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The application of fractionation techniques to protein purification at a process or industrial scale is reviewed. The principles of various chromatographic separations are described and the choice and selection of available matrices discussed. Gel filtration, ion-exchange hydrophobic interaction and affinity chromatographic techniques can all operate at process scale. HPLC is particularly suitable for process development and scale up without loss of efficiency. Magnetic separation, solvent partitioning and electrophoresis also offer potential for process scale operation. Recent developments also include improved matrices and the application of genetic engineering techniques to design proteins for efficient separation.

The nonantigenic interaction between a recombinant immunoglobulin G (IgG)-binding protein based on the B domain of Protein A from Staphylococcus aureus (termed SpA1) and the Fc fragment of rabbit IgG has been investigated. The contribution to binding of four putative hydrogen bond contacts between SpA1 and IgG-Fc were examined by the individual substitution of the residues in SpA1 involved in these interactions by others unable to form hydrogen bonds. It was found that the most important of the hydrogen bonds involved Tyr 18 which, when replaced by Phe, resulted in a twofold decrease in IgG-binding affinity. The residues of SpA1 proposed to make close, mainly hydrophobic, contacts with Fc were replaced by residues with potential electrostatic charge to establish the importance of the hydrophobic interaction in the complex. The IgG-binding affinities of the mutant proteins were compared to the wild-type protein by a competitive enzyme-linked immunosorbent assay. The replacement of individual hydrophobic residues by His generated a number of novel IgG-binding proteins with reduced binding affinity at pH 5.0 but which maintained strong binding affinities at pH 8.0. The elution profile of human IgG1-Fc (Fc fragment of human IgG1) from a column made from an immobilized two-domain mutant protein shows that the complex dissociates at a higher pH relative to that of the non-mutated protein thus offering favorable elution characteristics.

The gene encoding the tetrameric malate dehydrogenase (MDH) in a thermophilic Bacillus species (BI) has been cloned in an Escherichia coli plasmid. The nucleotide sequence of the gene, the first to be elucidated for a tetrameric MDH, shows the MDH subunit to contain 312 amino acids and have a molecular mass of 33648 Da, which confirms the experimentally determined value of about 35 kDa. Like the genomic DNA of BI, the MDH gene is relatively AT-rich; this contrasts with the generally GC-rich nature of the DNA of thermophilic Bacillus species. Comparison of amino acid sequences reveals that BI MDH bears greater structural similarity to lactate dehydrogenases (LDHs) than to other (dimeric) MDHs. MDHs and LDHs resemble each other in catalytic mechanism and several other respects. However, whereas MDHs in the majority of organisms are dimers, the tetrameric structure is favoured among LDHs. The stronger structural resemblance that BI MDH has to LDHs than to the dimeric MDHs provides some explanation as to why Bacillus MDH, unlike most other MDHs, is tetrameric. A 1 kb fragment containing the BI MDH gene, produced in a PCR, has been cloned into a high-expression E. coli plasmid vector. BI MDH synthesized from this clone constitutes about 47% of the total protein in cell extracts of the E. coli strain carrying the clone. MDH purified from BI and that purified from the E. coli strain carrying the MDH gene clone appear to be identical proteins by several criteria. A number of characteristics of the MDH have been elucidated, including the molecular masses of the native enzyme and the subunit, N-terminal amino acid sequence, isoelectric point, pH optimum for activity, thermostability, stability to pH, urea and guanidinium chloride and several kinetic parameters. Whereas the MDH is a stable tetramer in the pH range 5–7, it appears to be converted into a stable dimer at pH 3.5. This suggests that the dimer is a stable intermediate in the dissociation of the tetramer to monomers at low pH.

The nucleotide-binding fold of many NAD(+)-dependent dehydrogenases contains a conserved acidic amino acid residue which hydrogen-bonds with the 2′- and 3′-hydroxy groups of the adenine-ribose of the cofactor. This residue is highly conserved as aspartate in malate dehydrogenases, except in the thermophilic enzyme from Thermus aquaticus B (TaqMDH), which has glutamic acid-41 in the equivalent position. The catalytic mechanism was dissected to investigate the functional significance of this difference in TaqMDH with respect to a mutant enzyme where glutamic acid-41 was replaced by aspartic acid. The mutant enzyme was found to retain a high degree of protein structural stability to both thermal and chemical denaturation. When compared with the wild-type enzyme the mutant had a higher Km and Kd for both reduced and oxidized cofactors (NADH and NAD+) and a 2-3-fold increase in steady-state kcat in both assay directions. The rate-determining step for the reduction of oxaloacetate by wild-type TaqMDH was shown to be the rate of NAD+ release, which was about 2.5-fold higher for the mutant enzyme. This correlates well with the 1.8-fold higher steady-state kcat of the mutant enzyme and represents an improvement in the steady-state kcat of a thermophilic enzyme at moderate temperature by a conservative amino acid substitution which increases the rate of product release.

The stability of wild-type Escherichia coli malate dehydrogenase was compared with a mutant form of the enzyme with the amino acid residue at position 102 changed from arginine to glutamine. The mutation occurs on the underside of a mobile loop which closes over the active-site cleft on formation of the enzyme/cofactor/substrate ternary complex. The mutant enzyme is kinetically compromised while the wild-type enzyme is highly specific for oxaloacetate. The mutant enzyme was shown to be more resistant to irreversible thermal denaturation by thermal inactivation experiments and high-sensitivity differential scanning calorimetry than the wild-type enzyme. In contrast, resistance of both enzymes to reversible unfolding in guanidinium chloride was similar. Circular dichroic spectropolarimetry shows the secondary structures of the enzymes are similar but there is a demonstrable difference in tertiary structure. From the position of the mutation, it is conjectured that the substitution on a mobile surface loop results in partial closure of the loop and greater resistance to thermal inactivation of the mutant enzyme. However, molecular modelling combined with circular dichroic spectropolarimetry indicate that the mutation may have a more widespread effect on the structure than simply partial closure of the mobile surface loop as the environment of distant tyrosine residues is altered. Resistance of the wild-type enzyme to thermal inactivation can be increased by cofactor addition, which may have the effect of partial closure of the mobile surface loop, but has little effect on the mutant enzyme.

The stability and unfolding of an immunoglobulin (Ig) G binding protein based upon the B domain of protein A (SpAB) from Staphylococcus aureus were studied by substituting tryptophan residues at strategic locations within each of the three alpha-helical regions (alpha 1-alpha 3) of the domain. The role of the C-terminal helix, alpha 3, was investigated by generating two protein constructs, one corresponding to the complete SpAB, the other lacking a part of alpha 3; the Trp substitutions were made in both one- and two-domain versions of each of these constructs. The fluorescence properties of each of the single-tryptophan mutants were studied in the native state and as a function of guanidine-HCl-mediated unfolding, and their IgG binding activities were determined by a competitive enzyme-linked immunosorbent assay. The free energies of folding and of binding to IgG for each mutant were compared with those for the native domains. The effect of each substitution upon the overall structure and upon the IgG binding interface was modelled by molecular graphics and energy minimization. These studies indicate that (i) alpha 3 contributes to the overall stability of the domain and to the formation of the IgG binding site in alpha 1 and alpha 2, and (ii) alpha 1 unfolds first, followed by alpha 2 and alpha 3 together.

Unlike the European leech Hirudo medicinalis, the Asian jawed leech Hirudinaria manillensis is specialized for feeding on mammalian blood. In the salivary glands of both these leeches, there is a potent inhibitor of thrombin, called hirudin, which acts as an anticoagulant. We have reported previously the isolation and purification of a variant of hirudin, called bufrudin, from the head portions of Hirudinaria. In the present study, the complete amino acid sequence of bufrudin was determined by automated Edman degradation of peptide fragments generated after cleavage of protein with trypsin or thermolysin. Comparison of the primary structure of bufrudin, with hirudin HV1, show about 70% sequence identity with deletion of two amino acids, but the key amino acids at the C-terminus, involved in the inhibition of thrombin, are conserved. However, similar sequence comparison of bufrudin with hirullin P18, a hirudin variant isolated from the same leech species but from whole leech, instead of heads, reveals even less sequence identity of about 60%. From the amino acid sequence, it is suggested that the conformation of the C-terminal portion of bufrudin may be significantly different from hirullin P18, but similar to hirudin HV1, upon its interaction with thrombin. These results indicate that, as with Hirudo leech, various isoforms of hirudin also exist in Hirudinaria leech, with a significant change occurring in the structure of the molecule during the evolution of leeches.

The cell-surface proteins of the infective bacteria Streptococcus and Staphylococcus are probably involved in the process of injection. These proteins share many features including secretion signal peptides, cell-wall spanning regions, membrane anchor domains and repeated domains of various functions. These common features may have evolved by gene duplication and swapping of gene fragments.

X-ray crystallography predicts hydrogen-bonding interactions between the side chains of Thr198 and two other amino acid residues, Glu194 (adjacent to the catalytic His195) and Ser318 (on the alpha-H helix which rearranges on substrate binding). In order to investigate the contribution of this conserved amino acid residue, Thr198, two mutants of Bacillus stearothermophilus lactate dehydrogenase were created (Val198 and Ile198). The steady-state kinetic parameters for both mutant enzymes were very similar with increased substrate Km and reduced kcat when compared with the wild-type enzyme. The mutation Val198 allowed non-productive binding of pyruvate to the unprotonated form of His195. Steady-state kinetic parameters determined for the Val198 mutant enzyme in high solvent viscosity suggested both an altered rate-limiting step in catalysis and implicated Thr198 in allosteric activation by the effector fructose 1,6-bisphosphate (Fru1,6P2). A shift in the Fru1,6P2 activation constant for the Val198 mutant enzyme suggested that Thr198 stabilises the catalytically competent (Fru1,6P2-activated) form of the enzyme by 6.6 kJ/mol. However, Thr198 was not important for maintaining the thermal stability of the Fru1,6P2-activated form. Equilibrium unfolding in guanidinium chloride indicated that Thr198 contributes 17.2 kJ/mol subunits towards the tertiary structural stability. The results emphasise the importance of the side chain-hydroxyl group of Thr198 which is required for (a) productive substrate binding, (b) allosteric activation and (c) protein conformational stability. The characteristics of the B. stearothermophilus lactate dehydrogenase mutations reported here were significantly different from those of the same mutations made in the corresponding position of the analogous enzyme Thermus flavus malate dehydrogenase [Nishiyama, M., Shimada, K., Horinouchi, S.,Beppu, T. (1991) J. Biol. Chem. 266, 14294-14299].

The malate dehydrogenase from Escherichia coli has been specifically altered at a single amino acid residue by using site-directed mutagenesis. The conserved Arg residue at amino acid position 102 in the putative substrate binding site was replaced with a Gln residue. The result was the loss of the high degree of specificity for oxaloacetate. The difference in relative binding energy for oxaloacetate amounted to about 7 kcal/mol and a difference in specificity between oxaloacetate and pyruvate of 8 orders of magnitude between the wild-type and mutant enzymes. These differences may be explained by the large hydration potential of Arg and the formation of a salt bridge with a carboxylate group of oxaloacetate.

NAD(+)-dependent D-lactate dehydrogenase from Lactobacillus helveticus was purified to apparent homogeneity, and the sequence of the first 36 amino acid residues determined. Using forward and reverse oligonucleotide primers, based on the N-terminal sequence and amino acid residues 220-215 of the Lactobacillus bulgaricus enzyme [Kochhar, S., Hunziker, P. E., Leong-Morgenthaler, P.Hottinger, H. (1992) J. Biol. Chem. 267, 8499-8513], a 0.6-kbp DNA fragment was amplified from L. helveticus genomic DNA by the polymerase chain reaction. This amplified DNA fragment was used as a probe to identify two recombinant clones containing the D-lactate dehydrogenase gene. Both plasmids overexpressed D-lactate dehydrogenase (greater than 60% total soluble cell protein) and were stable in Escherichia coli, compared to plasmids carrying the L. bulgaricus and Lactobacillus plantarum genes. The entire nucleotide sequence of the L. helveticus D-lactate dehydrogenase gene was determined. The deduced amino acid sequence indicated a polypeptide consisting of 336 amino acid residues, which showed significant amino acid sequence similarity to the recently identified family of D-2-hydroxy-acid dehydrogenases [Kochhar, S., Hunziker, P. E., Leong-Morgenthaler, P.Hottinger, H. (1992) Biochem. Biophys. Res. Commun. 184, 60-66]. The physicochemical and catalytic properties of recombinant D-lactate dehydrogenase were identical to those of the wild-type enzyme, e.g. alpha 2 dimeric subunit structure, isoelectric pH, Km and Kcat for pyruvate and other 2-oxo-acid substrates. The kinetic profiles of 2-oxo-acid substrates showed some marked differences from that of L-lactate dehydrogenase, suggesting different mechanisms for substrate binding and specificity.

The effects of the antiarthritic drugs aurothiomalate (AuTm), aurothioglucose (AuTg), auranofin, its metabolite triethylphosphinegold(I)thioglucose (Et3PAuTg), and several related complexes on the growth of Pseudomonas putida were studied. Two strains were used, one of which (BK135) was more sensitive to Et3PAuTg (tolerant up to 4 microM) than the other (BK403; tolerant to at least 500 microM). Gold thiolate complexes and thiolate ligands alone had little effect on growth. Gold phosphine complexes increased the length of the lag phase of growth and reduced oxygen uptake. Marked changes in cellular morphology were determined by electron microscopy. Copper(II) compounds and aurothiomalate were synergistic in their growth inhibitory effects towards these bacteria. Experiments with 195Au suggested that a mechanism does not exist for the short term (minutes) uptake of gold by sensitive or resistant bacteria, but the resistant strain appeared to limit gold uptake over a longer term (hours).

We describe a new bonded-phase packing material, based upon surface-stabilised microparticulate silica, suitable for the rapid separation and purification of oligonucleotides. Columns packed with this material were demonstrated to give rapid separations of individual oligonucleotide species of up to 44 base units with high purity; agarose gel electrophoresis showed that the products were essentially single bands, with only trace quantities of the (n — 1)-mer present. Baseline resolution of the desired oligomer from (n±1)-mer was achieved under preparative loading conditions, where up to 200–300 μg of oligonucleotide could be separated. The separation was essentially independent of structure or sequence of the oligonucleotides. The retention mechanism of the oligonucleotides was investigated, and the results used to determine the optimum column configuration and separation conditions.

Evidence is presented to demonstrate that the Zn2+ metallo-enzyme glycerol dehydrogenase from the thermophile Bacillus stearothermophilus has one cysteine residue per subunit which is only available for reaction with thiol reagents in the metal-depleted form of the enzyme. Modification of the metal-depleted enzyme by methyl methanethiosulphonate prevents the reactivation of the enzyme by Zn2+ ions and induces dissociation of the oligomer into subunits. The rate of reaction of the cysteine residue with the thiol reagent DTNB is limited by a factor other than reagent concentration and it is proposed that the reagent only reacts with the cysteine residue in dissociated monomers. The enzyme has been labelled at the single cysteine residue by radioactive iodo[2-3H]acetic acid. Two radiolabelled peptides have been isolated and sequenced; one peptide is a component of the other. Spectroscopic evidence suggests that the cysteine residue is not involved in ligation of the essential metal ion. Chemical modification studies using the reagent diethylpyrocarbonate have suggested that two histidines are involved in the ligation of the metal.

For an enzyme to be employed as a reagent in any field, be it clinical chemistry or organic synthesis, it must first be purified to a degree that removes any other enzyme capable of catalysing undesirable side-reactions. This may or may not mean purification to homogeneity. For the enzyme to be commercially viable, purification must yield tens or hundreds of grams of protein. On this scale, the availability of sufficient starting material may pose a problem, particularly for animal tissues. For this and other reasons, bacteria may provide the most valuable source of enzymes. Bacteria are readily produced in enormous quantities and, by virtue of their diverse metabolism, contain many enzymes not present in other organisms. Furthermore, the enzymes from thermophilic species often show a desirable increase in stability under the arduous conditions which may be encountered in a reactor. The preparation of enzymes on this scale from bacteria, or any other source, can usually be accomplished by the application of the normal range of techniques available to the protein chemist. Thus precipitation methods, gel filtration, ion exchange and affinity chromatography are all valuable. A major difference between this and normal laboratory-scale scale purification is cost; large-scale enzyme purification requires a considerable investment in equipment, materials and manpower. This cost must be viewed in terms of the value of the product.

This study shows that the antigenicity of Erwinia chrysanthemi L-asparaginase can be reduced by site-directed mutagenesis. Ten B-cell epitopes of the enzyme were identified using synthetic hexapeptides and polyclonal antisera from rabbits and mice. The region 282GIVPPDEELP292 near the C-terminus was an immunodominant epitope. Binding of two hexapeptides (283IVPPDE288 and 287DEELPG292) to the antibodies was dependent on Pro285, and Pro286, since their replacement by almost any other amino acid resulted in reduced binding. The other residues were less important for binding the antibodies, as binding was relatively unaffected by amino acid substitutions. Three site-directed mutant enzymes, P285T (proline-285-->threonine etc.), P286Q and E288A, were expressed in Escherichia coli. The purified enzymes had subunit M(r) values of 35,000. The pI values of P285T, P286Q and the wild-type enzymes were 8.6, and that for the mutant E288A was 9.2. The kcat. and Km values for the mutants P286Q and E288A with L-asparagine and L-glutamine were comparable with those of the wild-type enzyme. The Km values for the mutant P285T with both substrates was similar to that of the wild-type enzyme, whereas the kcat. was reduced by 2-fold with L-asparagine and by 4-fold with L-glutamine. The change proline-->threonine reduced the antigenicity of the enzyme by 8-fold, as shown in sandwich e.l.i.s.a.s. using monoclonal antibodies raised against the wild-type enzyme.

The X-ray structure of lactate dehydrogenase (LDH) shows the side-chain carboxylate group of Asp-143 to be buried in the hydrophobic interior of the enzyme, where it makes hydrogen-bonding interactions with both the side-chain hydroxyl group of Ser-273 and the main-chain amide group of His-195. This is an unusual environment for a carboxylate side-chain as hydrogen bonding normally occurs with water molecules at the surface of the protein. A charged hydrogen-bonding interaction in the interior of a protein would be expected to be much stronger than a similar interaction on the solvent-exposed exterior. In this respect the side-chain carboxylate group of Asp-143 appears to be important for maintaining tertiary structure by providing a common linkage point between three discontinuous elements of the secondary structure, alpha 1F, beta K and the beta-turn joining beta G and beta H. The contribution of the Asp-143 side-chain to the structure and function of Bacillus stearothermophilus LDH was assessed by creating a mutant enzyme containing Asn-143. The decreased thermal stability of both unactivated and fructose-1,6-diphosphate (Fru-1,6-P2)-activated forms of the mutant enzyme support a structural role for Asp-143. Furthermore, the difference in stability of the wild-type and mutant enzymes in guanidinium chloride suggested that the carboxylate group of Asp-143 contributes at least 22 kJ/mol to the conformational stability of the wild-type enzyme. However, there was no alteration in the amount of accessible tryptophan fluorescence in the mutant enzyme, indicating that the mutation caused a structural weakness rather than a gross conformational change. Comparison of the wild-type and mutant enzyme steady-state parameters for various 2-keto acid substrates showed the mutation to have a general effect on catalysis, with an average difference in binding energy of 11 kJ/mol for the transition-state complexes. The different effects of pH and Fru-1,6-P2 on the wild-type and mutant enzymes also confirmed a perturbation of the catalytic centre in the mutant enzyme. As the side-chain of Asp-143 is not sufficiently close to the active site to be directly involved in catalysis or substrate binding it is proposed that the effects on catalysis shown by the mutant enzyme are induced either by a structural change or by charge imbalance at the active site.(ABSTRACT TRUNCATED AT 400 WORDS)

The Gin residue at amino acid position 102 ofBacillus stearothermophilus lactate dehydrogenase was replaced with Ser, Thr, Tyr, or Phe to investigate the effect on substrate recognition. The Q102S and Q102T mutant enzymes were found to have a broader range of substrate specificity (measured byk cat/K m) than the wild-type enzyme. However, it is evident that either Ser or Thr at position 102 are of a size able to accommodate a wide variety of substrates in the active site and substrate specificity appears to rely largely on size discrimination in these mutants. The Q102F and Q102Y mutant enzymes have low catalytic efficiency and do not show this relaxed substrate specificity. However, their activities are restored by the presence of an aromatic substrate. All of the enzymes have a very low catalytic efficiency with branched chain aliphatic substrates.

A fusion protein, consisting of the N-terminal 81 amino acids from an inactive bovine DNase I (Q38,E39-E38,Q39) and two sequential synthetic IgG-binding domains based upon domain B of Protein A from Staphylococcus aureus has been shown to bind to porcine IgG with a similar affinity and pH profile to Protein A. The same residue in each B domain (Tyr111 and Tyr169) has been mutated by cassette mutagenesis to Ser, Glu, His, Lys or Arg and the effect of the mutation on binding interactions with porcine IgG investigated. The evidence presented suggests that the interactions at the B domain are highly sensitive to the presence of a charged residue.

Expression of the Thermus aquaticus B malate dehydrogenase (MDH)-encoding gene ( mdh ), cloned in Escherichia coli , was initially at a relatively low level (0.1% of soluble cell protein) and was effected by read-through from the tac promoter in the plasmid vector used. An enhancement in expression to 0.4% of soluble cell protein was achieved by shortening the intervening sequence between the promoter and the translation start codon of mdh . An Nde I restriction site (5′;-CAT-ATG-3′) was engineered in the shortened fragment, which also changed the start codon from GTG to ATG. This resulted in an eightfold increase in expression, to 3.2% of soluble cell protein. Expression was further increased by subcloning the mdh gene via the engineered Nde I site, into two plasmid expression vectors, one carrying the E. coli trpP promoter and the other the E. coli mdhP promoter. In both these expression systems, 40–50% of the soluble cell protein was T. aquaticus MDH. This suggests that expression of the cloned T. aquaticus mdh in E. coli is enhanced predominantly by the optimisation of transcription and translation initiation signals. Moreover, the base composition of the coding region and the pattern of codon usage dictated by it appear to have little effect on expression. Heat treatment of the cell extract at 85°C further effected purification of T. aquaticus MDH to over 80% of the soluble cell protein. The MDHs purified to homogeneity from the high-expression clones were identical with the MDH isolated from T. aquaticus B cells with respect to all measured parameters.

A novel protein able to bind with high affinity to the Fc fragment of IgG from a variety of animals has been produced by a gene synthesis approach. The IgG binding is accomplished by the presence of a single or two consecutive domains based upon domain B from protein A of Staphylococcus aureus. The IgG-binding moiety is fused to a peptide containing 21, 53 or 81 amino acids derived from the N-terminus of bovine DNase I. The latter is present to guide the expression of the protein in Escherichia coli into an inclusion body. This facilitates the high expression and recovery of the IgG-binding domains. The binding activity of this fusion protein is very close to that of the native protein A. Site-directed mutagenesis of the fusion protein and subsequent identification of changed binding interactions is reported.

The human LDH-A and LDH-B cDNAs, containing the coding regions for the l -lactate dehydrogenase A4 (M) and B4 (H) polypeptides respectively have been cloned into Escherichia coli to place the cDNAs under the control of hybrid E. coli/Bacillus stearothermophilus transcriptional and translational signals. Human A4- and B4-isoenzymes are produced in E. coli cells harbouring the expression plasmids pHLDHA22 and pHLDHB10 at levels of 6.5 and 1.5% of the soluble protein of the cell, respectively. The tac promoter of these vectors was not induced by isopropyl β- d -thiogalactopyranoside. The A4 and B4 human isoenzymes synthesized in E. coli were purified to homogeneity and show the same properties as isoenzymes isolated from human tissue. The amino acid sequences of 12 N-terminal residues of the human isoenzymes synthesized in E. coli were determined to be identical to those deduced from the DNA sequence of the cloned cDNAs except that the N-terminal methionine was absent from both. However, in contrast to LDH made in human cells, acetylation of the N-terminal alanine does not take place in E. coli cells.

The composition of LB broth (tryptone, yeast extract and NaCl) was investigated by 1H, 31P-NMR spectroscopy, FPLC and gel electrophoresis. An enexpected finding was the high level of 2′,3′-cyclic nucleotides, detected by characteristic 31P-NMR resonances in the region 20–21 ppm, originating from the yeast component. 31P-NMR resonances for cyclic nucleotides were observed during the autolysis of Saccharomyces cerevisiae cells, and in model reactions of RNase with RNA.

Evidence is presented to demonstrate that the Zn2+ -depleted, inactive form of the glycerol dehydrogenase from Bacillus stearothermophilus exists in one of two possible conformations in equilibrium, the position of which is temperature sensitive. The conformation of the metal-depleted enzyme favoured by higher temperatures (20-40 degrees C) is able to bind Zn2+ and regain catalytic activity, whereas that favoured at lower temperatures (0-10 degrees C) is unable to bind metal ions and is thus inactive. This equilibrium is also pH dependent with a pK of 6.6. At pH 6.0, the equilibrium lies in favour of the form of the enzyme able to bind metal ions and exhibit activity.

Basic c -type cytochromes homologous with plant and animal mitochondrial cytochrome c have been isolated and purified from Rhodymenia palmata and Porphyra umbilicalis . The N -terminal regions have been analysed using a Beckman 890C automatic sequencer. When compared to animal cytochrome c , the Rhodymenia cytochrome c has an unblocked N -terminal tail of 10 amino acids, whereas Porphyra has an unblocked N -terminal tail of only a single amino acid.

The amino acid sequence of plastocyanin from marrow was determined. It consists of a single polypeptide chain of mol.wt. 10284 containing 99 amino acid residues. The sequence was determined by using a Beckman 890C automatic sequencer and by dansyl–phenyl isothiocyanate analysis of peptides obtained by the enzymic digestion of purified CNBr fragments. The sequence is in good agreement with the amino acid composition, except that fewer residues of glutamic acid were found in the sequence than were suggested by the composition. Evidence for histidine-37 was weaker than for the rest of the sequence. A ‘tree’ of phylogenetic affinities was constructed by using several higher-plant plastocyanin sequences.

Homogeneous β-lactamase (β-lactam hydrolase, E.C. 3.5.2.6) from Enterobacter cloacae P99, an enzyme that has an important function in antibiotic resistance, was prepared using a single cation-exchange chromatographic step with CM-Sepharose fast-flow. A 6-g amount of the enzyme was isolated from 5 kg of cell paste, with 84% of the enzyme activity in the cell homogenate being recovered by the single cation-exchange step. The specific activity of the β-lactamase was 587 U/mg protein. The relative molecular mass of the enzyme was determined to be 45 kDa by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate and the isoelectric point was 8.95.

The amino acid sequence of plastocyanin from lettuce ( Lactuca sativa L.) was determined by using a Beckman 890C automatic sequencer and by dansyl-phenylisothiocyanate analysis of peptides obtained by enzymic digestion of purified CNBr fragments. The protein consists of a single polypeptide chain of 99 residues, and shows close homology with other higher plant plastocyanins. The data are discussed in relation to the possible residues involved in the binding of copper in plastocyanin.

The amino acid sequence of spinach (Spinacia oleracea L.) plastocyanin was determined. It consists of a single polypeptide chain of 99 residues and has a sequence molecular weight of 10415. The sequence was determined by using a Beckman 890C automatic sequencer and by the dansyl--phenyl isothiocyanate analysis of peptides obtained by the enzymic digestion of purified CNBr fragments. Overlap through the two methionine residues was not shown. Sedimentation equilibrium in the ultracentrifuge gave a molecular weight for spinach plastocyanin of about 9000, in contrast with the value of 21000 reported previously by Katoh et al. (1962).

Glycerokinase from the thermophilic bacterium Bacillus stearothermophilus was found to have an affinity for several reactive, triazine dyes. The enzyme was found to have a strong affinity for several dyes when the dyes were immobilised on an agarose support matrix. Matrices based on cellulose, or polymers of acrylic acid, were less suitable. The study of the effect of various physicochemical properties is described, including ligand concentration, pH, ionic strength, column dimensions and flow-rate. Glycerokinase could be bound to Procion blue MX-3G-Sepharose at low ionic strength (pH 7.5) and was most readily recovered by substrate elution. This matrix was used to purify to homogeneity the enzyme derived from 1 kg of bacteria, in three steps, with an overall recovery of 44%.

The technology of microbial enzyme production has progressed rapidly over the past decade. The main application of industrial microbiology has been in the food and beverage market, but this emphasis is now changing as microbial enzymes become more widely used, particularly in the health-care field.

The complete amino acid sequence of Sambucus nigra L. plastocyanin has been determined. The protein consists of a single polypeptide chain of 99 amino acids and one atom of copper, giving a molecular weight of 10517. The sequence shows considerable similarity to that of Chlorella fusca plastocyanin.

Homogeneous plastocyanin was obtained from Cucurbita pepo L. The MW was 11 360 daltons based on 0.56% copper. Values of S°20W = 1.69 S and D°20W = 1.46 x10−6 cm2 sec−1 and V = 0.73 (amino acid analysis) or 0.74 (pycnometry) indicated 10 300 to 10 900 daltons. Gel chromatography on Sephadex G75 suggested 12 200 daltons. Amino acid analyses of two cultivars indicated 10928 to 11 000 daltons for 102 residues and one atom of copper, excluding 1.0% protein-bound carbohydrate. The single thiol group reacted slowly with 2-chloromercuri-4-nitrophenol but rapidly with mercuric acetate. The redox potential was +350 mV between pH 6.5 and 9.0 with a one-electron change and an ionizable group of pK 5.6. The isoelectric point was at pH 4.2. Light absorption maxima occurred at 253, 259, 264, 269, 278, 284, 460 (very weak), 597 and 775 nm. The best ratio E597:E278 was 0.87. Extinction coefficient at 597 nm was 4.75 x 103 l.mol−1 cm−1. Copper was reversibly removed and 83% restored to apoprotein during successive treatments with mercuric acetate, Sephadex G25 and glutathione. The oxidized protein was crystallized from 58 or 60%-saturated ammonium sulphate containing 2% dioxan at pH 4.0 to 4.5. Oxidized crystals in ammonium sulphate were reduced by ascorbic acid or photochemically with glycerol and then dissolved. Homogeneous ferredoxin from the same source had a MW of 11 400 daltons based on 0.98% iron and 11 045 by amino acid analysis. Two atoms each of iron and labile sulphide were recorded. Methionine was present. Aggregation occurred during sedimentation which indicated 20 500 daltons from S°20W = 2.44S; D°20W = 1 .0 x 10−6 cm2 sec−1 and V = 0.71. Gel chromatography indicated 12 000 to 16 000 daltons depending on media. Redox potential at 25° was −404 mV at pH 7.5. Light absorption maxima were at 277, 333, 422 and 462 nm. The best ratio E422:E277 nm was 0.51. The extinction coefficient at 422 nm was 9.8 x 10−3 l.mol−1 cm−1. The protein crystallized as red needles from 78% or 80%-saturtted (NH4)2SO4.

The amino acid sequence of plastocyanin from potato was determined. It consists of a single polypeptide chain of 99 residues, of molecular weight 10332. The sequence was determined by using a Beckman 890c sequencer and by dansyl–Edman analysis of peptides derived from purified CNBr fragments. The sequence shows considerable similarity with that of Chlorella fusca, and also with the C-terminal region of bacterial azurins.

3-Hydroxybutyrate dehydrogenase (EC 1.1.1.30) and malate dehydrogenase (EC 1.1.1.37) were purified to homogeneity on a large scale involving only two sequential affinity-chromatography steps on two triazine dye-Sepharose matrices. Recoveries of both enzymes were in excess of 60%. Malate dehydrogenase could also be purified by a combination of triazine dye affinity chromatography and gel filtration on Ultrogel AcA-44, but this offered no significant advantage over the purely affinity procedure.

The amino acid sequence of plastocyanin from broad bean was determined. It consists of a single polypeptide chain of 99 residues. The sequence was determined by using a Beckman 890C sequencer and by dansyl–phenyl isothiocyanate analysis of peptides obtained by the enzymic cleavage of purified cyanogen bromide fragments. Some parts of the sequence depend on the results of Edman degradation of peptides for which amino acid analyses were not obtained. The evidence for one overlap is not strong.

Summary: A strain of Pseudomonas putida resistant to low concentrations of cadmium (0.25 mM-Cd2+) adapted to growth in the presence of 3 mM-Cd2+ in a chemically defined medium. This increased resistance was rapidly lost if Cd2+ was omitted from the growth medium. P. putida concentrated 109Cd2+ actively. Mechanisms appeared to exist for expelling cadmium from cells during the lag and exponential phases and for continued growth in the presence of high intracellular Cd2+ concentrations. Cd2+-resistant cells adapted so as to control both the extent and rate of Cd2+ uptake when compared to control cells.

The amino acid sequences of the plastocyanins from Mercurialis perennis and Capsella bursa-pastoris have been determined. The amides at positions 64 and 68 in the Mercurialis sequence were positioned by ‘homology’ Both proteins are single polypeptide chains of 99 residues and are closely related to other higher plant plastocyanins.

Summary: Cadmium-adapted Pseudomonas putida exhibited a long lag phase (6 h) on incubation in a defined medium containing 3 mM-Cd2+. During this time extensive blebbing of the outer membrane was observed by electron microscopy and polyphosphate granules containing Cd2+ were present in the cells. Cells from exponential-phase cultures of cadmium-adapted P. putida were found in clusters. They were much smaller than control cells grown without cadmium, and contained electron-dense aggregates. Cadmium-adapted cells released more lipopolysaccharide and protein into the external medium than did control cells; the addition of Ca2+, but not Mg2+, to the medium prevented this increased release. Cadmium-adapted cells also showed greatly increased sensitivity towards certain antibiotics, including the aminoglycosides, cyclic polypeptides and doxycycline. It is suggested that this is related to changes in membrane structure.

Pseudomonas putida adapted to growth in 3 mM cadmium. The resistance mechanism involved complexation of cadmium in polyphosphate granules, changes in the structure of the cell membrane and induction of three cysteine-rich, low molecular weight proteins (3500-7000) containing 4 to 7 g-atoms per mole of cadmium, zinc, and copper. Each protein was produced during a different phase of growth, and the smallest protein (3500) was released into the environment when the cells lysed at the end of the exponential phase. The metal binding sites of the major protein were further characterized using a range of physical methods, including 113Cd NMR. The properties of the bacterial pseudothioneins are compared to those of metallothioneins. Images FIGURE 2.

An incompressible kieselguhr-agarose composite, derivatized with carboxymethyl groups, Macrosorb KAX-CM, is compared with a conventional agarose gel, CM-Sepharose Fast Flow, for the large-scale purification of l-asparaginase from Erwinia chrysanthemi. The Macrosorb KAX-CM has better pressure/flow characteristics than CM-Sepharose Fast Flow, but both matrices are identical on criteria of product purity. CM-Sepharose Fast Flow has a greater capacity than Macrosorb KAX-CM and enzyme is eluted in a smaller volume.

A new and novel method for determination of serum acetaminophen is described. The assay, which can be completed in less than 5 min, is based on the enzymatic hydrolysis of acetaminophen, with subsequent colorimetric detection of the aminophenol so produced. Various possible means of aminophenol estimation are described; the final reaction conditions have been optimized for maximum sensitivity and assay speed. This assay compares favorably with other available procedures; it requires only small sample volumes; it is rapid, simple, and highly specific for the parent drug; and it requires neither great technical ability nor expensive instrumentation.

There is much current interest in the metallothioneins. These are low molecular weight proteins (6–7,000 Daltons) which have a high cysteine (33%) and metal content (7 g atoms/mol). This, together with the absence of aromatic amino acids and histidine, makes this protein very unusual [1]. Metallothioneins have no defined functions, although many possible roles have been suggested including detoxification [2], and regulation of zinc [3] and copper metabolism [4]. Metallothioneins are widespread in mammals [1] and have also been isolated from fish [5], invertebrates [6], plants [7] and eukaryotic microorganismas such as yeast [8] and Neurosporra crassa [9]. N. crassa protein has been sequenced and found to have extensive homology with mammalian metallothionein [9]. Metallothioneins or similar proteins have never been isolated from bacteria. We have studied the mechanism of cadmium resistance in a gram negative bacterium and isolated novel cadmium binding proteins. The study of these bacterial proteins may provide new insight into the metabolism and probable function of metallothioneins. Three different cadmium-binding-proteins are produced by a cadmium-resistant strain of Pseudomonas putida, isolated from sewage sludge. The bacterium accumulates cadmium from the environment, and the internal concentration can reach 9 mM. Up to 30% of this cadmium is found in the cytoplasm. Three cadmium binding proteins were isolated from the cytoplasm of P. putida (fig.1). Each protein is produced during a different phase of the growth cycle (Fig2). The proteins have a high cysteine content and bind cadmium, copper and zinc. They are induced by the presence of Cadmium-binding-protein 1, molecular weight 7,000 (CdBP1 binds 7 g atoms metal/mol/, and CdBP2 (7,000 daltons) binds 3 g atoms metal/mol. CdBP3, is a much smaller molecule (3,800 daltons) and binds cadmium and copper (4.5 g atoms metal/mol). All three cadmium-binding proteins contain large amounts of cysteine, but only about half that commonly found in metallothioneins. CdBP3 has the highest cysteine content of 22%. Also typical of the metallothioneins is the high serine, glycine, and alanine contents of CDBP1 and CdBP2. CadBP2 also contains substantial amounts of lysine. Both CdBP2 and CdBP3 have few or no residues of arginine, leucine, isoleucine, or the aromatic amino acids. The major differences from metallothionein are the high glutamate content of CdBP2 and CdBP3. CdBP1 also contains substantial amounts of leucine, isoleucine and also four residues of aromatic amino acids. 1H NMR experiments at 400 MHz support the amino acid analysis, revealing the lack of aromatic amino acids in CdBP2 as well as the high cysteine content. The high metal-binding capacity of these proteins, but the lower content of cysteine, suggests that in addition to cysteine-SH other protein ligands may be involved in cadmium binding.

The amino acid sequence of plastocyanin from dock has been completed. It is a single polypeptide chain of 99 residues which is closely related to other plant plastocyanins. Compared to a preliminary sequence presented earlier, the completed sequence now shows two changes, at positions 53 and 92.

Glucokinase from Bacillus stearothermophilus was irreversibly inactivated by the reactive dichlorotriazinyl dye Procion Blue MX-3G at pH 8.0. The enzyme was protected from inactivation by the substrate MgATP. Kinetic data implied that the dye occupied the MgATP-binding site. The apparent Km values for MgATP and D-glucose were found to be 70 microM and 210 microM respectively, and the Kd of the pure reactive dye was 16 microM; 1 mol of the pure reactive dye bound to 1 mol of glucokinase subunit. The dye was shown to have potential as an affinity probe for glucokinase. Glycerokinase from the same bacterium was inactivated by Procion Blue MX-3G at high concentrations (5 mM), but only after a period of increased enzyme activity. Kinetic data indicated that the dye preferentially attacked the glycerol-binding site. The apparent Km values for MgATP and glycerol were found to be 38 microM and 13 microM respectively, and 4 mol of reactive dye could be bound to 1 mol of glycerokinase subunit. This was surprising in view of the MgATP-dependent elution of glycerokinase from immobilized Procion Blue MX-3G.

P. cepacia bacteria adapted to growth in a chemically defined medium containing millimolar concentrations of Au(I) thiolates including the antiarthritic drug Au(I) thiomalate. The bacteria became very large, accumulated polyhydroxybutyrate and gold, and excreted a yellow protein ("thiorin"), which caused foaming of the culture medium. Thiorin was shown by 1H-NMR, amino acid analysis, and gel filtration chromatography to be of low molecular weight (ca. 9500) and to contain predominantly Cys (oxidized), Glx, and Gly.