSG by 160-min refolding. For the refolding of 0.2 mg/mL lysozyme, with 0.6 mM n-hexanoyl cystamine and 1.12 mM residual DTT as redox agents, the activity recovery reaches as high as 93% after refolding for only 20 min. For ribonuclease A (RNase A) refolding, with 0.4 mM n-hexanoyl cystamine and 1.30 mM DTT, the recovery of activity reaches as high as 90% within 3 h. Thus, with n-octanoyl or n-hexanoyl cystamine as the oxidants, the necessity to remove excess DTT in the reduced and denatured protein solutions can be greatly alleviated. With a moderate hydrophobicity, n-hexanoyl cystamine is promising for application in oxidative protein refolding at an extensive concentration range. It is observed that in the oxidative refolding of 0.2 mg/mL lysozyme and RNase A, only about half of n-hexanoyl cystamine is needed when compared to cystamine to achieve the same kinetic effect.     

Initial protein concentration and residual denaturant concentration strongly affect the batch refolding of hen egg white lysozyme

The effects of several variables on the refolding of hen egg white lysozyme have been studied. Lysozyme was denatured in both urea, and guanidine hydrochloride (GuHCl), and batch refolded by dilution (100 to 1000 fold) into 0.1M Tris-HCl, pH 8.2, 1 mM EDTA, 3 mM reduced glutathione and 0.3 mM oxidised glutathione. Refolding was found to be sensitive to temperature, with the highest refolding yield obtained at 50°C. The apparent activation energy for lysozyme refolding was found to be 56 kJ/mol. Refolding by dilution results in low concentrations of both denaturant and reducing agent species. It was found that the residual concentrations obtained during dilution (100-fold dilution: [GuHCl]=0.06 mM, [DTT]=0.15 mM) were significant and could inhibit lysozyme refolding. This study has also shown that the initial protein concentration (1–10 mg/mL) that is refolded is an important parameter. In the presence of residual GuHCl and DTT, higher refolding yields were obtained when starting from higher initial lysozyme concentrations. This trend was reversed when residual denaturant components were removed from the refolding buffer.     

A sensitive method for measuring neutralizing antibodies to Bordetella pertussis toxin: optimized ADP-ribosylation of transducin

Pertussis toxin (PT), preactivated with 20 mm dithiothreitol (DTT), was incubated with different serum dilutions (1/10-1/200) before addition to the reaction mixture. Final concentrations of the reagents were: PT (50 ng/ml), dithiothreitol (DTT)(less than or equal to 0.3 mM), bovine transducin (2 micrograms/ml), ATP (1 mM). GTP (1 mM), lysophosphatidylcholine (LTC) (0.1 mg/ml), sodium acetate (NaAc) (0.1 M), Tris-HCl, pH 7.1 (0.06 M) and 32P-NAD+ (10 microCi 28 Ci/mM). The reaction was stopped by precipitation with 10% TCA (w/v), the pellet was collected and the samples were submitted to SDS-PAGE followed by autoradiography. ADP-ribosylation was detected as the radiolabelling of a protein band (m.w. approximately 40 kD) corresponding to the alpha-subunit of transducin. 32P-ADP incorporation was a linear function of PT concentration. By this assay quantitative differences in PT neutralizing antibodies were found between sera which were not revealed by measuring PT neutralizing antibodies by a Chinese hamster ovary (CHO) cell culture test or by an enzyme-linked immunosorbent assay (ELISA). The conditions of the ADP-ribosylation of bovine transducin have been optimized to permit detection of the enzymic activity of as low amounts of PT as 0.5 ng. This opens the possibility of the study of the presence and avidity of neutralizing antibodies to PT in post-vaccination sera without preceding purification and concentration of the antibodies and thus may provide a useful tool for evaluation of whooping cough vaccines.