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This paper reviews calibration-, uncertainty-, and recovery-related documents from 10 consensus-based organizations. The main points from each treatise are summarized. Also included is a critique of the various approaches, as well as recommendations for a statistically sound protocol that is more compatible with chromatographic data.

US Environmental Protection Agency (EPA) Method 331 determines perchlorate in drinking water using non-suppressed ion chromatography with tandem mass spectrometry. This study reports the results of calibration and recovery studies in reagent water, as well as of a recovery study in simulated drinking water (i.e., total dissolved solids are 500 mg/mL each of chloride, sulfate, and bicarbonate). The perchlorate concentrations in the study ranged from 0.05 to 64 ng/mL. At 95% confidence, the Hubaux-Vos detection limit (H-V DL) was 0.04 ng/mL for the calibration study and the simulated-drinking-water recovery study, and 0.03 ng/mL for the reagent-water recovery study. The lowest concentration minimum reporting level was 0.03 ng/mL for reagent water and 0.0 7 ng/mL for simulated drinking water, again at 95% confidence.

Improved ion-chromatographic approaches for measuring trace chloride in nitric acid are presented. Two columns, the IonPac Cryptand A1 and a higher-capacity Cryptand prototype, were tested and compared. Also, the use of a Continuously Regenerated Anion Trap Column (CR-ATC) was evaluated for its ability to purify electrolytically generated eluent. Nitric acid (70%) was used as the test matrix and chloride was used as the test analyte; prior to injection, the nitric acid was diluted to 0.7% for the A1 column and to 2.8% for the prototype column. Chloride could be quantified in only 20 min on either column; detection limits computed for 70% HNO3 (at 95% confidence, alpha = beta = 2.5%) were 1.8 and 1.5 ppm for the A1 and prototype columns, respectively. Results also showed that the CR-ATC was necessary for obtaining acceptable acid blanks.

Quantitative analysis of the brightener component bis (sodium-sulfopropyl) disulfide (SPS) in acidic copper plating baths poses a real challenge due to the complex chemical matrix containing large amounts of Cu(II) ion and sulfuric acid together with other organic additives and additive decomposition products. We developed a new ion-pair chromatography method to analyze micro-molar amounts of SPS directly in plating bath samples without the need for sample pre-treatment. Addition of tetra-N-methylammonium cation as ion-pairing agent to a methanol-sulfuric acid-water eluent increases the retention time of the anionic SPS2- on a C18 column sufficiently to separate this compound from Cu(II) ion and additive by-products.

To meet the needs of the laboratory-water market, a modified electro-deionization (EDI) module has been developed to produce Type 2 purified water. An EDI module consists of desalting and concentrating fluidic compartments that are both filled with anion and cation ion-exchange resins; an anode and a cathode electrode are at opposite ends. In the design in this research, the anode electrode is segmented into three parts and individual dc amperages are applied to each segment; the cathode electrode is a single common electrode. Critical to the performance and longevity of this type of EDI module are: (1) the optimization of the applied dc amperages and (2) the ionic mass balance (i.e., the concentrations of specific and total ions of the RO feedwater to the module compared to the concentrations in the water exiting the module via the desalting and concentrating compartments). To determine a suitable current for each electrode pair, a full-factorial experimental design was developed and employed. For the application of this combination of amperages, the critical parameter of specific-ion mass balance was determined using ion-chromatographic measurements.

A 54-year-old male was observed driving erratically. The subject displayed both horizontal and vertical nystagmus and poor balance on field sobriety tests (FST's). Further observations included slow movements, breathing, and speech, extremely poor coordination, lack of convergence, and pupils with slow reaction to light. The subject related he was seeing roadway lines doubled and felt a hallucinogenic effect. Breath alcohol results were negative. Blood tests found zolpidem and fluoxetine in addition to hydrocodone at therapeutic levels. This case is presented as an example of hallucinogenic effects from the combination of zolpidem and serotonin reuptake inhibitors.

The crystal structure of the mitochondrial NAD-malic enzyme from Ascaris suum, in a quaternary complex with NADH, tartronate, and magnesium has been determined to 2.0-A resolution. The structure closely resembles the previously determined structure of the same enzyme in binary complex with NAD. However, a significant difference is observed within the coenzyme-binding pocket of the active site with the nicotinamide ring of NADH molecule rotating by 198 degrees over the C-1-N-1 bond into the active site without causing significant movement of the other catalytic residues. The implications of this conformational change in the nicotinamide ring to the catalytic mechanism are discussed. The structure also reveals a binding pocket for the divalent metal ion in the active site and a binding site for tartronate located in a highly positively charged environment within the subunit interface that is distinct from the active site. The tartronate binding site, presumably an allosteric site for the activator fumarate, shows striking similarities and differences with the activator site of the human NAD-malic enzyme that has been reported recently. Thus, the structure provides additional insights into the catalytic as well as the allosteric mechanisms of the enzyme.

The variable-capacity Dionex Cryptand A1 column was used for the determination of low-ppm levels of chloride and sulfate in etchants comprised of acetic acid, nitric acid and phosphoric acid. All possible ratios of the three acids could be analyzed for chloride and sulfate, if the samples were first diluted 1:100. However, a suitable eluent program was found to be needed for each mixture. A proprietary formulation was chosen to undergo this suitability determination. The resulting gradient was 10 mM KOH with a step to 30 mM NaOH at 15 min, flow-rate=0.5 ml/min; column temperature=29 degrees C; sample loop=7.5 microl. Under these conditions, a low-ppm calibration study (using the proprietary mix as the matrix) was performed and the associated prediction intervals were determined. At 50 ppm (in the original etchant), the +/- prediction interval was +/- 7 ppm for chloride and +/- 20 ppm for sulfate, both at the 95% confidence level. This step gradient was found to be a good starting place for separating the five components in all other ratios of these three acids.

The structure of the Ascaris suum mitochondrial NAD-malic enzyme in binary complex with NAD has been solved to a resolution of 2.3 A by X-ray crystallography. The structure resembles that of the human mitochondrial enzyme determined in complex with NAD [Xu, Y., Bhargava, G., Wu, H., Loeber, G., and Tong, L. (1999) Structure 7, 877-889]. The enzyme is a tetramer comprised of subunits possessing four domains organized in an "open" structure typical of the NAD-bound form. The subunit organization, as in the human enzyme, is a dimer of dimers. The Ascaris enzyme contains 30 additional residues at its amino terminus relative to the human enzyme. These residues significantly increase the interactions that promote tetramer formation and give rise to different subunit-subunit interactions. Unlike the mammalian enzyme, the Ascaris malic enzyme is not regulated by ATP, and no ATP binding site is observed in this structure. Although the active sites of the two enzymes are similar, residues interacting with NAD differ between the two. The structure is discussed in terms of the mechanism and particularly with respect to previously obtained kinetic and site-directed mutagenesis experiments.

Semiconductor-grade hydrogen peroxide (30%) is analyzed for anion contamination to be certain the levels of each analyte do not exceed 30 ppb (w/w). This paper presents a reproducible, platinum-decomposition approach that uses ion chromatography to quantify the various analytes (fluoride, chloride, sulfate, bromide, nitrate, and phosphate). Important to the success of the method are: (1) use of disposable HDPE bottles for the digestion, (2) immersion of the bottles in a water bath, (3) careful re-mixing of the digesting peroxide after two (of six total) hours, and (4) careful clean-up and sample-handling procedures (to avoid contamination). Except for fluoride and nitrate, all analytes exhibited recoveries from 89.6 to 98.3%, with +/- prediction intervals (at the 95% confidence level) from 1.5 to 3.0 ppb. Fluoride's recovery was low (74.9%), but reproducible (+/- prediction interval at 95% confidence=2.0 ppb). Nitrate reeovery was 99.1%, but noisy (+/- prediction interval at 95% confidence=8.7 ppb); this imprecision was thought to be due to contamination from atmospheric nitrite. A Dionex DX 500 microbore ion chromatograph with AS15 column and 1-ml sample loop were used for all determinations; detection was by conductivity. Statistical analyses were performed using JMP software.

Two hydroxide-selective microbore analytical columns (the Dionex AS11 and AS15) were tested and compared for the quantitation of anionic species in 30% hydrogen peroxide. The ions of interest were fluoride, acetate, formate, chloride, bromide, nitrate, sulfate, and phosphate. Statistically sound calibration and spiking studies were carried out, investigating the range of a blank to 60 ppb. Prior to injection onto the separators, peroxides were loaded without pretreatment onto a concentrator column, which was then washed with deionized water to remove the matrix. Although retention times gradually decreased during the spiking studies, reliable quantitation was still achievable on both columns at the target concentration of 30 ppb. However, various resolution problems meant that the AS11 should not be recommended for this application.

Semiconductor etchants are concentrated-acid mixtures that are prepared under tight specifications. Assay procedures are needed to ensure that the proportion of each component is within a small percentage (usually 10 relative percent or less) of the target concentration. One such etchant class contains chromium trioxide, usually in combination with HF and HNO3, While several ion-chromatographic columns can be used to analyze most mineral acids, chromium(VI) presents a problem. This latter species is highly retained by many separators and may also degrade the resins. This paper compares two columns that showed potential for success with these assays: the AS 11 and the AS16 separators (both from Dionex). These columns permit elution of chromium(VI) as chromate in 25 min or less. A representative mixture of HF, HNO3 and CrO3 was used in the research. Simultaneous calibration studies were conducted and the data sets analyzed statistically. Also investigated was the effect on the columns of repeated exposure to chromate.

In the semiconductor industry, there is interest in determining borate at sub-ppb levels in ultrapure water, since borate is an early breakthrough ion from ion-exchange resin beds. Although dissolved silica is the most common species currently used to monitor the breakdown of the deionization systems, it is thought that borate probably breaks through earlier than silicate. To be of use as an early-warning indicator, borate must be determined at ppt levels. This paper discusses benchtop results with several new column products designed to deliver low-ppt detection limits for boron as borate. The system uses a prototype borate-specific concentrator column that is coupled to an ion-exclusion separator and suppressed-conductivity detection. The acidic eluent, containing mannitol, quantitatively elutes the borate from the concentrator. The analytical separation is performed using a specially designed ion-exclusion column. Data presented are from two multilevel calibration studies. Included is a discussion of detection-limit calculations and recommended formats for reporting results.

Calibration studies involve the preparation and analysis of replicates for multiple concentrations of standards. Curves that are fitted through the data are evaluated for their adequacy of fit. A helpful test is a lack-of-fit procedure, which is performed easily by most statistical software. When coupled with Radj2, the procedure differentiates between data that are not linear and those that are simply noisy. The test requires data from exact replicates of the various standard levels involved. However, in ppt-level ion chromatography, the above condition may be impossible to meet. With the common anions (e.g., chloride, nitrite), the working standards must be prepared by mass and all liquids must be poured; transfer pipets contaminate at these concentrations. Since it is virtually impossible to pour out the desired mass exactly, final concentrations will vary slightly. Consequently, a different approach is needed for lack-of-fit testing. This paper discusses reasonable alternatives and applies them to actual data.

The structure of the G protein Gialpha1 complexed with the nonhydrolyzable GTP analog guanosine-5'-(betagamma-imino)triphosphate (GppNHp) has been determined at a resolution of 1.5 A. In the active site of Gialpha1. GppNHp, a water molecule is hydrogen bonded to the side chain of Glu43 and to an oxygen atom of the gamma-phosphate group. The side chain of the essential catalytic residue Gln204 assumes a conformation which is distinctly different from that observed in complexes with either guanosine 5'-O-3-thiotriphosphate or the transition state analog GDP.AlF4-. Hydrogen bonding and steric interactions position Gln204 such that it interacts with a presumptive nucleophilic water molecule, but cannot interact with the pentacoordinate transition state. Gln204 must be released from this auto-inhibited state to participate in catalysis. RGS proteins may accelerate the rate of GTP hydrolysis by G protein alpha subunits, in part, by inserting an amino acid side chain into the site occupied by Gln204, thereby destabilizing the auto-inhibited state of Galpha.

The effect of Mg2+ binding on the conformation of the inactive GDP-bound complex of the heterotrimeric G protein alpha subunit Gi alpha 1 has been investigated by X-ray crystallography. Crystal structures of the Gi alpha 1.GDP complex were determined after titration with 5, 10, 100, and 200 mM Mg2+. Comparison of these structures with that of the Mg2+-free complex revealed Mg2+ bound at the same site as observed in the structure of the active, Gi alpha 1. GTP gamma S.Mg2+-bound complex of Gi alpha 1, with a similar coordination scheme except for the substitution of a water molecule for an oxygen ligand of the gamma-phosphate of Gi alpha 1.GTP gamma S. Mg2+. In contrast to the GDP.Mg2+ complex of Gt alpha and of other G proteins, switch I residues of Gi alpha 1 participate in Mg2+ binding and undergo conformational changes as a consequence of Mg2+ binding. Partial order is induced in switch II, which is disordered in the Mg2+-free complex, but no order is observed in the switch III region. This contrasts with the GDP.Mg2+ complex of Gt alpha in which both switch II and III switch are ordered. Mg2+ binding also induces binding of an SO42- molecule to the active site in a manner which may mimic a Gi alpha 1.GDP.PO42-.Mg2+ product complex. Implications of these findings are discussed.

1,3,5-Triazabicyclo[3.1.0]hexane-2,4-diones proved to be potent antineoplastic and cytotoxic agents in murine and human cancer cells. In L1210 lymphoid leukemia cells DNA synthesis was significantly suppressed over 60 min by the agents from 25 to 100 microM. DNA synthesis was blocked at multiple sites including DNA polymerase alpha, ribonucleoside reductase, dihydrofolate reductase, PRPP-amido transferase, and nucleoside kinases which would be additive overall in suppressing DNA synthesis. The DNA molecule itself did not appear to be at target of the agents since no alkylation of nucleotide bases, intercalation between base-pairs or cross-linking of strands occurred after 24 h incubation at 100 microM. Nevertheless, L1210 DNA fragmentation did occur after 24 h incubation at 100 microM which is usually associated with tumor cell apoptosis.

The Gly42 --> Val mutant of Gialpha1 was characterized structurally and biochemically to elucidate two important features of Gialpha1-catalyzed GTP hydrolysis. The crystal structure of the GTPgammaS-bound G42VGialpha1 protein demonstrates that the steric bulk of Val42 pushes the Gln204 residue into a catalytically incompetent conformation, providing a rationale for the diminished GTPase activity of this mutant. The same phenomenon may also account for the diminished GTPase activity of the homologous transforming Gly42 --> Val mutation in p21(ras). Similarly, the steric bulk of the unique Ser42 residue in Gzalpha may account for the comparatively slower rate of GTP hydrolysis by this Galpha subunit. The G42VGialpha1 subunit was also characterized structurally in its GDP.Pi- and GDP-bound states, providing a unique opportunity to view three "snapshots" of GTP hydrolysis. Hydrolysis of GTP to a transient GDP.Pi-bound intermediate is associated with substantial conformational changes in the switch II segment of the protein. Eventual release of Pi results in further removal of switch I from the active site and a highly mobile switch II segment. Despite their disparate biochemical properties, the structural similarity of G42VGialpha1 to the G203AGialpha1 mutant in the GDP.Pi-bound form suggests that both mutations stabilize a conformation of the GDP. Pi-bound protein that occurs only transiently in the wild-type protein. The structures of the GDP-bound forms of the wild-type and mutant proteins are similar.

Objective: We have become aware of several commercial products that, when orally ingested, will purportedly not only eliminate "toxins" from a person's system, but will also correct any urinary imbalances caused by excessive water consumption., Method: Unblinded study of one volunteer subject, tested weekly x 4 for 24-hour urine elimination of test drug under conditions of control, control plus 1200 mL water, Quick Flush', and Eliminator., Results: Each of the treatment protocols studied caused reductions of drug or metabolite concentrations as measured by gas chromatography-mass spectrometry in urine specimens collected up to 24 hours after ingestion of amphetamine, 9-carboxy-11-nor-delta-9-THC, benzoylecgonine, or codeine, yet the radioimmunoassay screening results demonstrated very little effect. Water alone was approximately as effective as the two commercial products in reducing the metabolite level. None of the treatment protocols employed in this study altered urinary pH, specific gravity, or creatinine concentration outside the normally accepted physiological range., Conclusions: Attempts to conceal drug abuse by water dilution are most likely to play a substantial role when concentrations are at or near the detection threshold for a particular assay such as the terminal stages of drug eliminations.

The crystallographic structure of the G protein heterotrimer Gi alpha 1(GDP)beta 1 gamma 2 (at 2.3 A) reveals two nonoverlapping regions of contact between alpha and beta, an extended interface between beta and nearly all of gamma, and limited interaction of alpha with gamma. The major alpha/beta interface covers switch II of alpha, and GTP-induced rearrangement of switch II causes subunit dissociation during signaling. Alterations in GDP binding in the heterotrimer (compared with alpha-GDP) explain stabilization of the inactive conformation of alpha by beta gamma. Repeated WD motifs in beta form a circularized sevenfold beta propeller. The conserved cores of these motifs are a scaffold for display of their more variable linkers on the exterior face of each propeller blade.

Crystallographic analysis of 2.2 angstrom resolution shows that guanosine triphosphate (GTP) hydrolysis triggers conformational changes in the heterotrimeric G-protein alpha subunit, Gi alpha 1. The switch II and switch III segments become disordered, and linker II connecting the Ras and alpha helical domains moves, thus altering the structures of potential effector and beta gamma binding regions. Contacts between the alpha-helical and Ras domains are weakened, possibly facilitating the release of guanosine diphosphate (GDP). The amino and carboxyl termini, which contain receptor and beta gamma binding determinants, are disordered in the complex with GTP, but are organized into a compact microdomain on GDP hydrolysis. The amino terminus also forms extensive quaternary contacts with neighboring alpha subunits in the lattice, suggesting that multimers of alpha subunits or heterotrimers may play a role in signal transduction.

Mechanisms of guanosine triphosphate (GTP) hydrolysis by members of the G protein alpha subunit-p21ras superfamily of guanosine triphosphatases have been studied extensively but have not been well understood. High-resolution x-ray structures of the GTP gamma S and GDP.AlF4- complexes formed by the G protein Gi alpha 1 demonstrate specific roles in transition-state stabilization for two highly conserved residues. Glutamine204 (Gln61 in p21ras) stabilizes and orients the hydrolytic water in the trigonal-bipyramidal transition state. Arginine 178 stabilizes the negative charge at the equatorial oxygen atoms of the pentacoordinate phosphate intermediate. Conserved only in the G alpha family, this residue may account for the higher hydrolytic rate of G alpha proteins relative to those of the p21ras family members. The fold of Gi alpha 1 differs from that of the homologous Gt alpha subunit in the conformation of a helix-loop sequence located in the alpha-helical domain that is characteristic of these proteins; this site may participate in effector binding. The amino-terminal 33 residues are disordered in GTP gamma S-Gi alpha 1, suggesting a mechanism that may promote release of the beta gamma subunit complex when the alpha subunit is activated by GTP.

Several different crystal forms of Gi alpha 1 have been grown and analyzed. Crystals of native protein containing bound GTP gamma S belong to space group P3(1)2(1) or P3(2)2(1) with cell dimensions a,b = 80.6 A and c = 106.3 A and diffract to a resolution of 1.9 A using synchrotron radiation. Crystals of native protein containing bound GDP belong to space group I4 with cell dimensions a,b = 121.3 A, and c = 67.7 A and diffract to 3.0 A. Data sets from crystals grown using mutant proteins have also been obtained and characterized.

We show that quantitative analysis of replicated, full-factorial crystal growth experiments and, by implication, similar studies of a wide variety of other phenomena, can be a powerful tool for analyzing macromolecular systems with complex, interacting dependencies on functionally significant factors. Bacillus stearothermophilus tryptophanyl-tRNA synthetase crystallizes in three different crystal forms depending on the ligands present under otherwise identical conditions. Comparison of crystallographic space groups for complexes with different ligands reveals that the three forms entail at least two very different families of packing arrangements that are correlated with specific changes in the enzyme ligation state. One is associated with the ligand-free enzyme, substrate ligands, and the binding of the activated amino acid; the other results from the presence of high ATP concentrations and/or the synthesis of the unusual acyl-transfer product, tryptophanyl-2'(3') ATP. Together with previous physico-chemical studies of aminoacyl-tRNA synthetases, these observations suggest that the two families are related, respectively, to the biochemical processes of amino acid activation and acyl transfer. Further evidence that the crystal polymorphism results from an underlying protein conformational polymorphism has now been obtained by quantitative analysis of how crystal growth depends on pH and the substrates tryptophan and ATP. The analysis consists first in showing that crystallization conditions for the unliganded protein are very favorable, suggesting that variation in crystal growth induced by pH and substrates under otherwise identical conditions is due to their effects on the protein conformation and not on incidental perturbations of crystal growth, per se. Next, crystal growth experiments are shown to be reproducible enough to support statistical analysis of quantitative scores assigned to the results. Finally, the observed variation in scores can be attributed at high confidence levels chiefly to three effects: that of pH alone, the synergistic effects of pH plus tryptophan, and of tryptophan plus ATP. These statistical inferences are consistent with other biochemical data, and support the conclusions based on crystal packing that representative stages of the enzyme mechanism have been trapped in the different crystal forms. The pH-tryptophan interaction implies that there is a pH-dependent conformational change favoring high affinity substrate binding at high pH. The pH-ATP interaction implies that a subsequent conformational change, not previously considered, occurs between tryptophan activation and acyl transfer.

Monoclinic crystals of Bacillus stearothermophilus tryptophanyl-tRNA synthetase grown in the presence of substrate tryptophan (space group P2(1)) display evidence of a low-resolution trigonal space group (P321). The origin and averaging transformations for the local 32 point group of this unusually clear sixfold non-crystallographic symmetry may be inferred without prior estimation of the electron density. This local symmetry was exploited in conjunction with solvent density contrast variation to determine the shape of the molecular envelope. X-ray intensities measured from crystals equilibrated in mother liquors of three different electron densities were used to estimate three parameters for each reflection: the modulus of the envelope transform, [Gh]; and components, Xh and Yh, relative to Gh, of the structure-factor vector for the transform of intramolecular density fluctuations. The moduli ([Gh]) behave somewhat like structure-factor amplitudes from small-molecule crystals, and estimation of their unknown phases was successfully carried out by statistical direct methods. Reflections to 18 A resolution, which obey rather well the symmetry of space group P321, were merged to produce an asymmetric unit in that space group. [Gh] values for the 34 strongest of these were phased using the small-molecule direct-methods package MITHRIL [Gilmore (1984). J. Appl Cryst. 17, 42-46]. The best phase set was expanded back to the P2(1) lattice and negative density was truncated to generate initial phases for all reflections to 18 A resolution. Phase refinement by iterative imposition of the local 32 symmetry produced an envelope with convincing features consistent with known properties of the enzyme. The envelope implies that the tryptophanyl-tRNA synthetase dimer is an elongated structure with an axial ratio of about 4:1, in which the monomers have two distinct domains of unequal size. The smaller of these occurs at the dimer interface, and resembles the nucleotide binding portion of the tyrosyl-tRNA synthetase. It may therefore contain the amino-terminal one hundred or so residues, including all three cysteines, previously suggested to comprise a nucleotide-binding domain in the tryptophanyl enzyme. A purely crystallographic test of the overall features of this envelope was carried out by transporting it to a tetragonal crystal form of the same protein in which the asymmetric unit is a monomer. The small domain fits snugly inside three mercury and one gold heavy-atom binding sites for this crystal form; and symmetry-related molecules provide excellent, but very different, lattice contacts in nearly all directions.

Pulmonary function tests were done in seven CAPD patients with acute peritonitis on the day of admission to the hospital and after recovery. Subsequently, the effect of dialysate infusion alone on lung function was studied in 19 patients initiated on CAPD and nine of these patients were restudied 7.6 +/- 4.1 months later. Peritonitis was associated with a 30% reduction in vital capacity and a significant fall in arterial oxygen tension (7 to 11 mmHg) (p less than 0.01). Dialysate infusion alone decreased functional residual capacity (FRC) and produced small changes in PaO2 which were more pronounced in the supine position. The decrease in PaO2 observed in changing from sitting prior to dialysate infusion, to supine after dialysate infusion was due to the development of airways closure at resting lung volumes. Follow-up studies in nine patients demonstrated a continued and significant fall in FRC with dialysate infusion, without however, any changes in PaO2. We conclude that peritonitis in CAPD patients is accompanied by significant changes in lung function which are probably due to a decrease in diaphragmatic mobility resulting in atelectasis and underventilation of dependent lung zones. In CAPD patients without peritonitis, dialysate infusion in the supine position produces significant changes in PaO2, but with time, compensatory mechanisms develop to abolish the changes in PaO2.

It has previously been shown that tryptophanyl-tRNA synthetase from Bacillus stearothermophilus crystallizes in different forms, depending on the substrates present during crystallization [Carter, C. W., Jr., & Carter, C. W. (1979) J. Biol. Chem. 254, 12219-12223]. Radiolabeling experiments show that the tetragonal crystals (type IV), grown in the presence of tryptophan and ATP, contain enzymatically formed 3'(2')-tryptophanyladenosine 5'-triphosphate (Trp-ATP). Trp-ATP is formed by acyl transfer of the tryptophanyl moiety of an acyladenylate intermediate, Trp-5'-AMP, to a second molecule of ATP bound in the site normally occupied by the 3' CCA terminus of tRNATrp. This compound is therefore a chemical marker in type IV crystals for that part of the tRNA binding site on the synthetase. Solution of this crystal structure, now in progress, may therefore provide useful information concerning the mechanism of aminoacylation of tRNATrp by this enzyme and may help locate its tRNA binding site.

We have prepared crystals of tryptophanyl-tRNA synthetase from Bacillus stearothermophilus complexed to tryptophan (type II*), and to tryptophanyl-3'(2')-ATP (type IV). The latter compound is a product analog, enzymatically synthesized by acyl transfer of tryptophan from the tryptophanyl-5'-AMP intermediate to a second molecule of ATP. It resembles the 3'-terminal fragment, tryptophanyl-3'(2')-adenosine, of Trp-tRNATrp. Both crystal forms diffract to high resolution. Although both forms are grown from 2 M K2HPO4, they are dramatically different in the shape of the unit cell and in space group symmetry. Type II* crystals are monoclinic (space group P21). However, low-resolution reflections obey the symmetry of space group P321, which indicates both the existence and the location of noncrystallographic symmetry in the monoclinic unit cell. Type IV crystals belong to space group P41212 (or its enantiomorph) and the unit cell is elongated along the fourfold screw axis. Analysis of molecular packing suggests that intermolecular contacts in the two crystal types are very different. Thus, the two structures may exhibit conformational differences related to catalysis by this enzyme. Solution of type II* and type IV crystal structures may provide representations resembling a Michaelis complex and an acyl transfer product complex.