Site distribution in resin beads as determined by confocal Raman spectroscopy.

Scanning confocal Raman spectroscopy was used to study the distribution of reactive sites within a resin bead used for solid-phase synthesis. The distribution of NH2 groups in aminomethylated polystyrene resin (APS) was determined by doping with varying amounts of 4-cyanobenzoic acid. The extent of loading was determined by both elemental analysis and ninhydrin assays. The spatial distribution of the coupled 4-cyanobenzamide within the bead was determined to an in-plane resolution of 1 microm and depth resolution of about 4 microm, using the strong Raman CN stretching vibrational transition at 2230 cm(-1). Dry and swollen beads were studied and the distribution was found to be essentially uniform throughout the bead in all cases.

Characterization of the electrostatic perturbation of a catalytic site (Cys)-S-/(His)-Im+H ion-pair in one type of serine proteinase architecture by kinetic and computational studies on chemically mutated subtilisin variants.

We have used two structurally well-characterized serine proteinase variants, subtilisins Carlsberg and BPN', to produce (Cys)-S-/(His)-Im+H ion-pairs by chemical mutation in well defined, different, electrostatic microenvironments. These ion-pairs have been characterized by pH-dependent rapid reaction kinetics using, as reactivity probes, thiol-specific time dependent inhibitors, 2,2'-dipyridyl disulfide and 4,4'-dipyrimidyl disulfide, that differ in the protonation states of their leaving groups in acidic media, computer modelling and electrostatic potential calculations. Both ion-pairs possess nucleophilic character, identified by the striking rate maxima in their reactions with 2,2'-dipyridyl disulfide in acid media. In the Carlsberg enzyme, the (Cys220)-S-/(His63)-Im+H ion-pair is produced by protonic dissociation with pKa 4.1 and its reactivity is not perturbed by any detectable electrostatic influence other than the deprotonation of His63 (pKa 10.2). In the BPN' enzyme, the analogous, (Cys221)-S-/(His64)-Im+H ion-pair is produced by protonic dissociation with pKa 5.1 and its reactivity is affected by an ionization with pKa 3.5 in addition to the deprotonation of His64 (pKa > or = 10.35). It is a striking result that calculations using finite difference solutions of the Poisson-Boltzmann equation provide a value of the pKa difference between the two enzyme catalytic sites (0.97) in close agreement with the value (1.0) determined by reactivity probe kinetics when a protein dielectric constant of 2 is assumed and water molecules within 5 A of the catalytic site His residue are included. The pKa difference is calculated to be 0.84 when the water molecules are not included and a protein dielectric constant of 20 is assumed. The calculations also identify Glu156 in the BPN' enzyme (which is Ser in the Carlsberg enzyme) as the main individual source of the pKa shift. The additional kinetically influential pKa of 3.5 is assigned to Glu156 by examining the non-covalent interactions between the 2-pyridyl disulfide reactivity probe and the enzyme active centre region.

Structure of chymopapain M the late-eluted chymopapain deduced by comparative modelling techniques and active-centre characteristics determined by pH-dependent kinetics of catalysis and reactions with time-dependent inhibitors: the Cys-25/His-159 ion-pair is insufficient for catalytic competence in both chymopapain M and papain.

Chymopapain M, the monothiol cysteine proteinase component of the chymopapain band eluted after chymopapains A and B in cation-exchange chromatography, was isolated from the dried latex of Carica papaya and characterized by kinetic and chromatographic analysis. This late-eluted chymopapain is probably a component of the cysteine proteinase fraction of papaya latex discovered by Schack [(1967) Compt. Rend. Trav. Lab. Carlsberg 36, 67-83], named papaya peptidase B by Lynn [(1979) Biochim. Biophys. Acta 569, 193-201] and partially characterized by Polgár [(1981) Biochim. Biophys. Acta 658, 262-269] and is the enzyme with unusual specificity characteristics (papaya proteinase IV) that Buttle, Kembhavi, Sharp, Shute, Rich and Barrett [Biochem. J. (1989) 261, 469-476] claimed to be a previously undetected cysteine proteinase eluted from a cation-exchange column near to the early-eluted chymopapains. A study of the time-dependent chromatographic consequences of thiol-dependent proteolysis of the components of papaya latex is reported. Chymopapain M was isolated by (i) affinity chromatography followed by separation from papain using cation-exchange f.p.l.c. on a Mono S HR5/5 column and (ii) cation-exchange chromatography followed by an unusual variant of covalent chromatography by thiol-disulphide interchange. The existence in chymopapain M of a nucleophilic interactive Cys/His catalytic-site system analogous to those in papain (EC 3.4.22.2) and other cysteine proteinases was deduced from the characteristics shape of the pH-second-order rate constant (k) profiles for its reactions with 2,2'-dipyridyl disulphide and ethyl 2-pyridyl disulphide. Analysis of the pH-k data for the reactions of chymopapain M with the 2-pyridyl disulphides and with 4,4'-dipyridyl disulphide permits the assignment of molecular pKa values of 3.4 and 8.7 to the formation and subsequent dehydronation of the Cys-S-/His-Im+H state of the catalytic site and reveals three other kinetically influential ionizations with pKa values 3.4, 4.3 and 5.6. The pH-dependences of kcat./Km for the hydrolysis of N-acetyl-L-Phe-Gly-4-nitroanilide at 25.0 degrees C and I0.1 M catalysed by chymopapain M and papain were determined. For both enzymes, little catalytic activity (5-7% of the maximal) develops consequent on formation of the catalytic site Cys-S-/His-Im+H ion-pair state (across pKa 3.4 for both enzymes). For papain, full expression of Kcat./Km for the uncharged substrate requires only the additional hydronic dissociation with pKa 4.2. By contrast, full expression of kcat./Km for chymopapain M requires additional hydronic dissociation with pKa values of 4.3 and 5.6.(ABSTRACT TRUNCATED AT 400 WORDS)

Clarification of the pH-dependent kinetic behaviour of papain by using reactivity probes and analysis of alkylation and catalysed acylation reactions in terms of multihydronic state models: implications for electrostatics calculations and interpretation of the consequences of site-specific mutations such as Asp-158-Asn and Asp-158-Glu.

1. The complex behaviour of papain (EC 3.4.22.2) in acidic media has been investigated by (a) stopped-flow reactivity probe kinetics using 4,4'-dipyrimidyl disulphide (I) and 2,2'-dipyridyl disulphide (II) as thiol-specific time-dependent inhibitors with markedly different susceptibilities to activation by hydronation (protonation) and (b) using the multitasking application program SKETCHER for the rapid evaluation of pH-dependent kinetic data by means of interactive manipulation of calculated curves. 2. The substantially lower basicity of (I) (pKa 0.91) than that of (II) (pKa 2.45) combined with retention of high reactivity permitted the pKa for the formation of the (Cys-25)-S-/(His-159)-Im+H ion-pair state of papain to be determined kinetically as 3.4, a value close to that (3.3) deduced by potentiometric difference titration [Lewis, Johnson and Shafer (1976) Biochemistry 15, 5009-5017] and lower than the value (approx. 4) often reported from pH-dependent kinetic studies. The higher values are now known to arise from inadequate data analysis that does not take account of other overlapping kinetically influential ionizations. 3. Re-evaluation of the extensive sets of pH-kcat/Km data for the hydrolysis of nine substrates by papain reported by Polgár and Halász (1978) (Eur. J. Biochem. 88, 513-521) by making use of SKETCHER, the known pKa value (3.4) from the reaction with compound (I) and two additional kinetically influential pKa values deduced from the reaction with compound (II) now permits the identification of the pH-dependent events in reactions of papain with inhibitors and substrates. 4. A major conclusion is that, whereas in reactions of simple alkylating agents and compound (I) full nucleophilic character of (Cys-25)-S-/(His-159)-Im+H is provided by hydronic dissociation with pKa 3.3-3.4, in catalysis relatively little catalytic competence is produced consequent upon ion-pair formation. Substantial catalytic competence requires further hydronic dissociation with pKa approx. 4, and for cationic substrates further enhancement is produced by hydronic dissociation with pKa approx. 5. 5. The present work, together with the kinetic analysis of reactions of papain in alkaline media reported by Mellor, Thomas, Topham and Brocklehurst [Biochem. J. (1993) 290, 289-296], defines the kinetically influential ionizations of papain as 3.4, 4.0, 5.0, 8.3 and 10.0 of which 3.4 and 8.3 relate to the formation and subsequent dehydronation of the ion-pair state.(ABSTRACT TRUNCATED AT 400 WORDS)

Catalytic antibody activity elicited by active immunisation. Evidence for natural variation involving preferential stabilization of the transition state.

1. The hydrolytic activity of IgG purified from (a) 13 samples of ovine antiserum collected from three animals during a two-year immunisation programme using a phosphate immunogen (comprising the amide conjugate bonded through the carboxy group of 4-nitrophenyl 4-carboxymethylphenyl hydrogen phosphate and amino groups of keyhole-limpet haemocyanin) and (b) a sample of ovine antiserum collected from another animal during an 18-week immunisation programme using an analogous sulphone immunogen (comprising the amide conjugate bonded through the amino group of 4-nitrobenzyl, 4-(4-aminobutoxy)benzyl sulphone and carboxyl groups of keyhole-limpet haemocyanin) were evaluated kinetically by using 4-nitrophenyl 4-(3-aza-2-oxoheptyl)phenyl carbonate and 4-nitrophenyl 4-(2-hydroxyethoxy)phenyl carbonate as substrates. 2. Catalytic activity was found in all 13 samples of anti-phosphate IgG but was absent in the sample of anti-sulphone IgG as well as in all samples of IgG isolated from the serum of non-immunised animals. These findings, taken together with the lack of catalytic activity of the anti-phosphate IgG towards the 2-nitrophenyl 4-(3-aza-2-oxoheptyl)phenyl carbonate, compel the view that the catalytic activity of the anti-phosphate IgG preparation is entirely antibody-mediated and is not due to contaminant hydrolytic enzymes. The fact that catalytic activity was found in all 13 samples of the anti-phosphate IgG provides the first evidence that it is possible, as a routine, to elicit a catalytic antibody response in a host animal via active immunisation. 3. The nature of the, albeit small, variation in the catalytic characteristics of the anti-phosphate IgG (increase in both kcat, the catalytic rate constant calculated as V/2[IgG] and kcat/Km, the apparent second-order rate constant for the overall catalysed conversion of substrate to products, with increase in Km suggests simultaneous improvement in transition state binding and deterioration in substrate binding as predicted from immunogen design and the postulated general mechanistic basis of antibody catalysis. 4. This interpretation is supported by the difference in the values of the dissociation constant Ki for the competitive inhibition by the transition-state analogue 4-methylphenyl 4-nitrophenyl hydrogen phosphate of reactions catalysed by two representative anti-phosphate IgG samples: for the catalysis with Km = 4.5 microM, Ki = 9 nM and for that with Km = 1.3 microM, Ki = 80 nM.(ABSTRACT TRUNCATED AT 400 WORDS)

Ionization characteristics of the Cys-25/His-159 interactive system and of the modulatory group of papain: resolution of ambiguity by electronic perturbation of the quasi-2-mercaptopyridine leaving group in a new pyrimidyl disulphide reactivity probe.

1. A new thiol-specific reactivity probe 4,4'-dipyrimidyl disulphide [compound (VII), m.p. 110 degrees C, pKa of its monohydronated form 0.91] was synthesized and used to resolve the ambiguity of interpretation of the behaviour of papain (EC 3.4.22.2) in alkaline media known to depend to varying extents on two ionizations with pKa values approx. 8.0-8.5 and > or = 9.5 respectively. 2. A new extensive pH-second-order rate constant (k) data set for the reaction of papain with 2-(acetamido)-ethyl 2'-pyridyl disulphide (IV) demonstrated the existence of a striking rate maximum at pH approx. 4, the independence of k around pH 8 and the increase in k with increase in pH across a pKa value of 10.0, behaviour similar to that of other 2-pyridyl disulphides (R-S-S-2-Py) that lack key substrate-like binding sites in R. 3. Although the simplest interpretation of the pKa value of 10.0 assigns it to the formation of (Cys-25)-S-/(His-159)-Im from the ion-pair state of the papain catalytic site, another interpretation may be conceived in which this pKa value is assigned to another group remote from the catalytic site, the state of ionization of which modulates catalytic-site behaviour. This alternative assignment is shown to require compensating effects in the pH region around 8 such that the formation of (Cys-25)-S-/(His-159)-Im across pKa 8.0-8.5 is without net kinetic effect in the reactions of simple 2-pyridyl disulphides such as compound (IV) and 2,2'-dipyridyl disulphide (II). 4. The lower basicity of compound (VII) relative to that of compound (II) (pKa 2.45) was predicted to diminish or abolish the compensation postulated as a possibility in reactions of 2-pyridyl disulphides because of the decreased effectiveness of reaction via a (His-159)-Im+H-assisted transition state. The characteristics of the pH-dependence of the reaction of papain with compound (VII) which are quite different from those for its reaction with compound (II) support both this prediction and the alternative assignment with a value of 8.3 for the pKa of the formation of (Cys-25)-S-/(His-159)-Im. 5. Evidence that the behaviour of papain towards both substrates and some substrate-derived time-dependent inhibitors is determined not only by the loss of the (Cys-25)-S-/(His-159)-Im+H ion-pair state by dehydronation with pKa 8.3 but also by another ionization of pKa approx. 10.0 is briefly discussed.

Catalytic-site characteristics of the porcine calpain II 80 kDa/18 kDa heterodimer revealed by selective reaction of its essential thiol group with two-hydronic-state time-dependent inhibitors: evidence for a catalytic site Cys/His interactive system and an ionizing modulatory group.

1. Four calpain II heterodimers (80 kDa/30 kDa, 80 kDa/29 kDa, 80 kDa/26 kDa and 80 kDa/18 kDa) were isolated from fresh porcine kidney by (NH4)2SO4 precipitation, chromatography on DEAE-Sepharose CL-6B and subsequently on Reactive Red 120/agarose followed by f.p.l.c. on a Q-Sepharose Hi-Load 16/10 column. 2. The major component (80 kDa/30 kDa) was used to provide the catalytically active calpain II 80 kDa/18 kDa heterodimer by treatment with CaCl2; titration with trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane (E64) in the presence of monothioglycerol showed the preparation to have 1.0 +/- 0.05 catalytic sites per molecule of heterodimer. 3. The 80 kDa/30 kDa heterodimer was separated from monothioglycerol and other low-molecular-mass material by gel filtration on Sephadex G-25 without loss of catalytic activity towards sulphanilic acid/azocasein in the presence of added Ca2+. On storage overnight at a concentration of 3 microM in KCl at 4 degrees C in the absence of Ca2+ the activator-free preparation still produced fully active 80 kDa/18 kDa heterodimer on addition of Ca2+. 4. Activator-free 80 kDa/30 kDa heterodimer (in the absence of Ca2+) reacts relatively slowly with ethyl 2-pyridyl disulphide at pH 5.9; over 5000 s five thiol groups per molecule react, all at similar rates. In the presence of 8 mM CaCl2 under otherwise identical conditions (and also in the pH range 3.8-10.4) an initial faster phase of reaction corresponding to approx. one thiol group per molecule of heterodimer is generated, but it is not cleanly separated from the subsequent slower reactions on the stopped-flow trace. This fast phase of reaction does not occur when E64-inactivated calpain II is substituted for active 80 kDa/18 kDa heterodimer. 5. Greatly improved resolution of the fast phase of reaction involving the catalytic-site thiol group was achieved by using 2,2'-dipyridyl disulphide (2-Py-S-S-2-Py) instead of ethyl 2-pyridyl disulphide. 6. The pH-dependence of the second-order rate constant (k) for the reaction of the catalytically active activator-free 80 kDa/18 kDa calpain II heterodimer with 2-Py-S-S-2-Py was studied by stopped-flow spectral analysis in the pH range approx. 3-8 without interference from reactions of other thiol groups. 7. The form of the pH-k profile establishes for the first time the existence of an interactive catalytic site system [probably containing a (Cys)-S-/(His)-Im+H ion pair] analogous to those present in monomeric non-Ca(2+)-activated cysteine proteinases.(ABSTRACT TRUNCATED AT 400 WORDS)

Evaluation of hydrogen-bonding and enantiomeric P2-S2 hydrophobic contacts in dynamic aspects of molecular recognition by papain.

1. 2-(N'-Acetyl-D-phenylalanyl)hydroxyethyl 2'-pyridyl disulphide (compound IV) (m.p. 59 degrees C; [alpha]D20 -6.6 degrees (c 1.2 in methanol)) was synthesized. 2. The results of a study of the pH-dependence of the second-order rate constant (k) for its reaction with the catalytic-site thiol group (Cys-25) of papain (EC 3.4.22.2) together with analogous kinetic data for the reactions of related time-dependent inhibitors, notably the L-enantiomer of compound (IV) (compound III) and the L- and D-enantiomers of 2-(N'-acetylphenylalanylamino)ethyl 2'-pyridyl disulphide (compounds I and II respectively), were used to assess the contributions of the (P1)-NH ... O = C < (Asp-158) and (P2) > C = O ... H-N-(Gly-66) hydrogen bonds and enantiomeric P2-S2 hydrophobic contacts in two manifestations of dynamic molecular recognition in papain-ligand association: (a) signalling to the catalytic-site region to provide for a (His-159)-IM(+)-H-assisted transition state and (b) the dependence of P2-S2 stereoselectivity on hydrogen-bonding interactions outside the S2 subsite. The analysis involved determination of the reactivities of individual ionization states of the reactions (pH-independent rate constants, k) and associated macroscopic pKa values and difference kinetic specificity energies (delta delta GKS = -RT1n(k1/k2), where k1 is the pH-independent second-order rate constant for reaction with one inhibitor and k2 is the analogous rate constant in the same ionization state for reaction with another inhibitor so that, when the structural change provides that k2 > k1, delta delta GKS is positive. 3. The kinetic data further illuminate the nature of the interdependence of binding interactions in papain first noted by Kowlessur, Topham, Thomas, O'Driscoll, Templeton & Brocklehurst [(1989) Biochem. J. 258, 755-764] in the S2 subsite, S1-S2 intersubsite and catalytic-site regions. Of particular note is the apparent dependence of the binding of the N-Ac-D-Phe moiety on the binding of the leaving group to (His-159)-Im+H and the fact that the resulting rate enhancement is more effective when (P1)-N-H is absent than when it is present. This result revealed by kinetic analysis goes beyond the conclusion suggested by model building that it is possible to make all of the binding contacts in complexes involving the D-enantiomers [(II) and (IV)] as in those involving the L-enantiomers [(I) and (III)].(ABSTRACT TRUNCATED AT 400 WORDS)

Variation in the P2-S2 stereochemical selectivity towards the enantiomeric N-acetylphenylalanylglycine 4-nitroanilides among the cysteine proteinases papain, ficin and actinidin.

1. Values of the kinetic specificity constant, kcat./Km, for the hydrolysis of N-acetyl-L-phenylalanylglycine 4-nitroanilide (I) and of its D-enantiomer (II) catalysed by ficin (EC 3.4.22.3) and by actinidin (EC 3.4.22.14) at pH 6.0, I 0.1 mol/l, 8.3% (v/v) NN-dimethylformamide and 25 degrees C were determined by using initial-rate data with [S] much less than Km and weighted nonlinear regression analysis as: for ficin, (kcat./Km)L = 271 +/- 6 M-1.s-1, (kcat./Km)D = 2.9 +/- 0.1 M-1.s-1, and for actinidin (kcat./Km)L = 13.3 +/- 0.7 M-1.s-1, (kcat/Km)D = 0.34 +/- 0.01 M-1.s-1.2. These data and analogous values for the corresponding reactions catalysed by papain (EC 3.4.22.2), (kcat./Km)L = 2064 +/- 31 M-1.s-1, (kcat./Km)D = 5.5 +/- 0.1 M-1.s-1, demonstrate marked variation in stereochemical selectivity for substrates (I) and (II) among the three cysteine proteinases with the following values for the index of stereochemical selectivity Iss = (kcat./Km)L/(kcat./Km)D: for papain, 375; for ficin 93; for actinidin 39. 3. Model building suggests ways in which, for the papain-catalysed reactions, binding interactions involving the extended acyl groups of the substrates may need to change as the reaction proceeds from adsorptive complex (ES) to tetrahedral intermediate (THI) before its rate-determining, general acid-catalysed collapse to acylenzyme intermediate. In particular, satisfactory alignment in the catalytic site at the THI stage of the acylation process appears to demand rotation of the substrate moiety about its long axis. 4. The different consequences of this rotation for the L- and D-enantiomers suggest that for closely related systems the greater the extent of this rotational adjustment the greater would be the value of Iss.5. For the actinidin-substrate combinations, model building suggests that even at the ES complex stage of catalysis it is not possible to approach optimized P2-S2 contacts and the three hydrogen-bonding interactions deduced for papain-ligand complexes in the absence of significant movement of protein conformation. Possible binding modes in which some of the interactions deduced for papain are relaxed are discussed. Consideration of postulated binding modes in the various transition states is shown to account for the order of reactivity reflected in values kcat./Km for the four reactions involving papain (Pap) and actinidin (Act) with the L- and D-enantiomeric substrates: Pap-L much greater than Act-L greater than Pap-D much greater than Act-D.(ABSTRACT TRUNCATED AT 400 WORDS)

A polyclonal antibody preparation with Michaelian catalytic properties.

1. 4-Nitrophenyl 4'-(3-aza-2-oxoheptyl)phenyl carbonate (I), an amide conjugate (XI) involving the carboxy group of 4-nitrophenyl 4'-carboxymethylphenyl phosphate and an amino group of keyhole-limpet haemocyanin, and a fluorescein derivative (XVII) were synthesized. 2. The conjugate (XI) was used as an immunogen with which to raise polyclonal antibodies in multigeneration cross-bred sheep; the fluorescent derivative (XVII) was used for the initial assessment of the antisera via binding assays monitored by fluorescence polarization; the carbonate ester (I) was used as a chromogenic substrate for the investigation of catalytic activity. 3. The IgG from the antiserum of sheep no. 270 was isolated by Na2SO4 precipitation and chromatography on Protein G-Sepharose. 4. This preparation of IgG catalysed the hydrolysis of the carbonate ester (I); the catalysis at pH 8.0 and 25 degrees C obeyed Michaelis-Menten kinetics with at least 25 turnovers, Km = 3.34 microM, and lower limits for kcat. of 0.029 s-1 and for kcat./Km of 8.77 x 10(3) M-1.S-1, on the unlikely assumption that the concentration of catalytic antibody is provided by twice the total IgG concentration (two sites per molecule); probable estimates of the fraction of the total IgG that is anti-haptenic IgG and of the fraction of this that is catalytically active suggest that the values of kcat./Km are actually very much larger than these lower limits. 5. The failure of the antibody preparation to catalyse the hydrolysis of the isomeric 2-nitrophenyl carbonate (II), which differs from compound (I) only in the position of the nitro substituent in the leaving group, compels the view that catalytic activity is due to antibody rather than contaminant enzyme; this conclusion is supported by (a) the failure of the following to discriminate effectively between the isomeric substrates (I) and (II): pig liver carboxylesterase, rabbit liver carboxylesterase (collectively EC 3.1.1.1), whole serum from a non-immunized sheep and whole serum from a sheep immunized with a derivative of 3-O-methylnoradrenaline and (b) the lack of catalytic activity in IgG preparations from sheep immunized with sulphoxide or sulphone analogues of immunogen (XI). 6. The various parameters used for the comparison of the kinetic characteristics of hydrolytic catalytic antibodies are discussed. 7. The characteristics of hydrolysis of compound (I) catalysed by the present polyclonal antibody preparation are shown to be substantially better in most respects than those of analogous reactions of two other carbonate esters catalysed by monoclonal antibodies.

Development and validation of a fluoroimmunoassay for urinary normetanephrine.

The development of a separation fluoroimmunoassay for urinary normetanephrine is described. Antiserum specific to normetanephrine was coupled, using cyanogen bromide, to magnetizable cellulose; and fluorescein labelled normetanephrine was synthesized from fluoresceinthiocarbamylethylene diamine and a previously described normetanephrine derivative. Using these reagents it was possible to construct a reproducible standard curve, covering a wide range of concentrations, and to accurately measure the concentration of this metabolite in acid-hydrolysed urine samples. Cross-reactivities of structurally similar compounds were low and the fluoroimmunoassay showed good correlation with an established gas-chromatographic assay. The procedure is rapid; it is possible to accurately determine the normetanephrine concentration of urine samples approximately 2 h after hydrolysis, resulting in an overall assay time of approximately 4 h. This is the first report of a non-isotopic immunoassay for normetanephrine.

Fluorescence polarization immunoassay of urinary vanillylmandelic acid.

This rapid fluorescence polarization immunoassay for urinary vanillylmandelic acid (VMA) involves use of our previously described antiserum and label and the program for 5-hydroxyindoleacetic acid in the Abbott TDx fluorimeter. Urine samples were measured directly, without pretreatment. The minimum detectable concentration was 0.3 mg/L, and the range of the standard curve was 0.3-200.0 mg/L. Precision, analytical recovery, and correlation of results with those by the Pisano method (Clin Chim Acta 1962;7:285-91) were all satisfactory. With this procedure one can determine the VMA concentration in 10 urine sample in 22 min. This is the first report of a clinical immunoassay for VMA and should greatly simplify screening for neural crest tumors.

Comparison of three different activation methods for coupling antibodies to magnetisable cellulose particles.

The periodate and 1,1'-carbonyldiimidazole activation methods were compared with the cyanogen bromide procedure for coupling antibodies to magnetisable cellulose/iron oxide solid-phase particles. Fluoroimmunoassays for quinine, primaquine, normetanephrine and cannabinoids were employed to assess the binding properties of such coupled solid phases. The cyanogen bromide and 1,1'-carbonyldiimidazole methods gave similar products in most cases, while the specific binding capacity of periodate coupled particles was between two and five times lower. Nevertheless, comparable standard curves could be obtained with solid phase coupled by each method. The periodate and 1,1'-carbonyldiimidazole methods are acceptable alternatives, notably for laboratories lacking the facility to handle the toxic cyanogen bromide.

Production and characterisation of antibodies to vanillylmandelic acid.

Antibodies were raised to the catecholamine metabolite vanillylmandelic acid (VMA). The side chain was protected and, after derivatisation through the 4-phenolic hydroxyl group, the hapten was coupled to KLH and the resultant conjugate deprotected. Sheep immunised with this immunogen responded with specific, high titre antibodies to VMA as assessed using a fluorescent label. Cross-reaction of the antiserum from one of the sheep was minimal, being under 1% with all naturally occurring related compounds tested. This report is the first to describe an effective antiserum for VMA which will permit measurement over the normal range in urine.