Monoclonal antibodies against tryptophanyl-tRNA-synthetase

Monoclonal antibodies designated as Am1 and Am2 were prepared against purified beef pancreas tryptophanyl-tRNA synthetase (EC 6.1.1.2). Both antibodies were able to bind the native enzyme in a solid-phase assay and to precipitate enzyme activity in immune complexes. Am2 inhibited the tryptophanyl-tRNA synthetase activity in ATP-[32P]pyrophosphate exchange and in tRNATrp aminoacylation reactions; Am1 had no influence on both the enzyme activity and the inhibiting action of Am2. Only Am2, not Am1, bound elastase-modified form of the enzyme which consists of two subunits shortened by 20 000 daltons from the N-end of the molecule. These results were interpreted as an evidence for non-overlapping of Am1 and Am2 antigenic determinants along the polypeptide chains of the enzyme.     

A mammalian tryptophanyl-tRNA synthetase shows little homology to prokaryotic synthetases but near identity with mammalian peptide chain release factor

Determination of the amino acid sequence of beef pancreas tryptophanyl-tRNA synthetase was undertaken through both cDNA and direct peptide sequencing. A full-length cDNA clone containing a 475 amino acid open reading frame was obtained. The molecular mass of the corresponding peptide chain, 53,728 Da, was in agreement with that of beef tryptophanyl-tRNA synthetase, as determined by physicochemical methods (54 kDa). Expression of this clone in Escherichia coli led to tryptophanyl-tRNA synthetase activity in cell extracts. The open reading frame included two sequences analogous to the consensus sequences, HIGH and KMSKS, found in class I aminoacyl-tRNA synthetases. The homology with prokaryotic and yeast mitochondrial tryptophanyl-tRNA synthetases was low and was limited to the regions of the consensus sequences. However, a 90% homology was observed with the recently described rabbit peptide chain release factor (eRF) [Lee et al. (1990) Proc. Natl. Acad. Sci. 87, 3508-3512]. Such a strong homology may reveal a new group of genes deriving from a common ancestor, the products of which could be involved in tRNA aminoacylation (tryptophanyl-tRNA synthetase) or translation termination (eRF).     

Development and characterization of a competitive polyclonal antibody enzyme-immunoassay for salmon insulin-like growth factor-II

This paper describes the development and validation of a competitive, polyclonal antibody enzyme-immunoassay (EIA) for the measurement of salmon and trout insulin-like growth factor-II (IGF-II). A polyclonal antiserum was raised against a synthetic peptide epitope, corresponding to amino acid residues 1-9 of the N-terminus of mature Atlantic salmon (Salmo salar) IGF-II. The antiserum was purified by hydrophobic charge induction chromatography (HCIC). The partially purified immunoglobulins were used in an enzyme-immunoassay system (EIA) resulting in a highly specific assay for salmon IGF-II with cross-reactivity of less than 0.01% for recombinant salmon IGF-I and recombinant salmon growth hormone (GH), and 5.57% for salmon insulin (sIns). The recombinant salmon IGF-II (rsIGF-II) standard curve limit of detection was 1.37 ng/ml with an EC(50) of 44.97+/-0.82 ng/ml. Intra- and interassay coefficients of variation were determined at 7.47% (n=15) and 7.42% (n=15), respectively. Added rsIGF-II was adequately recovered from acid-treated Atlantic salmon and rainbow trout (Oncorhynchus mykiss) plasma samples. Parallel dose-response inhibition curves were demonstrated for the plasma of both fish species tested. Circulating IGF-II levels of 22.26+/-2.66 and 18.24+/-1.43 ng/ml were determined for acid-treated plasma of normal adult Atlantic salmon and rainbow trout, respectively. This EIA should prove to be useful in the study of factors which influence circulating plasma levels of IGF-II in these fish species.     

Two-dimensional structure of beta-amyloid(10-35) fibrils

Beta-amyloid (Abeta) peptides are the main protein component of the pathognomonic plaques found in the brains of patients with Alzheimer's disease. These heterogeneous peptides adopt a highly organized fibril structure both in vivo and in vitro. Here we use solid-state NMR on stable, homogeneous fibrils of Abeta(10-35). Specific interpeptide distance constraints are determined with dipolar recoupling NMR on fibrils prepared from a series of singly labeled peptides containing (13)C-carbonyl-enriched amino acids, and skipping no more that three residues in the sequence. From these studies, we demonstrate that the peptide adopts the structure of an extended parallel beta-sheet in-register at pH 7.4. Analysis of DRAWS data indicates interstrand distances of 5.3 +/- 0.3 A (mean +/- standard deviation) throughout the entire length of the peptide, which is compatible only with a parallel beta-strand in-register. Intrastrand NMR constraints, obtained from peptides containing labels at two adjacent amino acids, confirm the secondary structural findings obtained using DRAWS. Using peptides with (13)C incorporated at the carbonyl position of adjacent amino acids, structural transitions from alpha-helix to beta-sheet were observed at residues 19 and 20, but using similar techniques, no evidence for a turn could be found in the putative turn region comprising residues 25-29. Implications of this extended parallel organization for Abeta(10-35) for overall fibril formation, stability, and morphology based upon specific amino acid contacts are discussed.     

Development of sensitive enzyme immunoassay for human nerve growth factor.

We prepared anti-recombinant human nerve growth factor (hNGF) antibody IgG and characterized its property immunologically. This antibody IgG reacted with some animal NGFs, especially with bovine NGF, on immunodiffusion analysis. Using this antibody IgG, we developed a sensitive two-site enzyme immunoassay (EIA) system for human NGF, based on the biotin-streptoavidin system. NGF at a concentration as low as 0.02 pg/well (corresponding to 8 x 10(-19) mol) could be measured with high reproducibility. The sensitivity of this EIA was equal to that of our EIA for mouse NGF. With this EIA, the detection limit of other mammalian NGFs was reduced in parallel with the degree of decrease in amino acid sequence homology between them and hNGF.     

Secondary structural analysis of the Na(+),K(+)-ATPase and its Na(+) (E(1)) and K(+) (E(2)) complexes by FTIR spectroscopy

The Na(+),K(+)-ATPase is an integral membrane protein which transports sodium and potassium cations against an electrochemical gradient. The transport of Na(+) and K(+) ions is presumably connected to an oscillation of the enzyme between the two conformational states, the E(1) (Na(+)) and the E(2) (K(+)) conformations. The E(1) and E(2) states have different affinities for ligand interaction. However, the determination of the secondary structure of this enzyme in its sodium and potassium forms has been the subject of much controversy. This study was designed to provide a quantitative analysis of the secondary structure of the Na(+),K(+)-ATPase in its sodium (E(1)) and potassium (E(2)) states in both H(2)O and D(2)O solutions at physiological pH, using Fourier transform infrared (FTIR) with its self-deconvolution and second derivative resolution enhancement methods, as well as curve-fitting procedures. Spectroscopic analysis showed that the secondary structure of the sodium salt of the Na(+),K(+)-ATPase in H(2)O solution contains alpha-helix 19.8+/-1%, beta-sheet 25.6+/-1%, turn 9.1+/-1%, and beta-anti 7.5+/-1%, whereas in D(2)O solution, the enzyme shows alpha-helix 16.8+/-1%, beta-sheet 24.5+/-1.5%, turn 10.9+/-1%, beta-anti 9.8+/-1%, and random coil 38.0+/-2%. Similarly, the potassium salt in H(2)O solution contains alpha-helix 16.6+/-1%, beta-sheet 26.4+/-1.5%, turn 8.9+/-1%, and beta-anti 8.1+/-1%, while in D(2)O solution it shows alpha-helix 16.2+/-1%, beta-sheet 24.5+/-1.5%, turn 10.3+/-1%, beta-anti 9.0+/-1%, and random coil 40+/-2%. Thus the main differences for the sodium and potassium forms of the Na(+),K(+)-ATPase are alpha-helix 3.2% in H(2)O and 0.6% in D(2)O, beta-sheet (pleated and anti) 1.5% in H(2)O and random structure 2% (D(2)O), while for other minor components (turn structure), the differences are less than 1%.     

Synthesis and characterization of a chimeric peptide derived from fasciculin that inhibits acetylcholinesterase.

Fasciculins are peptides isolated from mamba (Dendroaspis) venoms which exert their toxic action by inhibiting acetylcholinesterase (AChE). They contain a characteristic triple stranded antiparallel beta-sheet formed by residues 22-27, 34-39 and 48-53. A chimeric peptide named Fas-C, encompassing most of these sequences was synthesized using SPPS/Boc-chemistry and characterized chemically, structurally and functionally. Fas-C has two disulfide bridges, formed sequentially using dual cysteine protection. SDS-PAGE patterns, HPLC profiles and MS proved the peptide identity. Circular dichroism indicated the presence of 13.6% and 41.6% of beta-sheet and beta-turn, respectively, comparable to values observed in the native toxin. An inhibitory effect on eel AChE was displayed by the peptide (Ki71.6 +/- 18.3 microM), although not reaching the affinity level of the parent native toxin (Ki 0.3 nM). It is confirmed that the principal binding region of fasciculin to AChE resides within loop II.     

Preparation of antibodies against a novel immunosuppressant, FTY720, and development of an enzyme immunoassay for FTY720

FTY720 (1) is a novel immunosuppressant (immunomodulator), derived from ISP-I (2: myriocin and thermozymocidin). To clarify the pharmacokinetic properties of 1, antibodies against 1 were prepared and a competitive enzyme immunoassay (EIA) was developed. Two kinds of haptens, 3 and 4, for 1 were synthesized and coupled to ovalbumin (OVA). Rabbits were immunized with 3-OVA or 4-OVA, and corresponding antibodies were obtained. Both antibodies recognized the 2-amino-2-(2-phenylethyl)propane-1,3-diol moiety in 1. Using the anti-3-OVA antibody, a competitive EIA for 1 was developed and evaluated. The range of quantification by the EIA was 0.06-10 ng/mL. The application of the EIA has enabled us to measure the FTY720 concentration in serum after oral administration of 1 (1mg/kg) to rats.     

Solution structure of delta-Am2766: a highly hydrophobic delta-conotoxin from Conus amadis that inhibits inactivation of neuronal voltage-gated sodium channels

The three-dimensional (3D) NMR solution structure (MeOH) of the highly hydrophobic delta-conotoxin delta-Am2766 from the molluscivorous snail Conus amadis has been determined. Fifteen converged structures were obtained on the basis of 262 distance constraints, 25 torsion-angle constraints, and ten constraints based on disulfide linkages and H-bonds. The root-mean-square deviations (rmsd) about the averaged coordinates of the backbone (N, C(alpha), C) and (all) heavy atoms were 0.62+/-0.20 and 1.12+/-0.23 A, respectively. The structures determined are of good stereochemical quality, as evidenced by the high percentage (100%) of backbone dihedral angles that occupy favorable and additionally allowed regions of the Ramachandran map. The structure of delta-Am2766 consists of a triple-stranded antiparallel beta-sheet, and of four turns. The three disulfides form the classical 'inhibitory cysteine knot' motif. So far, only one tertiary structure of a delta-conotoxin has been reported; thus, the tertiary structure of delta-Am2766 is the second such example. Another Conus peptide, Am2735 from C. amadis, has also been purified and sequenced. Am2735 shares 96% sequence identity with delta-Am2766. Unlike delta-Am2766, Am2735 does not inhibit the fast inactivation of Na+ currents in rat brain Na(v)1.2 Na+ channels at concentrations up to 200 nM.     

Aldehyde and ketone substrate analogues inhibit the collagenase of Clostridium histolyticum

The collagenase from Clostridium histolyticum is a mixture of several collagenases, all of which are zinc metalloproteases. This enzyme catalyzes the cleavage of the X-Gly peptide bond in the repeating sequence of collagen: -Gly-Pro-X-Gly-Pro-X-. Thus the S3, S2, and S1 subsites on the enzyme appear to be occupied by the sequence -Gly-Pro-X- and the S1', S2', and S3' subsites also by -Gly-Pro-X-. Short peptides up to and including N alpha-acyltetrapeptides containing the repeat sequence do not detectably inhibit the enzyme (IC50 greater than 10 mM). However, peptide aldehydes of the form aminoacyl-X-glycinal, presumably occupying the S1, S2, ..., Sn subsites, are inhibitors. The most potent of these was Pro6-Gly-Pro-glycinal, with an IC50 of 340 +/- 70 microM. The single peptide aldehyde investigated, which could occupy the S1' and S2' subsites, 4-oxobutanoyl-L-proline, did not inhibit collagenase (IC50 greater than 20 mM). The peptide ketone 5-benzamido-4-oxo-6-phenylhexanoyl-Pro-Ala (XXV), which could occupy the S1-S3' subsites, inhibits collagenase with an IC50 of 120 +/- 50 microM, over 80-fold more potently than its parent peptide analogue benzoyl-Phe-Gly-Pro-Ala (XXIII). The alcohol analogue of XXV, 5-benzamido-4-hydroxy-6-phenylhexanoyl-Pro-Ala (XXVI), is over 60-fold less potent with an IC50 of 8 +/- 2mM. Extending the peptide ketone XXV to occupy the S2-S3' subsites gave 5-(N alpha-carbobenzoxy-L-prolinamido)-4-oxo-6-phenylhexanoyl-Pro -Ala (XXVII). Surprisingly, XXVII had an IC50 of only 5.2 +/- 2 mM.(ABSTRACT TRUNCATED AT 250 WORDS)     

Synthesis, cloning and expression of the single-chain Fv gene of the HPr-specific monoclonal antibody, Jel42. Determination of binding constants with wild-type and mutant HPrs.

The monoclonal antibody Jel42 is specific for the Escherichia coli histidine-containing protein, HPr, which is an 85 amino acid phosphocarrier protein of the phosphoenolpyruvate:sugar phosphotransferase system. The binding domain (Fv) has been produced as a single chain Fv (scFv). The scFv gene was synthesized in vitro and coded for pelB leader peptide-heavy chain-linker-light chain-(His)(5) tail. The linker is three repeats from the C-terminal repetitive sequence of eukaryotic RNA polymerase II. This linker acts as a tag; it is the antigen for the monoclonal antibody Jel352. The codon usage was maximized for E.coli expression, and many unique restriction endonuclease sites were incorporated. The scFv gene incorporated into pT7-7 was highly expressed, yielding 10-30% of the cell protein as the scFv, which was found in inclusion bodies with the leader peptide cleaved. Jel42 scFv was purified by denaturation/renaturation yielding preparations with K(d) values from 20 to 175 nM. However, based upon an assessment of the amount of active refolded scFv, the binding dissociation constant was estimated to be 2.7 +/- 2.0 nM compared with 2.8 +/- 1.6 and 3.7 +/- 0.3 nM previously determined for the Jel42 antibody and Fab fragment respectively. The effect of mutation of the antigen HPr on the binding constant of the scFv was very similar to the properties determined for the antibody and the Fab fragment. It was concluded that the small percentage ( approximately 6%) of refolded scFv is a true mimic of the Jel42 binding domain and that the incorrectly folded scFv cannot be detected in the binding assay.     

The immune response to GHRH, relationship to conformation

Antibodies to GHRH1-44, GHRH1-29, and proinsulin were induced in guinea pigs. GHRH1-44 forms 7 S and 10 S complexes with antibodies. It is a divalent antigen. The sequence 30-44 bound 85% of the antibodies to GHRH1-44 with high affinity (3.8 +/- 0.9 x 10(9) l/mol). The fragment 1-29 bound with low affinity (0.6 +/- 0.3 x 10(9) l/mol) 15% of the antibodies (2p less than 0.001). Antibodies to GHRH1-29 had low affinity towards the native hormone (0.4 +/- 0.2 x 10(9) l/mol) and the region 1-29 (0.3 +/- 0.2 x 10(9) l/mol). Antibodies to proinsulin bound linear C-peptide with lower affinity (0.3 +/- 0.2 x 10(9) l/mol) than the C-peptide loop in proinsulin (3.4 +/- 0.9 x 10(9) l/mol). It is concluded that the conformation of the epitopes on the sequence 1-29, recognized during the immune response, i.e. on the cell membrane, is different from the conformation of GHRH1-29 or GHRH1-44 in aqueous solution.     

cDNA cloning and membrane topology of the rabbit gastric H+/K(+)-ATPase alpha-subunit.

We have cloned and sequenced a cDNA for the rabbit gastric proton-potassium pump (H+/K(+)-ATPase) alpha-subunit. The deduced peptide contains 1035 amino acids (Mr 114,201) and shows 97% sequence identity with the respective rat and hog proteins. A monoclonal antibody 146-14 has been shown previously to react with the extracytoplasmic side of the catalytic H+/K(+)-ATPase subunit and here we show that the epitope is in the region between amino acids 855 and 902 (the numbering of the H+/K(+)-ATPase catalytic subunit throughout the paper refers to the rabbit sequence). The localization of this epitope in conjunction with previously observed trypsin cleavage sites in the C-terminal one third of the enzyme and the hydrophobicity plot of the deduced peptide sequence are evidence for a structural model for the alpha-subunit of the H+/K(+)-ATPase which contains at least ten membrane spanning segments, similar to that deduced for the Ca(2+)-ATPase of sarcoplasmic reticulum.     

A new sandwich enzyme immunoassay for measurement of plasma pre-beta1-HDL levels

Pre-beta1-HDL, a putative discoid-shaped high density lipoprotein (HDL) of approximately 67-kDa mass that migrates with pre-beta mobility in agarose gel electrophoresis, contains apolipoprotein A-I (apoA-I), phospholipids, and unesterified cholesterol. It participates in the retrieval of cholesterol from peripheral tissues. In this study we established a new sandwich enzyme immunoassay (EIA) for measuring plasma pre-beta1-HDL using mouse anti-human pre-beta1-HDL monoclonal antibody (MAb 55201) and goat anti-human apoA-I polyclonal antibody. MAb 55201 reacted with apoA-I in lipoprotein [A-I] with molecular mass less than 67 kDa, and with pre-beta1-HDL separated by nondenaturing two-dimensional electrophoresis, whereas it did not react with apoA-I in alpha-HDL. Pre-beta1-HDL levels measured by this method declined when incubated at 37 degrees C for 2 h, whereas this decrease was not observed in the presence of 2 mM lecithin:cholesterol acyltransferase inhibitor 5,5'-dithiobis (2-nitrobenzoic acid). To clarify the clinical significance of measuring pre-beta1-HDL by this method, 47 hyperlipidemic subjects [male/female 22/25; age 55 +/- 14 years; body mass index 25 +/- 4.5 kg/m(2); total cholesterol (TC) 245 +/- 64 mg/dl; triglyceride (TG) 232 +/- 280 mg/dl; HDL cholesterol (HDL-C) 51 +/- 23 mg/dl] and 25 volunteers (male/female 15/10; age 36 +/- 9.3 years; body mass index 23 +/- 3.5 kg/m(2); TC 183 +/- 28 mg/dl; TG 80 +/- 34 mg/dl; HDL-C 62 +/- 15 mg/dl) were involved. Plasma pre-beta1-HDL levels were significantly higher in hyperlipidemic subjects than in volunteers (39.3 +/- 10.1 vs. 22.5 +/- 7.5 mg/ml, P < 0.001) whereas plasma apoA-I levels did not differ (144.2 +/- 28.4 vs. 145.3 +/- 16.3 mg/dl).These results indicate that this sandwich EIA method specifically recognizes apoA-I associated with pre-beta1-HDL.     

The use of N-[beta-(4-diazophenyl)ethyl]maleimide as a heterobifunctional agent in developing enzyme immunoassay for neurotensin

A heterobifunctional crosslinking agent N-[beta-(4-diazophenyl)ethyl]maleimide (DPEM) was newly synthesized and characterized to possess the maleimide group with a stability greater than that previously reported for N-(4-diazophenyl)maleimide. Using the peptide hormone neurotensin (NT) as a model hapten, DPEM was used in the conjugation reaction with bovine serum albumin (BSA) and with beta-D-galactosidase (beta-Gal) in developing an enzyme immunoassay (EIA) for NT. The NT-DPEM-BSA conjugate elicited anti-NT antibodies in rabbits and the NT-beta-Gal conjugate behaved as an enzyme marker of NT in the EIA. The EIA developed double antibody was reproducible and sensitive in detecting NT at concentrations as low as 30 fmol per tube. The specificity of anti-NT serum seems to be primarily toward the carboxy-terminal region of NT, showing cross-reactions with such NT fragments as NT2-13, NT8-13, and NT1-8 for 120, 22, and less than 0.1%, respectively. The utility of this assay was also demonstrated by measuring the NT immunoreactivity in several rat organs. DPEM could be useful for developing EIAs for other peptide hormones (even those which contain neither a free amino group nor a free carboxyl group), using the imidazole, phenolic, or indole group(s) of amino acids as a binding site for carrier proteins.     

Determination of the spatial structure of insectotoxin 15A from Buthus erpeus by (1)H-NMR spectroscopy data]

The solution structure of insectotoxin 15A (35 residues) from scorpion Buthus eupeus was determined on the basis of 386 interproton distance restraints 12 hydrogen-bonding restraints and 113 dihedral angle restraints derived from 1H NMR experiments. A group of 20 structures was calculated with the distance geometry program DIANA followed by the restrained energy minimization with the program CHARMM. The atomic RMS distribution about the mean coordinate position is 0.64 +/- 0.11 A for the backbone atoms and 1.35 +/- 0.20 A for all atoms. The structure contains an alpha-helix (residues 10-20) and a three-stranded antiparallel beta-sheet (residues 2-5, 24-28 and 29-33). A pairing of the eight cysteine residues of insectotoxin 15A was established basing on NMR data. Three disulfide bridges (residues 2-19, 16-31 and 20-33) connect the alpha-helix with the beta-sheet, and the fourth one (5-26) joins beta-strands together. The spatial fold of secondary structure elements (the alpha-helix and the beta-sheet) of the insectotoxin 15A is very similar to those of the other short and long scorpion toxins in spite of a low (about 20%) sequence homology.     

Evidence for Specific Polypeptide Chain Folding in Myelin Basic Protein from Reactions Between Fragments of the Protein and Monoclonal Antibodies

The specificities of two monoclonal IgM antibodies (18.25 and 21.14.2) evoked in mice with guinea pig myelin basic protein were examined and interpreted in terms of a specific folding of the protein's polypeptide chain. Studies with guinea pig and rabbit myelin basic protein fragments showed that a region encompassing the central Phe-Phe (87-88) sequence is obligatory, but not sufficient, for reactivity with antibody 18.25. Appreciable reactivity was observed for rabbit peptides 22-95 and 45-151, and lower, but significant, reactivity was shown by peptide 32-95. Only very weak reactivity was seen with peptide 44-95. No reactivity was observed with peptide 1-95 after its lysine residues were acetylated, acetamidinated, or guanidinated. These results have been interpreted in terms of a polypeptide chain folding that creates an epitope within sequence Val-Val-His-Phe-Phe-Lys-Asn-Ile-Val (84-92). The specific conformation of this epitope, which includes probably the Lys-89 and possibly the Asn-90 and Val-92 side chains, could be formed by the association of sequence 84-92 with either sequence Ile-Leu-Asp-Ser-Ile-Gly-Arg-Phe-Phe (37-45) or with sequence Val-Leu-Ser-Arg-Phe (108-112) to form beta-sheet structures essentially identical with those that appear to be present in the intact BP [Martenson R.E.J. Neurochem. 46, 1612-1622 (1986)]. The second monoclonal antibody, no. 21.14.2, reacts only with guinea pig myelin basic protein and fragments containing the species-restricted sequence Arg-Ala-Asp-Tyr-Lys-Ser-Lys (129-135).(ABSTRACT TRUNCATED AT 250 WORDS)     

Use of a phosphotyrosine-antibody pair as a general detection method in homogeneous time-resolved fluorescence: application to human immunodeficiency viral protease

A homogeneous time-resolved fluorescence (HTRF) assay has been developed for human immunodeficiency viral (HIV) protease. The assay utilizes a peptide substrate, differentially labeled on either side of the scissile bond, to bring two detection components, streptavidin-cross-linked XL665 (SA/XL665) and a europium cryptate (Eu(K))-labeled antiphosphotyrosine antibody, into proximity allowing fluorescence resonance energy transfer (FRET) to occur. Cleavage of the doubly labeled substrate by HIV protease precludes complex formation, thereby decreasing FRET, and allowing enzyme activity to be measured. Potential substrates were evaluated by HTRF with the best results being obtained using (LCB)K4AVSQNbeta-NapPIVpYA(NH2) and Eu(K)-pY20 where the peptide titrated with an EC50 of 7.7 +/- 0.3 nM under optimized detection conditions. Using these HTRF detection conditions, HIV protease cleaved the substrate in 50 mM NaOAc, 150 mM KF, 0.05% Tween 20, pH 5.5, with apparent first-order kinetics with a Km of 37.8 +/- 8.7 microM and a kcat of 0.95 +/- 0.07 s-1. Examination of the first-order rate constant versus enzyme concentration suggested a Kd of 9.4 +/- 2.7 nM for the HIV protease monomer-dimer equilibrium. The HTRF assay was also utilized to measure the inhibition of the enzyme by two known inhibitors.