Free Amino Terminus Research Articles

A rapid method for determination of endoproteinase substrate specificity: specificity of the 3C proteinase from hepatitis A virus.

The preferred amino acid residues at the P'1 and P'2 positions of peptide substrates of the 3C proteinase from hepatitis A virus (HAV-3C) have been determined by a rapid screening method. The enzyme was presented with two separate mixtures of N-terminal acetylated peptides, which were identical in sequence except for the amino acids at the P'1 or P'2 positions, where a set of 15 or 16 amino acids was introduced. Enzyme-catalyzed hydrolysis of the peptide mixtures generated free amino termini, which allowed direct sequence analysis by Edman degradation. The relative yield of each amino acid product in the appropriate sequencing cycle gave the amount of each substrate mixture component hydrolyzed. This allowed the simultaneous evaluation of the relative kcat/Km values for each component in the mixture. The peptide substrates preferred by the HAV-3C proteinase in the P'1 mixture were glycine, alanine, and serine. The enzyme has little specificity at P'2; only arginine and proline peptides were excluded as substrates. This method provides a rapid determination of the preferred residues for a peptide substrate and should be applicable to other endoproteinases.

A POTENT INHIBITOR OF OSTEOCLASTIC BONE RESORPTION WITHIN A HIGHLY CONSERVED PENTAPEPTIDE REGION OF PARATHYROID HORMONE-RELATED PROTEIN; PTHrP[107–lll

We have recently shown that a carboxyl-terminal sequence of parathyroid hormone-related protein, PTHrP[107-139], is a potent direct inhibitor of osteoclastic bone resorption. We now report that this bone resorption inhibitory activity, which we have called osteostatin, is entirely contained within the highly conserved pentapeptide PTHrP[107-111]. Our results indicate that processing at residue 106 and a free amino terminus is required for full activity of the peptide. The retroinverted peptide is considerably less potent than the parent peptide. The retention of full biological activity in such a short fragment was unexpected. This data provides the basis for the development of further analogs with potential therapeutic use in disorders associated with increased osteoclastic bone resorption.

Reduced peptide bond pseaadopeptide analogues of neurotensins binding and biological activities, and in vitro metabolic stability

A series of pseudopeptide analogues of neurotensin was produced by systematically replacing the five peptide bonds in neurotensin-(8–13) with CH 2NH (Ψ, reduced) bonds. All these analogues were synthesized with a free amino terminus (H derivatives) and with a N-terminai tert-butyloxycarbonyl group (Boc derivatives). The compounds were screened in vitro for agonist or antagonist activity and for metabolic stability by testing (1) their ability to inhibit the binding of radiolabelled neurotensin to homogenates of newborn mouse brain; (2) their ability to contract isolated guinea-pig ileum preparations; and (3) their degradation in the presence of rat brain homogenates. All the analogues bound to the mouse brain neurotensin receptor and all exhibited agonist activity in the guinea-pig ileum assay. Only the H- and Boc-[Ψ8,9] derivatives were at least as potent as their parent compounds neurotensin-(8–13) and Boc-neurotensin-(8–13) in the binding and biological assays. All the other pseudopeptide analogues with reduced bonds at position 9–10, 10–11, 11–12 and 12–13 showed a marked reduction in potency ranging from 2 to 4 orders of magnitude. All the derivatives that were protected at their N terminus either by the presence of a Boc group or by the presence of a reduced bond at position 8–9 and 9–10 were slowly degraded by rat brain homogenates. The other derivatives were, in contrast, quite rapidly degraded. There was a good correlation between binding and biological potencies for those analogues that were resistant to degradation. Interestingly, the degradation-resistant H-[Ψ8,9] compound exhibited higher binding and biological potency then neurotensin. It is therefore expected that this analogue will produce highly potent and long-lasting neurotensin-like effects in vivo, and preliminary experiments indicate that this is indeed the case.

The complete amino acid sequence of bullfrog ( Rana catesbeiana) parvalbumin pI4.97

The primary structure of the parvalbumin (pI4.97) from bullfrog, Rana catesbeiana, skeletal muscle has been determined. It is composed of 110 amino acid residues and a free amino terminus, and has a molecular mass of 11919. The amino add sequences which are thought to be functionally important sites are also conserved in the bullfrog parvalbumin. The calculated phylogenic tree indicates that this parvalbumin belongs to the α group of parvalbumins. The mutation rate of parvalbumin was fairly rapid in frogs compared to mammals. The subdivergence of frogs is also discussed.

Electrostatics of a peptide at a membrane/water interface. The pH dependence of melittin association with lipid vesicles

The association of the peptide melittin with small unilamellar DMPC vesicles was studied as a function of pH. The results are discussed quantitatively assuming a water-membrane partition equilibrium. Electrostatic surface charging is taken into account as more and more of the strongly basic peptide accumulates at the bilayer/water interface. The data could be well described in terms of a Gouy-Chapman approach involving an effective interfacial charge well below the actual physical charge carried by the individual peptide molecules. The partition coefficient turned out to be pH invariant, so that one can exclude deprotonation reactions upon insertion of the peptide into the bilayer. The effective interfacial charge per associated melittin molecule decreased over a broad range of pH (pH 7 to pH above 10). Contributions of the free amino terminus and of the arginine residues could be determined by comparing with results obtained using modified melittin (N-terminally formylated and fully acetylated). The data suggest approximately equal fractional contributions of the amino terminus and the three lysines to the effective interfacial charge. The two arginines contribute less. Thus, they may be located farther away from the interface or be closely associated with counter-ions. The analysis is extended to the effect of different ionic strengths.

The main immunogenic region of the nicotinic acetylcholine receptor. Identification of amino acid residues interacting with different antibodies.

In myasthenia gravis a highly conserved area of the nicotinic receptor (AcChR) dominates the autoantibody response (main immunogenic region, MIR), and it is formed by residues within the sequence segment 67-76 of the AcChR alpha-subunit. We have studied the binding of eight anti-MIR mAb to synthetic peptides containing the sequence segment 67-76 of the human alpha-subunit, and peptide analogues containing single residue substitutions of this sequence. We used also a peptide where both Asp70 and Asp71 were substituted by glycine residues. The binding of six anti-MIR mAb was strongly influenced by several substitutions. All these mAb required residues Asn68, and Pro69 for binding. Five of them required also Asp71 and Tyr72. Substitution of Asp70, which is an Ala residue in Torpedo AcChR, was irrelevant for the binding of an anti-Torpedo and an anti-Electrophorus mAb, and moderately reduced the binding of an anti-human mAb (no. 203). Substitution of Trp67 moderately reduced the binding of some of these mAbs. A mAb of this group (the antihuman mAb no. 198) bound in a manner only slightly influenced by ionic strength, whereas the binding of the other five mAb of this group was very sensitive to the ionic strength. Two anti-Electrophorus MIR mAb bound similarly to all peptide analogues in low ionic strength. At high ionic strength only the peptide analogue where Asp 70 was changed to a Gly residue bound significantly. This may indicate that the Electrophorus MIR has an uncharged residue at this position, as does Torpedo AcChR. Residues at position 73, 74, 75, and 76 were of little or no importance for the binding of all anti-MIR mAb. A free amino terminus was essential for the binding of most mAb. The results of competition experiments between different peptides and native AcChR for mAb binding were consistent with those obtained in direct binding experiments.

Protein Structural Requirements and Properties of Membrane Binding by γ-Carboxyglutamic Acid-containing Plasma Proteins and Peptides

Abstract The membrane-binding characteristics of a number of modified vitamin K-dependent proteins and peptides showed a general pattern of structural requirements. The amino-terminal peptides from human prothrombin (residues 1-41 and 1-44, 60:40) bovine factor X (residues 1-44), and bovine factor IX (residues 1-42), showed a general requirement for a free amino-terminal group, an intact disulfide, and the tyrosine homologous to Tyr44 of factor X for membrane binding. Consequently, the peptide from factor IX did not bind to membranes. Any of several modifications of the amino terminus, except reaction with trinitrobenzenesulfonic acid, abolished membrane binding by the factor X and prothrombin peptides. Calcium, but not magnesium, protected the amino terminus from chemical modification. The requirement for a free amino terminus was also shown to be true for intact prothrombin fragment 1, factor X, and factor IX. Although aggregation of the peptide-vesicle complexes greatly complicated accurate estimation of equilibrium binding constants, results with the factor X peptide indicated an affinity that was not greatly different from that of the parent protein. The most striking difference shown by the peptides was a requirement for about 10 times as much calcium as the parent proteins. In a manner similar to the parent proteins, the prothrombin and factor X peptides showed a large calcium-dependent quenching of tryptophan fluorescence. This fluorescence quenching in the peptides also required about 10 times the calcium needed by the parent proteins. Thus, the 1-45 region of the vitamin K-dependent proteins contained most of the membrane-binding structure but lacked component(s) needed for high affinity calcium binding. Protein S that was modified by thrombin cleavage at Arg52 and Arg70 showed approximately the same behavior as the amino-terminal 45-residue peptides. That is, it bound to membranes with overall affinity that was similar to native protein S but required high calcium concentrations. These results suggested that the second disulfide loop of protein S (Cys47-Cys72) and prothrombin (Cys48-Cys61) were involved in high affinity calcium binding. Since factor X lacks a homologous disulfide loop, an alternative structure must serve a similar function. A striking property of protein S was dissociation from membranes by high calcium. While this property was shared by all the vitamin K-dependent proteins, protein S showed this most dramatically and supported protein-membrane binding by calcium bridging.

In-vitro biosynthesis of multiple preproglucagons results from acetylation of the primary translation products.

In-vitro translation of anglerfish islet mRNA revealed three glucagon precursors (preproglucagons): one with Mr 16,000 and two with Mr 14,000. The two Mr 14,000 precursors were well separated upon isoelectric focusing gels (pI values of 7.2 and 7.3), but had identical peptide maps. Translation of hybrid-selected Mr 14,000 preproglucagon mRNA in the presence of microsomal vesicles revealed that both precursors were processed to the same proglucagon. Northern blot analysis detected two mRNA species encoding Mr 14,000 precursor. A full-length Mr 14,000 preproglucagon cDNA was subcloned into a transcription vector, and coupled in-vitro transcription-translation was performed; surprisingly, both Mr 14,000 precursors were synthesized. To test whether acetylation of the free amino terminus generated the more acidic precursor, acetylase activity was partially inactivated with the inhibitor S-acetonyl-CoA, and acetyl-CoA was depleted by addition of oxaloacetate and citrate synthetase. Under these conditions, the level of the most basic preproglucagon was greatly enhanced, but when exogenous acetyl-CoA was added, the acidic form predominated. We conclude that acetylation generates the acidic precursor, and we discuss the implications of our findings for the biogenesis of other peptide hormones.

Escherichia coli S-adenosylmethionine decarboxylase. Subunit structure, reductive amination, and NH2-terminal sequences.

S-Adenosylmethionine decarboxylase is one of a small group of enzymes that use a pyruvoyl residue as a cofactor. Histidine decarboxylase from Lactobacillus 30a, the best studied pyruvoyl-containing enzyme, has an (alpha beta)6 subunit structure with the pyruvoyl moiety linked through an amide bond to the NH2-terminal of the larger alpha subunit (Recsei, P. A., Huynh, Q. K., and Snell, E. E. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 973-977). To examine potential structural analogies between the two enzymes, we have isolated and partially characterized S-adenosylmethionine decarboxylase. The purified enzyme comprises equimolar amounts of two subunits of Mr = 14,000 and 19,000 (by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and has a native molecular weight of 136,000 (by gel filtration). Approximately 4 mol of [methyl-3H] adenosylmethionine are incorporated per mol of enzyme (Mr = 136,000) when the enzyme is inactivated with this substrate and NaCNBH3. These data suggest an (alpha beta)4 structure with 1 pyruvoyl residue for each alpha beta pair. The two subunits have been separated by reversed-phase high performance liquid chromatography after reduction and carboxymethylation. The smaller subunit (beta) has a free amino terminus. The amino terminus of the larger subunit (alpha) appears to be blocked by a pyruvoyl group; this subunit can be sequenced only after this group is converted to an alanyl residue by reduction with sodium cyanoborohydride in the presence of ammonium acetate. This work suggests that S-adenosylmethionine decarboxylase is structurally much more similar to histidine decarboxylase than previously thought.

Group separation of peptides by ligand-exchange chromatography with a Sephadex containing N-(2-pyridylmethyl)glycine

The synthesis of 2-hydroxy-3[ N-(2-pyridylmethyl)glycine]propyl Sephadex ether—a new chelating resin—is described. This resin has been employed in the form of its Cu 2+ complex to separate peptides, as a group, from α-amino acids and NaCl. Ninety-seven ligands of different structures were separated chromatographically at room temperature. It was shown that two structural parameters of the ligands control the separation process, namely, the presence of ligand donor groups and the possibility for them to form chelate rings of suitable size. Separation of peptides from α-, γ-, δ-amino acids, N-acetyl derivatives of amino acids (except N-Ac-Trp), and NaCl is possible if the peptides fulfill the following structural requirements: the peptide molecule must have a free terminal amino group; a carbonyl group (of the peptide linkage) must be situated in the α- or β-position of the free amino group; and the peptide may not contain an imidazole residue (except Gly-Gly-His). A relationship was found between the log k′ and the corresponding p K HL H, log K CuL Cu, and log K CuBipyL CuBipy values. Interpretation of the different k′ values was possible based on the different basicities of the terminal amino groups and on the structures of the different side chains of the peptides.

Selective isolation of free or blocked amino-terminal peptides from protein digests by high performance liquid chromatography: application to the crystal protein of Bacillus thuringiensis

A method for the selective isolation of amino-terminal peptides from enzymatic digests of proteins using high performance liquid chromatography (HPLC) is described. The rationale behind this method is based on the condition that after citraconylation of the free amino groups of the protein, followed by enzymatic digestion and reaction with fluorodinitrobenzene only peptides derived from the amino terminus will not contain a dinitrophenyl (Dnp) group. After removal of citraconyl groups at acid pH, the peptide mixture was subjected to HPLC using an Altex Ultrasphere octadecylsilane column. The strong adsorption of the Dnp group retards the elution of all peptides, except the amino-terminal peptides which do not contain a Dnp group. A shallow gradient employing increasing acetonitrile to 28% over 30 min was used to elute the peptides. For all test proteins, only peptides derived from the amino terminus eluted in the shallow portion of the gradient. As the procedure involves the reversible blocking of any free amino terminus, peptides are isolated with their amino-termini free. However, proteins which are naturally blocked at their amino terminus will yield amino-terminal peptides with the blocking groups intact. The demonstrated sensitivity of the method is in the 1- to 5-nmol range of protein. Application of this procedure to the crystal protein from Bacillus thuringiensis var. kurstaki, which was solubilized at pH 10.5 in 2% β-mercaptoethanol, gave multiple amino-terminal peptides. However, prior treatment of the crystals with 8 M urea at pH 3, followed by solubilization at pH 10.5, gave only one major amino-terminal peptide. These results show that there is probably only one major protein present in the crystals and that the proteolysis results from processes occurring upon solubilization and not by the action of the proteases within the intact crystal.

Primary structure of copper-zinc superoxide dismutase from Neurospora crassa.

The complete amino acid sequence of copper-zinc superoxide dismutase from Neurospora crassa is reported. The subunit consists of 153 amino acids and has a Mr of 15,850. The primary structure was determined by automated and manual sequence analysis of peptides obtained by digestions of the carboxymethylated and aminoethylated enzyme with trypsin and thermolysin. The protein is devoid of tryptophan and methionine and displays a free amino terminus. Comparison of the amino acid sequence with those from human erythrocyte, bovine erythrocyte, horse liver, swordfish liver, and yeast copper-zinc superoxide dismutases reveals a high degree of sequence homology among the six enzymes. Most prominently, the regions containing the amino acid residues participating in the metal-binding and the half-cystine residues forming the intramolecular disulfide bridge are highly conserved. The invariant amino acids Pro 74 and Asp 76 of the four vertebrate and yeast superoxide dismutases were found to be substituted by arginine and alanine, respectively, in the Neurospora enzyme. These radical substitutions occurring in the zinc ligand region, known to form a characteristic loop structure in bovine erythrocyte copper-zinc superoxide dismutase (Tainer, J. A., Getzoff, E. D., Beem, K. M., Richardson, J. S., and Richardson, D. C. (1982) J. Mol. Biol. 160, 181-217), however, do not affect the catalytic properties of the Neurospora enzyme.

Exopeptidase-high-performance liquid chromatography peptide mapping of small peptides

We report here a rapid and quantitative peptide mapping procedure based on a combination of immobilized exopeptide digestion and high-performance liquid chromatographic (HPLC) separation of enzymatic products. Immobilized amino-peptidase M (E.C. 3.4.11.2) and carboxypeptidase Y (E.C. 3.4.12.1) were chosen because of their broad specificity. Products of enzymatic degradation of proctolin, for example, were separated on C 18 or C 1 reversed-phase HPLC columns with a trifluoroacetic acid-acetonitrile gradient elution. Peaks corresponding to peptide fragments were collected, hydrolyzed in propionic acid-hydrochloric acid, and identified by amino acid analysis. This method is limited, of course, to peptides with free carboxyl or amino termini and by the sensitivity of the amino acid analysis method employed.

A general procedure for the end labeling of proteins and positioning of amino acids in the sequence.

This paper reports a procedure for the specific radiolabeling of the amino termini of proteins. By Edman degradation, a protein is protected at all lysine amino groups while retaining a free amino terminus and such a modified protein is end-labeled by an amino group-specific reagent (radioiodinated Bolton-Hunter reagent). Partial proteolyses with a variety of specific amino acid cleaving reagents generate a series of fragments which predict the location of the specific amino acids in the primary structure. The amino acids determined so far include Arg, Asp, Cys, Glu, Met, Trp, and Asn-Gly. The procedure is demonstrated on beta-galactosidase and lambda immunity 434 repressor protein. One of the uses of the procedure, the identification and localization of point mutations within the sequence, is illustrated using lambda immunity 434 repressor protein.

Amino group environments and metal binding properties of carbon-13 reductively methylated bovine alpha-lactalbumin.

13C NMR spectroscopy has been used to study the amino group environments and metal binding properties of 13C reductively methylated bovine alpha-lactalbumin. Bovine alpha-lactalbumin is a Ca2+ metalloprotein containing 12 lysyl amino groups and a free amino terminus. All 13 amino groups can be 13C-dimethylated without altering Ca2+ binding or biological activity. pH titrations (chemical shift vs. pH) of this dimethylated protein reveal unique behavior for each of the 13 amino groups. The pKa values for the lysyl amino groups range from 9.1 to 10.8 while the pKa for the N-terminal amino group is 8.3. This relatively high pKa (by 1 pH unit) for the N-terminal supports its interaction in an ion pair as proposed by Warme et al. [Warme, P. K., Momany, F. A., Rumball, S. V., Tuttle, R. W., & Scheraga, H. A. (1974) Biochemistry 13, 768-782]. Carbon-13 NMR studies further show that the removal of Ca2+ from the high-affinity binding site results in a conformational change, with the disruption of the N-terminal ion pair interaction (pKa decreased to 7.4). The study of Zn2+ binding to Ca2+-saturated protein suggests that Zn2+ binds initially at a low-affinity Ca2+ site while maintaining the N-terminal ion pair interaction. The further addition of Zn2+ leads to the disruption of this ion pair forming a presumed apoprotein-like conformation. Finally on the basis of the specific effects of added Mn2+ on the 13C NMR spectra of the methylated protein, a low-affinity divalent metal binding site is proposed about 7.5 A from the amino terminus.

The carboxyterminus of the hemagglutinin-neuraminidase of Newcastle disease virus is exposed at the surface of the viral envelope.

The amino-terminal and the carboxy-terminal amino acids of the hemagglutinin-neuraminidase glycoprotein of the Ulster strain of Newcastle disease virus have been analyzed before and after proteolytic activation of the precursor HNo (Mr approximately 82K). The amino termini of HNo and of the large cleavage fragment HN (approximately 74K) obtained by in vivo and in vitro proteolysis could not be sequenced by Edman degradation. This indicates that in both instances the amino termini are blocked. The carboxy termini of HNo and HN are different as demonstrated by end-point digestion with carboxypeptidase A. Furthermore, a small cleavage fragment (approximately 9K) of HNo that was removed from the virion after trypsin treatment could be purified by HPLC. In contrast to HN, this fragment displays a free amino terminus susceptible to Edman degradation. These data indicate that conversion of HNo involves removal of a 9K glycopeptide from the carboxy-terminal end. Thus, it has to be concluded that, unlike most other viral glycoproteins, the hemagglutinin-neuraminidase is inserted in the envelope with its carboxy terminus exposed at the surface of the virus particle.

Dual-detector methods for selective identification of prolyl residues and amide-blocked n-terminal groups in chromatographically separated peptides

On-line methods have been developed to screen for peptides containing proline residues or for the presence of an N-terminal blocking group such as an amide. Both methods combine UV detection with post-column reactions, producing a fluorescent product. The fluorescamine post-column reaction is used to detect peptides lacking a free amino terminus. Common blocking groups that can be detected are acetyl, formyl and internally cyclized amino acids such as pyroglutamic. Such groups prevent coupling of phenyl isothiocyanate to the peptide and prevent Edman sequence analysis. The two step, hypochlorite-o-phthalaldehyde post-column reaction detects the presence of proline in the peptide. Proline cannot be readily detected in peptide hydrolyzates by some popular methods of amino acid analysis. These techniques should enhance the success of sequence and composition analysis of collected peptides.

Protease inhibitors implicate metalloendoprotease in synaptic transmission at the mammalian neuromuscular junction.

Metalloendoproteases have been implicated in the calcium-dependent exocytosis of histamine from mast cells and in the calcium-dependent fusion of myoblasts. Because metalloendoproteases have also been identified in nervous tissue, we investigated the possibility that these proteases may be involved in neurotransmitter release at mammalian synapses. End-plate potentials were recorded intracellularly from mouse diaphragm/phrenic nerve preparations in vitro. The amplitude of the endplate potentials were reduced by as much as 90% during bath application of phosphoramidon, a specific inhibitor of metalloendoproteases, and by carbobenzoxy-dipeptide-amide synthetic substrates for metalloendoproteases. Only those synthetic dipeptides in which the amino group of the peptide bond was provided by a bulky hydrophobic amino acid, such as phenylalanine or leucine, which are substrates for metalloendoproteases, reduced synaptic transmission. Synthetic substrates in which proline or glycine provided the amino group of the peptide bond, which are not metalloendoprotease substrates, had little or no effect on the amplitude of end-plate potentials. The ability of synthetic substrates to reduce synaptic transmission was also dependent on the amino acid that provided the carboxyl group of the peptide bond, with glycine being more effective than tyrosine or serine. In addition, synthetic dipeptides with free carboxyl or amino termini, which have a low affinity for metalloendoproteases, also had little effect on synaptic transmission. The inhibition of synaptic transmission by phosphoramidon and the synthetic substrates occurred within 2 to 3 min and was completely reversible. Neither phosphoramidon nor the synthetic substrates altered the dose-response characteristics of the postsynaptic membrane to bath-applied carbachol. These results suggest that synaptic transmission requires the activity of a metalloendoprotease in the presynaptic nerve terminal and that proteolysis may be an important step during neurotransmitter exocytosis.

The primary structure of myoglobin from pacific green sea turtle ( Chelonia mydas caranigra)

The amino acid sequence of the main component myoglobin from skeletal muscle of Pacific green sea turtle ( Chelonia mydas caranigra) has been determined. The globin is 153 residues in length and has a free amino-terminus. The heme-binding and internal residues are as found in mammalian myoglobins. Ten substitutions are observed between this myoglobin and that from map turtle. About 38, 52, 47 and 86 substitutions are noted in comparison with the myoglobins of other reptiles, mammals, birds and fish, respectively. The inferred pattern of structural stabilization and conservation of two loci are typical of tetrapod myoglobin.

Determination of polypeptide amino acid sequences from the carboxyl terminus using angiotensin I converting enzyme.

A method for sequence analysis of polypeptides starting at the carboxyl terminus is described that utilizes degradation of the polypeptide into dipeptides with angiotensin I converting enzyme. Dipeptides were identified by gas chromatography-mass spectroscopy. Dipeptide alignment was achieved by replicate digestion of the polypeptide after modification at the carboxyl terminus either by chemical or enzymatic removal of one residue or by addition of a single residue. The addition reaction involved coupling of L-alpha-aminobutyric acid under conditions described herein which yielded essentially complete conversions. Unlike sequence determination methods that commence from the polypeptide amino terminus, this procedure does not require that a polypeptide have a free amino terminus for successful application. A number of polypeptides with varying chain lengths (up to 49 residues), containing among them most of the common amino acids, have been successfully analyzed in amounts as low as 5 nmol.