Qurra-tul-Ann Afza Gardner

Studies on the regioselectivity and kinetics of the action of trypsin on proinsulin and its derivatives using mass spectrometry.

Human M-proinsulin was cleaved by trypsin at the R(31)R(32)-E(33) and K(64)R(65)-G(66) bonds (B/C and C/A junctions), showing the same cleavage specificity as exhibited by prohormone convertases 1 and 2 respectively. Buffalo/bovine M-proinsulin was also cleaved by trypsin at the K(59)R(60)-G(61) bond but at the B/C junction cleavage occurred at the R(31)R(32)-E(33) as well as the R(31)-R(32)E(33) bond. Thus, the human isoform in the native state, with a 31 residue connecting C-peptide, seems to have a unique structure around the B/C and C/A junctions and cleavage at these sites is predominantly governed by the structure of the proinsulin itself. In the case of both the proinsulin species the cleavage at the B/C junction was preferred (65%) over that at the C/A junction (35%) supporting the earlier suggestion of the presence of some form of secondary structure at the C/A junction. Proinsulin and its derivatives, as natural substrates for trypsin, were used and mass spectrometric analysis showed that the k(cat.)/K(m) values for the cleavage were most favourable for the scission of the bonds at the two junctions (1.02±0.08×10(5)s(-1)M(-1)) and the cleavage of the K(29)-T(30) bond of M-insulin-RR (1.3±0.07×10(5)s(-1)M(-1)). However, the K(29)-T(30) bond in M-insulin, insulin as well as M-proinsulin was shielded from attack by trypsin (k(cat.)/K(m) values around 1000s(-1)M(-1)). Hence, as the biosynthetic path follows the sequence; proinsulin→insulin-RR→insulin, the K(29)-T(30) bond becomes shielded, exposed then shielded again respectively.

Characterization and bioassay of post-translationally modified interferon α-2b expressed in Escherichia coli

Examples of N-terminal acetylation are rare in prokaryotic systems, but in this study, we report one such example in which N-terminal Cys residue of recombinant human interferon α-2b produced in Escherichia coli is a favourite site for N(α)-acetylation. The recombinant protein following Q-sepharose chromatography gave a single band on PAGE analysis. However, on reverse phase HPLC the material separated into three peaks. These were characterized by mass spectrometric techniques as: (a) the direct translation product of the gene retaining the N-terminal methionine, (b) a species from which the methionyl residue had been removed by E. coli methionyl aminopeptidase to give the native interferon α-2b and (c) in which the N-terminal Cys residue of the latter contained an acetyl group. Tryptic digestion of interferon α-2b gave fragments linking Cys(1) to Cys(98) and Cys(29) to Cys(138), while that of N(α)-acetyl-interferon α-2b gave the Cys(1)-Cys(98) fragment with an additional mass of 42 attributed to an acetylated N-terminal. Bioassay of the derivatives showed that N(α)-acetyl-interferon α-2b had 10% of the activity of interferon α-2b. The results suggest that the lower activity derivative seen here in E. coli may also be produced when the protein is produced in yeast.

Mechanistic and stereochemical studies of glycine oxidase from Bacillus subtilis strain R5.

Glycine oxidase gene from a strain of Bacillus subtilis was cloned and expressed in Escherichia coli. The purified enzyme was found, by mass spectrometry, to have a protein M(r) of 40763 (value of 40761.6 predicted from DNA sequence) and a FAD prosthetic group M(r) of 785.1 (theoretical value of 785.5). Glycine oxidase optimally catalyzes the conversion of glycine and oxygen into glyoxylate, hydrogen peroxide, and ammonia. Using samples of [2-RS-(3)H(2),2-(14)C]-, [2-R-(3)H,2-(14)C]-, and [2-S-(3)H,2-(14)C]glycine, we found that in the overall process H(Si) is removed. Incubation of the enzyme with [2-RS-(3)H(2),2-(14)C]glycine under anaerobic conditions, when only the reducing half of the reaction can occur, led to the recovery of 98.5% of the original glycine, which had the same (3)H:(14)C ratio as the starting substrate. The primary isotope effect was studied using [2-(2)H(2)]glycine, and we found that the specificity constants, k(cat)/K(M), for the protio and deuterio substrates were 1.46 x 10(3) and 1.05 x 10(2) M(-1) s(-1), respectively. Two alternative mechanisms for FAD-containing oxidases that involve either the intermediacy of a FADH(2)-imino acid complex or an amino acid covalently linked to FAD, formed via a carbanion, have been considered. The current knowledge of the mechanisms is reviewed, and we argue that a mechanism involving the FADH(2)-imino acid complex can be dissected to satisfactorily explain some of puzzling observations for which the carbanion mechanism was originally conceived. Furthermore, our results, together with observations in the literature, suggest that the interaction of glycine with the enzyme occurs within a tight ternary complex, which is protected from the protons of the medium.

Inventory of 'slow exchanging' hydrogen atoms in human proinsulin and its derivatives: Observations on the mass spectrometric analysis of deuterio-proteins in D2O

Secondary structure elements of human proinsulin and of its tryptic products were compared by H/D exchange, in a single-pot, using mass spectrometry. Human proinsulin containing an N-terminal methionine, M-proinsulin, was engineered and converted into a perdeuterio derivative, which using an optimized mass spectrometric protocol and manual calculations gave a mass of 9669.6 (+/-1) Da showing the replacement, with deuterium of 146.4 from a total of 149 exchangeable hydrogen atoms (83 from amides and 66 from side-chains). Tryptic digestion of the perdeuterio-M-proinsulin, followed by the transfer of the digest from a deuterio- into a protio-medium showed, at the earliest time of analysis, that of the 27 (+/-1) D atoms retained in M-proinsulin, 24 (+/-1) were found in the insulin nucleus, M-insulin-RR, and 4.2 (+/-1) in the C-peptide-KR. A temporal analysis of the fate of D atoms in these species showed that whereas the C-peptide-KR rapidly exchanged its deuterium, losing all by 6 h, the loss of D atoms from M-proinsulin and M-insulin-RR was gradual and in each case, 12 deuterium atoms survived exchange for 72 h. At all time intervals the loss of D atoms from M-proinsulin mirrored that from M-insulin-RR plus the C-peptide-KR, suggesting that the secondary-structure elements of M-proinsulin are largely conserved in its two component parts.

TK1299, a highly thermostable NAD(P)H oxidase from Thermococcus kodakaraensis exhibiting higher enzymatic activity with NADPH.

Seven nicotinamide adenine dinucleotide oxidase homologs have been found in the genome of Thermococcus kodakaraensis. The gene encoding one of them, TK1299, consisted of 1326 nucleotides, corresponding to a polypeptide of 442 amino acids. To examine the molecular properties of TK1299, the structural gene was cloned, expressed in Escherichia coli and the gene product was characterized. Molecular weight of the recombinant protein was 49,375 Da when determined by matrix-assisted laser desorption/ionization time-of-flight and 300 kDa when analyzed by gel filtration chromatography indicating that it existed in a hexameric form. The enzyme was highly thermostable even in boiling water where it exhibited more than 95% of the enzyme activity after incubation of 150 min. TK1299 catalyzed the oxidation of NADH as well as NADPH and predominantly converted O₂ to H₂O (more than 75%). K(m) value of the enzyme towards NADH and NADPH was almost same (24 ± 2 μM) where as specific activity was higher with NADPH compared to NADH. To our knowledge this is the most thermostable and unique NAD(P)H oxidase displaying higher enzyme activity with NADPH.

Preventing the N-terminal processing of human interferon α-2b and its chimeric derivatives expressed in Escherichia coli

We have previously shown that human interferon α-2b (IFN) produced in Escherichia coli (E. coli) is heterogeneous at the N-terminal, with three major species (Ahsan et al., 2014). These are: (a) the direct translation product of the gene retaining the N-terminal methionine, (b) a species from which the methionyl residue has been removed by E. coli methionyl aminopeptidase to give the native interferon α-2b and (c) in which the N-terminal Cys residue of the latter contains an acetyl group. In this paper we overcome this heterogeneity, using engineered interferon derivatives with phenylalanine residue directly downstream of the N-terminal methionine (Met-Phe-IFN). This modification not only prevented the removal of the N-terminal methionine by E. coli methionyl aminopeptidase but also the subsequent N-acetylation. Critically, Met-Phe-IFN had enhanced activity in a biological assay. N-terminal stabilization was also achieved by fusing human cytochrome b5 at the N-terminal of interferon (b5-IFN-chimera). In this case also, the protein was more active than a reciprocal chimera with cytochrome b5 at the C-terminal of interferon (Met-IFN-b5-chimera). This latter protein also had a heterogeneous N-terminal but addition of phenylalanine following Met, (Met-Phe-IFN-b5-chimera), resolved this problem and gave enhanced biological activity.

Gene cloning and characterization of glycine oxidase from newly isolated Bacillus subtilis strain R5

The gene encoding the glycine oxidase from Bacillus subtilis strain R5 (goxR) was cloned and expressed in Escherichia coli. The gene consisted of 1,110 nucleotides that encoded a protein (GoxR) of 369 amino acid residues with a molecular mass of 40,761 Da. The GoxR exhibited 98.6% identity with glycine oxidase from B. subtilis strain 168. Gene expression and purification of the recombinant GoxR were performed. The recombinant GoxR existed in a homotetramer form. The recombinant protein effectively catalyzed the oxidation of glycine and d-alanine. The specific activity of the purified recombinant GoxR was 0.96 U/mg when glycine was used as a substrate and 1.0 U/mg when d-alanine was substrate. The enzyme displayed its highest activity at pH 8.0 and at a temperature of 50°C. The activation energy of the reaction catalyzed by the enzyme was calculated to be 26 kJ/mol. The enzyme activity was significantly inhibited in the presence of organic solvents. No enhancement of enzyme activity was observed in the presence of metal cations. The experimental results presented in this study demonstrate that the enzyme was a bonafide glycine oxidase.

First cloning and characterization of aspartate aminotransferase from river buffalo (Bubalus bubalis)

Aspartate aminotransferase catalyzes the transfer of an amino group from L-aspartate to α-oxoglutarate. We have purified cytosolic isozyme, to apparent homogeneity, from the heart of river buffalo. In order to clone the enzyme total RNA was isolated and the cDNA encoding complete polypeptide of 413 amino acids was amplified by reverse transcriptase polymerase chain reaction. The cDNA and the deduced amino acid sequence exhibited 98.2% and 99.5% identities, respectively, with that of cow. The cDNA was overexpressed in Escherichia coli and the gene product was obtained in enzymologically active form. The recombinant enzyme was about 40% of the total cell proteins. The purified recombinant enzyme displayed specific activity, Km, temperature and pH stability values similar to that of the native enzyme. Edman degradation and electrospray ionization mass spectra showed that the recombinant enzyme consisted of three species having N-terminal sequences of MAPPSIF, APPSIF and PPSIF, although the second one was the predominant species. Conditions are also described, which in the mass spectral analysis give either the three species corresponding to the apo- or the holo-enzyme; the latter retaining pyridoxal phosphate bound to the protein. To our knowledge this is the first report on sequence determination, cloning and characterization of aspartate aminotransferase from river buffalo.

Escherichia coli signal peptidase recognizes and cleaves archaeal signal sequence

Tk1884, an open reading frame encoding α-amylase in Thermococcus kodakarensis, was cloned with the native signal sequence and expressed in Escherichia coli. Heterologous gene expression resulted in secretion of the recombinant protein to the extracellular culture medium. Extracellular α-amylase activity gradually increased after induction. Tk1884 was purified from the extracellular medium, and its molecular mass determined by electrospray ionization mass spectrometry indicated the cleavage of a few amino acids. The N-terminal amino acid sequence of the purified Tk1884 was determined, which revealed that the signal peptide was cleaved between Ala26 and Ala27 by E. coli signal peptidase. To the best of our knowledge, this is the first report describing an archaeal signal sequence recognized and cleaved by E. coli signal peptidase.

Effect of Milking Method, Diet, and Temperature on Venom Production in Scorpions

Abstract In the present study, two common buthid scorpions, i.e., Androctonus finitimus (Pocock, 1897) (Scorpiones: Buthidae) and Hottentota tamulus (Fabricus, 1798) (Scorpiones: Buthidae), were maintained in the laboratory for venom recovery. The aim of study was to compare the quantity and quality of venom extracted from scorpions by manual and electrical method. We also recorded the effect of diet and temperature on venom production. Results of our study revealed that electrical method yielded good quality and higher quantity of venom as compared to manual method. The quantity of venom by two studied species differed statistically. We recorded the effect of food on venom production by providing different prey items to the scorpions and found that grasshopper nymphs and adults were the best diet for the scorpions to get maximum yield of venom as compared to other prey types (house crickets, house flies, and moths). Production of venom and activity of scorpions was found to be associated with temperature. During winter season, venom recovery was comparatively low as compared to the hottest part of year; when venom milking and activity of scorpions both were increased.