Cerebral protection during deep hypothermic circulatory arrest is predicated on efficient and complete cerebral cooling. Institutions approach cooling quite differently. We compared two different ...cooling strategies in terms of measured jugular venous bulb saturations in 39 infants undergoing deep hypothermic cardiopulmonary bypass to evaluate the effect of institutional cooling practices on jugular venous bulb saturation, an indirect measure of cerebral cooling efficiency.
The patients were grouped based on the method of core cooling. In group A (n = 17), core cooling was achieved rapidly by setting the water bath temperature of the heat exchanger at 4° to 5°C, and the patient was cooled until rectal temperature and nasopharyngeal temperature were 15°C or lower. In group B (n = 22), the heat exchanger was initially set at 18°C and slowly lowered to 12°C. Hypothermic temperatures of 12°C were maintained until the nasopharyngeal temperature was 18°C or less and the rectal temperature was 20°C or lower. Once cooling was complete, blood samples were analyzed by cooximetry for determination of arterial oxygen saturation and jugular venous bulb saturation.
In group A, the measured jugular venous bulb saturation was 98.0% ± 0.9% and the oxygen saturation to jugular venous bulb saturation difference was 0.3% ± 0.5%, measured at the time that institutional cooling objectives were achieved (total cooling time, 15.0 ± 0.45 minutes). In group B, jugular venous bulb saturation was 86.2% ± 12% and the oxygen saturation to jugular venous bulb saturation difference was 10.8% ± 12.2%, measured at the time that institutional cooling objectives were achieved (total cooling time, 17.5 ± 1.1 minutes) (p < 0.01).
Differences in cardiopulmonary bypass cooling techniques may alter the rate at which jugular bulb saturations rise. We believe this represents an indirect measure of the efficiency of brain cooling and therefore of cerebral protection.
The primary goal of this study was to gain a better understanding of the effect of environment and ionic strength on the p
K values of histidine residues in proteins. The salt-dependence of p
K ...values for two histidine residues in ribonuclease Sa (RNase Sa) (pI=3.5) and a variant in which five acidic amino acids have been changed to lysine (5K) (pI=10.2) was measured and compared to p
K values of model histidine-containing peptides. The p
K of His53 is elevated by two pH units (p
K=8.61) in RNase Sa and by nearly one pH unit (p
K=7.39) in 5K at low salt relative to the p
K of histidine in the model peptides (p
K=6.6). The p
K for His53 remains elevated in 1.5
M NaCl (p
K=7.89). The elevated p
K for His53 is a result of screenable electrostatic interactions, particularly with Glu74, and a non-screenable hydrogen bond interaction with water. The p
K of His85 in RNase Sa and 5K is slightly below the model p
K at low salt and merges with this value at 1.5
M NaCl. The p
K of His85 reflects mainly effects of long-range Coulombic interactions that are screenable by salt. The tautomeric states of the neutral histidine residues are changed by charge reversal. The histidine p
K values in RNase Sa are always higher than the p
K values in the 5K variant. These results emphasize that the net charge of the protein influences the p
K values of the histidine residues. Structure-based p
K calculations capture the salt-dependence relatively well but are unable to predict absolute histidine p
K values.
The p
K values of the titratable groups in ribonuclease Sa (RNase Sa) (pI=3.5), and a charge-reversed variant with five carboxyl to lysine substitutions, 5K RNase Sa (pI=10.2), have been determined ...by NMR at 20
°C in 0.1
M NaCl. In RNase Sa, 18 p
K values and in 5K, 11 p
K values were measured. The carboxyl group of Asp33, which is buried and forms three intramolecular hydrogen bonds in RNase Sa, has the lowest p
K (2.4), whereas Asp79, which is also buried but does not form hydrogen bonds, has the most elevated p
K (7.4). These results highlight the importance of desolvation and charge–dipole interactions in perturbing p
K values of buried groups. Alkaline titration revealed that the terminal amine of RNase Sa and all eight tyrosine residues have significantly increased p
K values relative to model compounds.
A primary objective in this study was to investigate the influence of charge–charge interactions on the p
K values by comparing results from RNase Sa with those from the 5K variant. The solution structures of the two proteins are very similar as revealed by NMR and other spectroscopic data, with only small changes at the N terminus and in the α-helix. Consequently, the ionizable groups will have similar environments in the two variants and desolvation and charge–dipole interactions will have comparable effects on the p
K values of both. Their p
K differences, therefore, are expected to be chiefly due to the different charge–charge interactions. As anticipated from its higher net charge, all measured p
K values in 5K RNase are lowered relative to wild-type RNase Sa, with the largest decrease being 2.2 pH units for Glu14. The p
K differences (p
K
Sa−p
K
5K) calculated using a simple model based on Coulomb's Law and a dielectric constant of 45 agree well with the experimental values. This demonstrates that the p
K differences between wild-type and 5K RNase Sa are mainly due to changes in the electrostatic interactions between the ionizable groups. p
K values calculated using Coulomb's Law also showed a good correlation (
R=0.83) with experimental values. The more complex model based on a finite-difference solution to the Poisson–Boltzmann equation, which considers desolvation and charge–dipole interactions in addition to charge–charge interactions, was also used to calculate p
K values. Surprisingly, these values are more poorly correlated (
R=0.65) with the values from experiment. Taken together, the results are evidence that charge–charge interactions are the chief perturbant of the p
K values of ionizable groups on the protein surface, which is where the majority of the ionizable groups are positioned in proteins.
The abundant surface glycolipid, lipophosphoglycan (LPG), of Leishmania promastigotes is composed of phosphosaccharide repeating units linked via a phosphosaccharide core to a conserved lyso ...alkylphosphatidylinositol membrane anchor. It is shown in this paper that monoclonal antibodies (mAbs) directed against LPG also react with an acid phosphatase secreted by L. mexicana promastigotes. Acid phosphatase purified by column chromatography (apparent Mr = 100,000) reacts in immunoblots with the anti-LPG mAb AP3 and another mAb, L3.13, which does not recognize LPG. mAb L3.13 was used to purify the enzyme by affinity chromatography. The resulting glycoprotein has the same molecular weight and binds AP3 on immunoblots. The secreted phosphatase is non-covalently associated with a high molecular weight, galactose-containing glycan or proteoglycan that is recognized by both AP3 and L3.13. In addition to acid phosphatase, other parasite proteins appear to be modified by LPG epitopes.
