[Ergonomic technology. Design study]. / Tehnologia ergonomica. Studiu de design.

The paper deals with domains and technological developments and related supports that enhance the rehabilitation process. Ergonomic Technology, Rehabilitation Engineering, Accessibility and Assistive technology are factors involved in promoting a greater independence for people with disabilities by designing and developing new devices with improved design and functionality. Results of a device design study for people with disabilities are presented.

[Signification of biorhythms for human performance assessment]. / Semnificatia bioritmurilor in evaluarea performantei umane.

The paper revisits the main features of physical performance from a chrono-medical perspective. Classic and up-to-date evaluation tests (electromyography, neuromuscular efficiency, rating of perceived exertion, ERGOJUMP tests, torque) are mentioned together with their interpretation against the general biorhythm behavioural background of the organism. Conclusions point out the importance of considering biorhythm influence upon different physiological variables or parameters in order to further improve overall human psycho somatic condition and performance.

[Descriptive motor sciences]. / Motricitate descriptiva.

The paper presents a new approach to human movements and positions using Descriptive Geometry, a discipline used in 2D and 3D description of object positions in space. Some particular positions of the human body are depicted using a reference system formed by three orthogonal planes: horizontal, vertical and lateral. Positions of arms are described using precise terms used in Descriptive Geometry. The use of precise terms in human positions description can improve communication and understanding of more complex positions in space.

Application of high-precision isotope ratio monitoring mass spectrometry to identify the biosynthetic origins of proteins.

Isotope ratio monitoring (IRM) mass spectrometry was used to measure the relative abundance of stable isotopes in several samples of adult human hemoglobin expressed in E. coli, yeast, and human blood. The results showed significant differences in the distribution of (15)N and (13)C isotopes among hemoglobin samples produced in these organisms. This indicates that IRM mass spectrometry can be used in forensic protein chemistry to identify the origin of protein expression.

Predicting the reactivity of proteins from their sequence alone: Kazal family of protein inhibitors of serine proteinases.

An additivity-based sequence to reactivity algorithm for the interaction of members of the Kazal family of protein inhibitors with six selected serine proteinases is described. Ten consensus variable contact positions in the inhibitor were identified, and the 19 possible variants at each of these positions were expressed. The free energies of interaction of these variants and the wild type were measured. For an additive system, this data set allows for the calculation of all possible sequences, subject to some restrictions. The algorithm was extensively tested. It is exceptionally fast so that all possible sequences can be predicted. The strongest, the most specific possible, and the least specific inhibitors were designed, and an evolutionary problem was solved.

Application of native-state electrospray mass spectrometry to identify zinc-binding sites on engineered hemoglobin.

We report the utility of native-state mass spectrometry to detect zinc ion binding to the engineered hemoglobin rHb52. Various preparations of this recombinant hemoglobin had significantly different oxygen affinities. Detailed characterization of denatured globins did not show any difference between analyzed hemoglobin molecules. However, when solutions of intact hemoglobin pseudotetramers were analyzed by native-state electrospray mass spectrometry, a significant shift in the mass spectrum was observed, indicating labile modification of hemoglobin. Using collision-induced dissociation (CID), we found a mass gain of 63 Da located on the beta-globin. EDTA treatment of modified hemoglobin prior to the infusion removed the modification and restored the predicted oxygen affinity. Ion-trap fragmentation of the +8 charged ion of modified beta-globin showed a neutral loss of 96+/-1 Da, consistent with neutral loss of zinc sulfide. These findings indicated zinc binding to the beta-globin through a cysteine residue. Involvement of Cys93 was confirmed by kinetics of cysteine residue reactivity with dithiodipyridine and peptide mapping. Presence of zinc was confirmed by ICP-MS metal analysis.

Assessing the relative stabilities of engineered hemoglobins using electrospray mass spectrometry.

An ion trap mass spectrometer equipped with an electrospray source was used to examine the relative thermodynamic stabilities of various hemoglobins with respect to both tetramer dissociation and hemin dissociation. The results demonstrated that the stability of hemoglobin molecules can be differentiated by the amount of applied collision-induced dissociation (CID) energy necessary to break up the intact tetramer into its constituent globins. The stability of the intact tetramer was affected by single mutations in the beta-globins. The stabilities of the constituent hologlobins were assessed via trap CID of selected ions. The results demonstrated the importance of the contributions of the hologlobin components to the stability of the intact tetramer. Genetic fusion of two alpha-globins, through the introduction of a single glycine residue between the C-terminus of one alpha-chain and the N-terminus of the second, significantly increased the stability of the hemoglobin pseudo-tetramer. Chemical crosslinking of the beta-globins in addition to genetic fusion of alpha-globins further stabilized the hemoglobin molecule. A dihemoglobin molecule produced by the genetic fusion of two di-alpha-globins with a flexible linker demonstrated a decreased stability relative to the corresponding monohemoglobin.

Nickel-catalyzed N-terminal oxidative deamination in peptides containing histidine at position 2 coupled with sulfite oxidation.

Peptides containing histidine at position 2 were observed to undergo spontaneous N-terminal oxidative deamination in aqueous solution in the presence of Ni(II), sulfite, and ambient oxygen. The reaction resulted in the formation of a free carbonyl on the N-terminal alpha-carbon (alpha-ketoamide) and was catalytic with respect to nickel. This oxidative deamination was confirmed by 13C NMR, 1H NMR, mass spectrometry, and chemical tests. No evidence of modification of histidine was found. It was demonstrated that the nickel-dependent N-terminal oxidative deamination also occurred in His-2 peptides using potassium peroxymonosulfate (oxone) as an oxidant. When oxone was used, oxygen was not required for the deamination to proceed. The results suggest that both nickel-catalyzed reactions (sulfite and oxygen, and oxone) produce an imine intermediate that spontaneously hydrolyzes to form the free carbonyl. These findings may provide a physiologically relevant model for oxidative carbonyl formation in vivo, as well as a useful method for producing a site-specific carbonyl on peptides and proteins.

Acute and long-term stability studies of deoxy hemoglobin and characterization of ascorbate-induced modifications.

The reaction of ascorbate with recombinant hemoglobin (rHb1.1) in the presence of differing partial pressures of oxygen was studied. In the presence of 15 000 ppm (1.5%) residual oxygen, ascorbate/oxygen-mediated reactions resulted in an increased rate of autoxidation, modification of the beta-globin, increased oxygen affinity and decreased maximum Hill coefficient. One of the observed modifications to the beta-globin was a 72 Da addition to its N-terminus. Detailed characterization indicates the modification was an imidazolidinone type structure. Thorough deoxygenation of the hemoglobin solution to <150 ppm of oxygen prior to addition of ascorbate was required to prevent these modifications. Addition of ascorbate to the deoxy hemoglobin (deoxyHb) at pH 8 induced aggregation, eventually leading to precipitation. No such precipitation was observed at pH 7. Long-term storage of the hemoglobin was carried out by addition of ascorbate to deoxyHb at pH 7. The level of methemoglobin remained at <2% for up to 1 year at 4 degreesC, with no detectable precipitation of the protein. Modifications similar to those observed by the acute studies were observed over the 1-year period and correlated with disappearance of the added ascorbate.

Carbamylation of cysteine: a potential artifact in peptide mapping of hemoglobins in the presence of urea.

Peptide mapping is a useful technique for identifying posttranslational modifications. However, sometimes artifacts can be introduced during the mapping procedure which can be misleading in identifying the origin and nature of the modifications. During peptide mapping of unalkylated hemoglobins with Staphylococcus aureus V8 proteinase, we found a significant level of carbamylated cysteines. Carbamylation was not detected if recombinant human hemoglobin (rHb1.1) was alkylated prior to digestion. Our experiments indicated that this modification was an artifact of the digestion procedure in which the slightly acidic conditions promoted the reaction of cysteine sulfhydryls with residual cyanate derived from urea. Carbamylmercaptans were found to be stable under acidic conditions but were unstable in base. The extent of cysteine carbamylation can be moderated by the use of scavengers.

Glutaraldehyde modification of recombinant human hemoglobin alters its hemodynamic properties.

Many cell-free hemoglobin solutions designed as oxygen-carrying therapeutics produce a hypertensive effect in animals. The response is likely due to oxidation of nitric oxide by hemoglobin. Since the site of oxidation may lie outside the vascular compartment, we tested the hypothesis that polymerization of hemoglobin, rHb1.1, by glutaraldehyde would attenuate the hypertensive response. Two products of the cross-linking reaction were isolated, a glutaraldehyde-derivatized monomer (mono-glxrHb) and a glutaraldehyde cross-linked polymer (poly-glxrHb), and evaluated for their effects on systemic hemodynamics in conscious rats. Administration of rHb1.1 caused a mean arterial pressure elevation of approximately 20 mm Hg and an increase in total peripheral resistance of approximately 30%. Administration of mono-glxrHb induced changes in mean arterial pressure and vascular resistance that were significantly diminished relative to those observed with rHb1.1. Poly-glxrHb elicited a mean arterial pressure response that was further reduced compared with that obtained with mono-glxrHb and a change in vascular resistance that was the same as the response to mono-glxrHb. These results suggest that rHb peripheral vasoconstriction elicited by rHb1.1 is significantly attenuated by glutaraldehyde modification of the hemoglobin monomer and that the effect of glutaraldehyde polymerization is likely due to surface modification and/or intramolecular cross-linking, rather than an increase in molecular size.

Identification of a nickel(II) binding site on hemoglobin which confers susceptibility to oxidative deamination and intramolecular cross-linking.

Complexation of Ni(II) with native state recombinant hemoglobin is shown to produce NH2-terminal deamination and globin cross-linking in the presence of the oxidant potassium peroxymonosulfate (OxoneTM). Both the oxidative deamination and cross-linking are exclusive to the beta chains. Recombinant hemoglobin mutants have been created to identify protein sequence requirements for these reactions. It was found that His-2 of the beta globin is required for redox active Ni(II) complexation, oxidative deamination, and cross-linking. The oxidative deamination results in the formation of a free carbonyl in place of the NH2-terminal amine of the beta chain. Most cross-linking of the beta globin occurs intramolecularly, forming beta globin dimers. Structural characterization of the beta globin dimers indicates the presence of heterogeneous cross-links within the central hemoglobin cavity between the NH2 terminus of one beta chain and the COOH-terminal region of the other.

High-fidelity translation of recombinant human hemoglobin in Escherichia coli.

Coexpression of di-alpha-globin and beta-globin in Escherichia coli in the presence of exogenous heme yielded high levels of soluble, functional recombinant human hemoglobin (rHb1.1). High-level expression of rHb1.1 provides a good model for measuring mistranslation in heterologous proteins. rHb1.1 does not contain isoleucine; therefore, any isoleucine present could be attributed to mistranslation, most likely mistranslation of one or more of the 200 codons that differ from an isoleucine codon by 1 bp. Sensitive amino acid analysis of highly purified rHb1.1 typically revealed < or = 0.2 mol of isoleucine per mol of hemoglobin. This corresponds to a translation error rate of < or = 0.001, which is not different from typical translation error rates found for E. coli proteins. Two different expression systems that resulted in accumulation of globin proteins to levels equivalent to approximately 20% of the level of E. coli soluble proteins also resulted in equivalent translational fidelity.

Incorporation of norvaline at leucine positions in recombinant human hemoglobin expressed in Escherichia coli.

We report here a novel finding that norvaline can be incorporated in place of leucine in recombinant human hemoglobin expressed in Escherichia coli. The presence of the norvaline was confirmed by several analytical methods such as amino acid analysis, peptide mapping, electrospray mass spectrometry, and Edman protein sequencing. It appears that substitution is distributed across both the beta- and di-alpha-globins in purified recombinant hemoglobin. The level of misincorporation correlated with the ratio of the free norvaline/leucine pool available in the cell culture. This suggests that the incorporation of norvaline for leucine occurs through misaminoacylation of tRNALeu, similar to the misincorporation of norleucine for methionine found in many recombinant proteins expressed in E. coli.

Mapping of recombinant hemoglobin using immobilized trypsin cartridges.

A tryptic mapping procedure has been developed for a recombinant hemoglobin (rHb1.1) using an immobilized trypsin cartridge. Apohemoglobin is passed through the trypsin cartridge and the products of the digestion are captured directly onto an in-line C18 reversed-phase column. The peptides are then separated using a gradient elution. This new procedure is rapid and reproducible and can be fully automated. The total time of analysis is less than 2 h. The mapping of apohemoglobins produced an unexpected isomerization of two peptides: beta8,9 (K66-K82) and alpha8,9 (K61-K90). It appears that the isomerization may occur through transpeptidation followed by proteolysis at a newly generated site next to the site of ligation. This mapping procedure can be a useful tool for research and routine analysis of proteins.

Binding of amino acid side-chains to S1 cavities of serine proteinases.

The P1 or primary specificity residue of standard mechanism canonical protein inhibitors of serine proteinases, inserts into the S1 primary specificity cavity of the cognate enzyme upon enzyme-inhibitor complex formation. Both natural evolution and protein engineering often change the P1 residue to greatly alter the specificity and the binding strength. To systematize such results we have obtained all 20 coded P1 variants of one such inhibitor, turkey ovomucoid third domain, by recombinant DNA technology. The variants were extensively characterized. The association equilibrium constants were measured at pH 8.30, 21 (+/-2) degrees C, for interaction of these variants with six well characterized serine proteinases with hydrophobic S1, cavities. The enzyme names are followed by the best, worst and most specific coded residue for each. Bovine chymotrypsin A alpha (Tyr, Pro, Trp), porcine pancreatic elastase (Leu/Ala, Arg, Ala), subtilisin Carlsberg (Cys, Pro, Glu), Streptomyces griseus proteinase A (Cys, Pro, Leu) and B (Cys, Pro, Lys) and human leukocyte elastase (Ile, Asp, Ile). The data set was merged with Ka values for five non-coded variants at P1 of turkey ovomucoid third domain obtained in our laboratory by enzymatic semisynthesis. The ratios of the highest to the lowest Ka for each of the six enzymes range from 10(6) to 10(8). The dominant force for binding to these pockets is the hydrophobic interaction. Excess steric bulk (too large for the pocket), awkward shape (Pro, Val and Ile), polarity (Ser) oppose interaction. Ionic charges, especially negative charges on Glu- and Asp- are strongly unfavorable. The Pearson pro duct moment correlations for all the 15 enzyme pairs were calculated. We suggest that these may serve as a quantitative description of the specificity of the enzymes at P1. The sets of Streptomyces griseus proteinases A and B and of the two elastases are strongly positively correlated. Strikingly, chymotrypsin and pancreatic elastase are negatively correlated (-0.10). Such correlations can be usefully extended to many other enzymes and to many other binding pockets to provide a general measure of pocket binding specificity.

Recombinant protein sequences can trigger methylation of N-terminal amino acids in Escherichia coli.

Recombinant human hemoglobin rHb1.1 has been genetically engineered with the replacement of the wild-type valine residues at all N-termini with methionine, an Asn 108 Lys substitution on the beta globins, and a fusion of the two alpha globins with a glycine linker. When rHb1.1 was expressed in Escherichia coli, methylation of the N-terminal methionine of the alpha globin was discovered. Another mutant has been engineered with the alpha globin gene coding for N-terminal methionine followed by an insertion of alanine. Characterization of expressed hemoglobin from this variant revealed a methylated N-terminal alanine that occurred through two posttranslational events: initial excision of the N-terminal methionine, followed by methylation of alanine as the newly generated N-terminus. No methylation was observed for variants expressed with wild-type valine at the N-terminus of the alpha globin. The methylation of N-terminal amino acids was attributed to a specific protein sequence that can trigger methylation of proteins expressed in E. coli. Here we demonstrate that proline at position 4 in the protein sequence of alpha globin seems an essential part of that signaling. Although N-terminal methylation has been observed previously for native E. coli proteins with similar N-terminal sequences, methylation of the recombinant globins has allowed further delineation of the recognition sequence, and indicates that methylation of heterologous proteins can occur in E. coli.

Amino acid sequences of ovomucoid third domains from 27 additional species of birds.

Ovomucoids consist of a single polypeptide chain which is composed of three tandem Kazal domains. Each Kazal domain is an actual or putative protein inhibitor of serine proteinases. Ovomucoid third domains were already isolated and sequenced from 126 species of birds (Laskowski et al., 1987, 1990). This paper adds 27 new species. A number of generalizations are made on the basis of sequences from 153 species. The residues that are in contact with the enzyme in enzyme-inhibitor complexes are strikingly hypervariable. While the primary specificity residue, P1, is the most variable; substitutions occur predominantly among aliphatic, hydrophobic residues. Consensus sequences for an avian ovomucoid third domain, for a b-type Kazal domain (i.e., a COOH terminal domain of multidomain inhibitors) and for a general Kazal domain are given. Finally, the individual new sequences are briefly discussed.

Effect of Elicitation and Changes in Extracellular pH on the Cytoplasmic and Vacuolar pH of Suspension-Cultured Soybean Cells.

We have employed both (31)P nuclear magnetic resonance spectroscopy and two intracellular fluorescent pH indicator dyes to monitor the pH of the vacuole and cytoplasm of suspension-cultured soybean cells (Glycine max Merr cv Kent). For the (31)P nuclear magnetic resonance studies, a flow cell was constructed that allowed perfusion of the cells in oxygenated growth medium throughout the experiment. When the perfusion medium was transiently adjusted to a pH higher than that of the ambient growth medium, a rapid elevation of vacuolar pH was observed followed by a slow (approximately 30 minute) return to near resting pH. In contrast, the concurrent pH changes in the cytoplasm were usually fourfold smaller. These data indicate that extracellular pH changes are rapidly communicated to the vacuole in soybean cells without significantly perturbing cytoplasmic pH. When elicitors were dissolved in a medium of altered pH and introduced into the cell suspension, the pH of the vacuole, as above, quickly reflected the pH of the added elicitor solution. In contrast, when the pH of either a polygalacturonic acid or Verticillium dahliae elicitor preparation was adjusted to the same pH as the ambient medium, no significant change in either vacuolar or cytoplasmic pH was observed during the 35 minute experiment. These results were confirmed in experiments with pH-sensitive fluorescent dyes. We conclude that suspension-cultured soybean cells do not respond to elicitation by significantly changing the pH of their vacuolar or cytoplasmic compartments.

Amino acid sequences of ovomucoid third domain from 25 additional species of birds.

Ovomucoids were isolated from 25 avian species other than the 101 studied in Laskowski et al. (1987, Biochemistry 26, 202-221). These were subjected to limited proteolysis with an appropriate enzyme, and connecting peptide extended ovomucoid third domains were isolated and sequenced to the end in a protein sequencer. Of the 25 new sequences, 13 duplicate ones were already known, and 12 are unique. Probably the most striking findings are a Pro14----Ser14 replacement in weka, an Ala14----Thr15 replacement in Bulwer's pheasant, the discovery of two additional amino acid residues Ile18 and Gly18 at the P1 reactive site position in Kalij pheasant and tawny frogmouth, respectively, and the first finding of a negative (Glu34) rather than positive (Lys34 or Arg34) amino acid residue at the NH2 terminus of the alpha helix in caracara ovomucoid third domain. These results complete the determination of all the sequences of ovomucoid third domains in the four species genus Gallus, in the five species genus Syrmaticus, and in the two species genera Aix and Pavo.