NMR investigation of the solution conformation of oxidized flavodoxin from Desulfovibrio vulgaris. Determination of the tertiary structure and detection of protein-bound water molecules.

Desulfovibrio vulgaris flavodoxin has been investigated with a combination of homo- and hetero-nuclear two-dimensional and three-dimensional NMR spectroscopy. The analysis of NOE, hydrogen exchange and J-coupling data led to a set of 1349 NOE, 63 hydrogen bond and 109 backbone phi-angle restraints which were used to determine the solution structure of the oxidized flavodoxin applying the distance geometry program DIANA combined with restrained energy minimization methods. Flavodoxin in solution consists of a five-stranded parallel beta-sheet which is pairwise flanked by four alpha-helices. The solution structure has been compared with the known crystal structure. While the global fold is identical, differences have been detected concerning local conformations. In addition, protein-bound water molecules have been localized by NOE effects which were detected in NMR experiments avoiding solvent suppression. The locations of these water molecules have been compared with those found in the X-ray structure.

Homonuclear and heteronuclear NMR studies of oxidized Desulfovibrio vulgaris flavodoxin. Sequential assignments and identification of secondary structure elements.

Recombinant Desulfovibrio vulgaris flavodoxin (molecular mass 16.3 kDa) was produced in Escherichia coli. The oxidized protein has been investigated with a combination of homonuclear and heteronuclear two-dimensional and heteronuclear three-dimensional NMR spectroscopy. Sequence-specific assignment of all backbone and most of the side chain 1H and 15N resonances has been obtained. The secondary structure has been inferred from the pattern of sequential, medium-, and long-range NOEs, together with information about slowly exchanging amide hydrogens and HN-H alpha spin-spin coupling constants. In solution, flavodoxin consists of a five-stranded parallel beta-sheet and four alpha-helices. Residues 3-9, 32-36, 52-58, 87-96, and 123-128 are involved in the beta-sheet whereas the a-helical regions comprise residues 13-28, 69-76, 104-114, and 134-148. Several proton resonances of the bound flavin mononucleotide cofactor have been assigned. NOE contacts between the prosthetic group and the apoprotein have been detected.

Confirmation of high blood flow rates through 150 mu filter/high-flow tubing.

Many new products designed to assist in rapid blood infusion are appearing. Some highly touted and routinely used devices for intravenous (IV) infusion have recently been shown to be, at least in part, defective. A tubing with an in-line 150 mu filter (150 mu High-Flow Blood Filter; Saftifilter Blood Administration Sets; Cutter Biological, Berkeley, CA 94710) has recently been introduced to facilitate rapid blood transfusion. It is claimed that at least 8.5 units of blood can be rapidly run through each set before replacement is necessary. To test this under simulated clinical conditions, four sets of ten random units of outdated erythrocytes at 4 to 9 degrees C were each admixed with 250 mL 70 degrees C 0.9 NaCl and infused through the system under a constant 300 mmHg pressure. Two sets infused through unmodified tubing flowed at an average of 25 mL/sec (1500 mL/min) before there was an appreciable slowing of the flow rate. Two sets with 8 Fr catheters attached infused at an average of 22 mL/sec (1320 mL/min) before there was an appreciable slowing of the flow rate. Even after the flow slowed, the 9th and 10th units infused at an average greater than 10 mL/sec (600 mL/min). The tubing/filter exceeded the manufacturer's published claims. This tubing/filter appears to be one element that could be an effective component of a high-flow infusion system.

Rapid admixture blood warming: technical advances.

The technique of rapid admixture blood warming of cold erythrocyte units is designed to warm erythrocyte units rapidly (less than 30 sec) while simultaneously providing saline for dilution. However, questions have been raised about the recommended use of a standard 250-ml bolus of 70 degrees C admixture saline, the uniformity and speed of blood unit warming, the difficulties inherent in keeping saline bags at 70 degrees C, and the safety of the methodology. To answer these questions, a series of tests were performed and modifications of the technique were introduced. The mean weight of 1000 successive units of erythrocytes for adult infusion was 305 g (range 220 to 410). The maximum temperature was 44 degrees C, using an internal temperature probe (1-cm temperature gradations; 2-sec recording intervals) when the smallest unit was admixed with a 250 ml 70 degrees C saline bolus; the largest unit had a minimum temperature of 30 degrees C. Plasma Hgb, osmotic fragility, and K of the minimum size erythrocyte unit showed no significant deviation from its control. Both thermographic photographs and the internal temperature recordings of the erythrocyte units demonstrated that solely due to fluid turbulence, uniform mixing occurs within approximately 30 sec of beginning the admixture process. Inverting the blood units caused a thermal layering of fluids and an unacceptable maximum blood temperature of 50 degrees C. There was no difference between the mixing time or efficacy in the presence of standard or large-bore iv tubing or additional in-line filters. Volumes of the 250-ml saline bags for admixture decreased markedly with deviations in electrolyte composition after greater than 2 wk at 70 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)

Red blood cell survival following admixture with heated saline: evaluation of a new blood warming method for rapid transfusion.

We studied the in vivo survival of packed red blood cells (RBC's) which had been warmed using the new technique of admixture with high-temperature saline. Packed RBC's from five normal male subjects were stored in CPDA-1 at 4 degrees C for 14 days. They were then warmed via admixture with an equal amount of saline heated to 70 degrees C. Osmotic fragility, and supernatant hemoglobin and potassium levels of the warmed RBC's were not significantly different from baseline values. Aliquots of the warmed RBC's were labeled with 51Chromium and transfused into autologous donors. Mean radiolabeled RBC survival at 24 hours was 90.2% (S.D. 6.2%), and mean radiolabeled RBC survival time was 25.3 days (S.D. 2.7 days). These results are within the normal range for RBC's stored for 14 days. This study suggests that RBC survival after transfusion is not impaired by admixture blood warming using saline at 70 degrees C.

Flow characteristics of admixed erythrocytes through medex tubing with a pall filter.

Resuscitation with fluid and blood components is the mainstay of therapy for hypovolemic patients. This study evaluated the flow rate and resultant temperature of 6 degrees C erythrocytes admixed with warmed saline passing through a new commercial large bore tubing. The tubing is 183 cm long and is 0.57 cm in diameter. The effect on the outflow fluid temperature when catheters of various sizes were added to the system distally was also assessed. The admixed solution temperature averaged 36.2 degrees C and the outflow temperature of the mixture from the distal tubing averaged 34.9 degrees C. There was an average drop in temperature over the length of the tube of 1.5 degrees C. The filter in the Medex Hi-Flow Trauma Quad system collapsed, severely restricting fluid flow, after only four units. The problem of filter clogging was overcome by the in-line addition of a Pall filter. Addition of this in-line filter had a negligible effect on the flow rate. The flow rate with the Pall filter in-line averaged 1,150 mL/min. As the catheters that were added distally to the system diminished in size, there was a predictable decrease in the admixed fluid flow rate. A warmed saline-erythrocyte solution may be very rapidly infused through commercial large-bore tubing modified with an in-line filter. The size of the catheters used determines the ultimate flow rate.

Red cell tolerance of admixture with heated saline.

Red cell stability in the face of thermal stress has been evaluated only in the setting of prolonged incubation. This study was conducted to determine red cell tolerance of rapid mixture with heated saline, which exposes red cells to heat only until thermal equilibration, which is a matter of seconds. Half-units of 35-day-old red cells stored in CPDA-1 were mixed at 6 to 10 degrees C in the blood container with an equal weight of 60, 70, or 80 degrees C saline. This resulted in mean mixture temperatures of 30.9, 37.5, and 42.6 degrees C, respectively. Controls consisted of the same mixture, but with 6 to 10 degrees C saline. The red cells in the mixtures were assessed for osmotic fragility, and the supernatant was examined for plasma hemoglobin and potassium. Neither osmotic fragility curves nor supernatant hemoglobin or potassium changed significantly with saline temperatures of 60 or 70 degrees C. When 80 degrees C saline was used, osmotic fragility, supernatant hemoglobin, and potassium all increased significantly (p less than 0.01) over control values. Red cells tolerate rapid mixture with 70 degrees C saline without hemolysis or change in osmotic fragility.