The basis for the development of a fuselage evacuation time for a ditched helicopter.

HYPOTHESIS: When a helicopter ditches or crashes in water, unless the buoyancy bags are inflated, it commonly sinks inverted. Thus, crew and passengers must make an underwater escape. It is postulated that later passengers in the escape sequence do not have the breath-holding ability to conduct a successful escape, particularly if the water is cold. This contributes to the 20-50% mortality rate in survivable accidents. METHODS: There were 132 immersed subject evaluations which were conducted in daylight and darkness to measure escape times from a helicopter underwater escape trainer, configured to the Super Puma, seated for 15 and 18 passengers. The subjects were highly experienced instructors or Navy clearance divers. RESULTS: The time from when each subject's head disappeared underwater until each subject surfaced and total fuselage evacuation time were measured and any problems hampering escape were noted. Breath-holding for the last subject out ranged from 28 to 92 s. An emergency breathing system was used by a minimum of four subjects each time and a maximum of 11 subjects in one condition. The buoyancy of the survival suit was the principal component that hampered escape. CONCLUSION: Breath-holding times were too long for the later subjects to escape without resorting to an EBS, in spite of the fact that they were highly trained. For regular crew and passengers flying over water, this would explain the high mortality, etc. Therefore, a new helicopter standard should be developed requiring fuselage design to accommodate total evacuation within 20 s from underwater. For current helicopters, where this cannot be achieved, passengers should be provided with some form of air supply, or, after ditching, the helicopter should be modified so that it will stay afloat on its side and retain an air space in the cabin.

Method for determining paracetamol in whole blood by chronoamperometry following enzymatic hydrolysis.

A method is proposed for the determination of paracetamol in whole undiluted blood, based on the enzymatic hydrolysis of the drug to p-aminophenol, which is then measured by chronoamperometry at a glassy carbon electrode. Hydrolysis of the paracetamol prior to electro-oxidation is shown to alleviate problems that arise from high background currents in the whole blood and so ensures a good linear correlation (r greater than 0.99) between the current and the paracetamol concentration. Recovery experiments and comparison with a reference method based on spectrophotometry suggest that the electrochemical assay only measures that proportion of paracetamol that is not bound to serum albumin.

Identification of lysine at the active site of human 5-aminolaevulinate dehydratase.

Reduction of human 5-aminolaevulinate dehydratase with NaBH4 in the presence of 14C-labelled substrate led to complete loss of catalytic activity and to incorporation of label into the enzyme protein. By comparison with authentic lysyl-aminolaevulinic acid, prepared chemically, the modified active-site amino acid obtained by acid hydrolysis was shown to be lysine. Sequencing of a CNBr-cleavage peptide isolated from the inactivated 14C-labelled enzyme revealed that the lysine was present within the sequence M-V-K-P-G-M.

Purification and properties of 5-aminolaevulinate dehydratase from human erythrocytes.

A new procedure for the isolation of homogeneous human 5-aminolaevulinate dehydratase (porphobilinogen synthase, EC 4.2.1.24) is described in which the enzyme is purified 35000-fold and in 65-74% yield. The specific activity of the purified enzyme, 24 units/mg, is the highest yet reported. An efficient stage for the removal of haemoglobin is incorporated in the method, which has general application to the purification of other erythrocyte enzymes. The erythrocyte dehydratase (Mr 285 000) is made up of eight apparently identical subunits of Mr 35 000. The enzyme is sensitive to oxygen, and its activity is maintained by the presence of thiols such as dithioerythritol. Zn2+ is obligatory for enzyme activity, the apoenzyme being essentially inactive (approximately equal to 12% of control) when assayed in buffers devoid of Zn2+. Addition of Zn2+ to the apoenzyme restores activity as long as the sensitive thiol groups are fully reduced; optimal stimulation occurs between 100 and 300 microM-Zn2+. The human enzyme is inhibited by Pb2+ in a non-competitive fashion [KiI (dissociation constant for E X S X Pb2+ complex) = 25.3 +/- 3.0 microM; KiS (dissociation constant for E X Pb2+ complex) = 9.0 +/- 2.0 microM]. Modification of thiol groups, inactivation by oxidation, alkylation or reaction with thiophilic reagents demonstrates the importance of sensitive thiol groups for full enzymic activity.

Mechanism of action of 5-aminolaevulinate dehydratase from human erythrocytes.

Purified 5-aminolaevulinate dehydratase (porphobilinogen synthase, EC 4.2.1.24) from human erythrocytes was incubated initially with limiting amounts of 5-amino [5-14C]laevulinate in a rapid-mixing apparatus. The single-turnover reaction with respect to the bound labelled 5-aminolaevulinate was completed by the addition of unlabelled 5-aminolaevulinate and the resulting radioactive porphobilinogen was isolated and degraded. The 14C label was found to be located predominantly at C-2 of the product, demonstrating that, of the two substrate molecules participating in the reaction, the 5-aminolaevulinate molecule initially bound to the enzyme provides the propionic acid 'side' of the porphobilinogen. The same enzyme-[14C]substrate species that yields regiospecific porphobilinogen may be trapped by reaction with NaBH4, showing that the substrate molecule initially bound to the enzyme does so in the form of a Schiff base. A conventional incubation with 5-amino[5-14C]laevulinate yielded porphobilinogen with an equal distribution of the label between C-2 and C-11. The reaction mechanism of the human erythrocyte 5-aminolaevulinate dehydratase thus follows the same course as that of other dehydratases studied in our laboratory by using single-turnover techniques.

Investigation of the effect of metal ions on the reactivity of thiol groups in human 5-aminolaevulinate dehydratase.

The reaction of human 5-aminolaevulinate dehydratase with 5,5'-dithiobis-(2-nitrobenzoic acid) (Nbs2) results in the release of 4 molar equivalents of 5-mercapto-2-nitrobenzoic acid (Nbs) per subunit. Two of the thiol groups reacted very rapidly (groups I and II), and their rate constants were determined by stopped-flow spectrophotometry; the other two thiol groups (groups III and IV) were observed by conventional spectroscopy. Titration of the enzyme with a 1 molar equivalent concentration of Nbs2 resulted in the release of 2 molar equivalents of Nbs and the concomitant formation of an intramolecular disulphide bond between groups I and II. Removal of zinc from the holoenzyme increased the reactivity of groups I and II without significantly affecting the rate of reaction of the other groups. The reactions of the thiol groups in both the holoenzyme and apoenzyme were little affected by the presence of Pb2+ ions at concentrations that strongly inhibit the enzyme, suggesting that Zn2+ and Pb2+ ions may have independent binding sites. Protein fluorescence studies with Pb2+ and Zn2+ have shown that the binding of both metal ions results in perturbation of the protein fluorescence.

Mechanism of action of beta-oxoacyl-CoA thiolase from rat liver cytosol. Direct evidence for the order of addition of the two acetyl-CoA molecules during the formation of acetoacetyl-CoA.

Single-turnover enzyme reactions were employed with beta-oxoacyl-CoA thiolase purified from rat liver cytosol to determine the order of binding of the two acetyl-CoA molecules to the enzyme during the formation of acetoacetyl-CoA. Equimolar quantities of [1-14C]acetyl-CoA and enzyme were mixed initially in a rapid mixing device and the reaction was quenched by addition of an excess of unlabelled acetyl-CoA. Degradation of the resulting acetoacetyl-CoA revealed that the larger proportion of the radioactivity was in C-3. In the converse experiment, in which unlabelled acetyl-CoA was mixed with enzyme and the reaction was quenched with [1-14C]acetyl-CoA, radioactivity was incorporated preferentially into C-1. Similar results were obtained when [14C]acetyl-enzyme complex isolated by gel filtration was reacted with unlabelled acetyl-CoA, the radioactivity appearing largely in C-3. These findings lead to the conclusion that of the two molecules of acetyl-CoA that are bound by the enzyme and converted into acetoacetyl-CoA, it is the one giving rise to C-3 and -4 that is bound initially to the enzyme in the form of the acetyl-enzyme intermediate complex.