A comparison of liquid hydrocarbon calibration standards in piston cylinders and standard cylinders with eductor tubes.

Natural gas liquid standards consist of various components primarily in the C1-C6 range; they are available in three types of cylinder packages: piston cylinders and two other types of standard cylinders with eductor tubes. The three cylinder packages have different operation conditions and thus a sample may behave differently in each of the packages. The piston cylinder maintains the components in a single liquid phase at a constant pressure, while the standard cylinders maintain the components as a two-phase mixture. Typically, the components may distribute between the two phases in different concentrations depending on the various thermodynamic variables, such as temperature, pressure, and volume. This study will examine the integrity of the sample in the three cylinder packages during a controlled cylinder depletion. The results for the various cylinders will be compared with a theoretical model of the experiments.

Changes in the functional properties of bovine hemoglobin induced by covalent modification with polyethylene glycol.

Polyethylene glycol conjugation to proteins and peptides (PEGylation) has been shown to promote increased retention time in the circulation as well as to blunt immune or allergic reactions. PEGylated bovine hemoglobin (PEG-Hb) is being explored in human clinical trials as an oxygen delivering agent for the sensitization of solid tumors to radiation therapy. In this study the functional properties of PEG-Hb were compared to those of bovine hemoglobin (Hb), the mutant human hemoglobin Rothchild and bovine hemoglobin crosslinked between the beta chains. The rate of heme transfer from Hb to serum albumin at pH 9.0 was greatly increased by PEGylation, suggesting destabilization of the heme-globin linkage and of the bonds between alpha beta dimers. Measurement of oxygen binding equilibrium showed that the oxygen affinity of Hb became unusually dependent on temperature and Hb concentration after PEGylation. Evidence is presented to suggest that PEGylation of lysine beta-81 at the entrance to the central cavity of the Hb tetramer might be responsible for these observations. The alterations of the functional properties of Hb induced by PEGylation are consistent with the beneficial effects of PEG-Hb in exchange transfusion and radiation sensitization models of human conditions.

Coupled reactions in hemoglobin. Heme-globin and dimer-dimer association.

Five different human hemoglobins were used to test the postulate that dissociation of hemoglobin (Hb) tetramers into alpha beta dimers and dissociation of heme from globin are linked reactions. Spectrophotometric measurements of the initial rate of heme transfer from Hb to serum albumin were made over a 3000-fold range of Hb concentration and yielded the heme-globin dissociation rate constant for tetramers and that for dimers. The tetramer-dimer dissociation constant (K4,2) could then be calculated from the rate constant at intermediate concentrations. The values obtained for the five hemoglobins, spanning a 250-fold range in K4,2, were in good agreement with those found by direct methods. The relation between this new linkage reaction of hemoglobin and the classical ones, such as the reciprocal relation between the binding of oxygen and protons, is discussed briefly.

Quantitative transformation of hemoglobin into stable tetramers.

A method is described for the preparation of human or bovine hemoglobin with a covalent bridge, formed by bispyridoxal-tetraphosphate, between the beta chains. The yield is 95% of the total hemoglobin. The location of the two molecules of bispyridoxal-tetraphosphate in the tetramer has been established. The functional properties of the cross-linked hemoglobin, its stability, and particularly, the simplicity of the method for its preparation, make it a promising candidate for an acellular blood substitute.

Hemoglobin tetramers stabilized by a single intramolecular cross-link.

A specific intramolecular cross-link was introduced into bovine and human hemoglobin by reaction of the deoxyhemoglobin with the dialdehyde, bispyridoxal tetraphosphate (bisPL)P4, followed by reduction with NaBH4. The yield of cross-linked hemoglobin is 80% in both cases, using 1 mol of (bisPL)P4 per mol of Hb. The crosslink is confined to the beta chains, where it connects the N-terminal residue (valine and methionine, respectively) to a lysine on the other beta chain across the central cavity. The stereochemical requirements for the reaction were probed by using a rigid analogous cross-linking reagent, as well as with a mutant Hb, which has a shorter distance between the residues to which the cross-link is attached. Introduction of the cross-link into human and bovine Hb results in a five-fold and four-fold reduction in the oxygen affinity and a decrease in the Bohr Effect by 1/3 and 1/2, respectively. Oxygenation remains cooperative, albeit with a decreased Hill coefficient. The cross-linked hemoglobins are oxidized more rapidly to the ferric form, but their resistance to heat denaturation is increased. The stability of the link between the beta chains and their hemes is 10 times greater in both cross-linked hemoglobins that in their native counterparts. The possible application of this chemical modification for the preparation of hemoglobin-based blood substitutes is discussed.

The stability of the heme-globin linkage in some normal, mutant, and chemically modified hemoglobins.

The rates and equilibria of heme exchange between methemoglobin and serum albumin were measured using a simple new spectrophotometric method. It is based on the large difference between the spectrum of methemoglobin and that of methemealbumin at pH 8-9. The rate of heme exchange was found to be independent of the albumin concentration and inversely proportional to the hemoglobin (Hb) concentration. Taken together with the finding that the rate was 10 times greater for Hb Rothschild, which is completely dissociated into alpha beta dimers and 10 times smaller for two cross-linked hemoglobins, the subunits of which cannot dissociate, this showed that the rate of dissociation of heme from alpha beta dimers is very much greater than from tetramers. Conditions were found for the attainment of an equilibrium distribution of hemes between beta globin and albumin. The equilibrium distribution ratio, R = methemealbumin/albumin/methemoglobin/apohemoglobin, for hemoglobin A was 3.4 with human and 0.005 with bovine serum albumin. Both the rates of exchange and the R values of HbS and HbF were the same as that for HbA. The equilibrium distribution ratio for Hb Rothschild was 7 times greater than that for HbA whereas that of one but not the other of the cross-linked hemoglobins was 10 times smaller. The structural bases for these differences are analyzed.

Functional properties of a new crosslinked hemoglobin designed for use as a red cell substitute.

A new crosslinking agent, bis-pyridoxal tetraphosphate, (bis-PL)P4, was used to prevent dissociation of the hemoglobin (Hb) tetramer. Yields in excess of 75 percent of intramolecularly crosslinked (bis-PL)P4Hb have been obtained using stoichiometric amounts of the crosslinking reagent. Some functional properties of (bis-PL)P4Hb have been evaluated in vitro and in vivo. Oxygen affinity was substantially reduced (p50 = 31 torr at 37 degrees C, pH 7.4, pCO2 = 40 torr), while the Bohr coefficient was -0.27 of H+ per mol of O2. Owing to its right-shifted dissociation curve, (bis-PL)P4Hb still yielded a p50 of 15 torr at a low temperature (16 degrees C), as compared with only 3 torr for normal adult Hb (HbA). Clearance of (bis-PL)P4Hb from plasma was significantly delayed (t1/2 = 171 min, at a dose of 0.2 g/kg of body weight compared with that of HbA (t1/2 = 54 min). Heart rate, mean arterial blood pressure, and respiration remained stable or returned to normal values within hours after bolus injection of the hemoglobin. The (bis-PL)P4Hb was not excreted in the urine, in contrast to HbA (21% of the total dose of HbA appeared in the urine within the first 2 hrs). These results show that the covalent beta-beta crosslink prevents the renal excretion of (bis-PL)P4Hb, thereby significantly prolonging vascular retention. These properties, together with an increased ability to unload O2, make (bis-PL)P4Hb a promising new candidate as a red cell substitute.

Bis-pyridoxal polyphosphates: a new class of specific intramolecular crosslinking agents for hemoglobin.

A series of compounds related to bis-pyridoxal phosphate has been synthesized and used to crosslink deoxyhemoglobin. The yield of crosslinked hemoglobin increased dramatically from about 15% for the di- or triphosphate to about 70% for the tetraphosphate. The site of attachment of the intramolecular crossbridge was found to be from the N-terminal amino group of one beta chain to lysine 82 of the other. Since the distance between these residues is only 11A, the bis-pyridoxal tetraphosphates probably have a "stacked" conformation. The crosslinked hemoglobins bind oxygen cooperatively but with a greatly decreased affinity. The increased ability to unload oxygen together with the stabilization of the tetramer qualifies them as promising cell-free blood substitutes.

p-Aminobenzoylpolyglutamates with hydrophobic end groups. A new class of inhibitors of hemoglobin S polymerization.

We have synthesized and tested a new series of compounds that inhibit the polymerization of deoxyhemoglobin S by noncovalent interaction. They consist of three structural elements: A p-aminobenzoyl residue to anchor the compound in the central cavity between the beta chains, a number of glutamates in gamma linkage to provide tight binding, and one or two hydrophobic amino acid residues which block the intermolecular hydrophobic interaction of valine beta 6. The most active compound was p-aminobenzoyl-(gamma-Glu)5-Phe-Phe. It increases the solubility of deoxy-HbS by a factor of 1.3 at a concentration of only 5-6 mM and is effective even in the presence of physiological concentrations of 2,3-diphosphoglycerate.

Binding of diphosphoglycerate and ATP to oxyhemoglobin dimers.

The relative affinity of diphosphoglycerate and ATP for hemoglobin dimers and tetramers can be measured under conditions where the protein is in large molar excess over the polyphosphate. Binding of both compounds to dimers was about 25 times stronger than to tetramers in the case of the three low-spin hemoglobins, oxyhemoglobin, carboxyhemoglobin and cyanomethemoglobin. The mutation in hemoglobin Kansas leads to an increased dissociation into alpha beta dimers. The increase in diphosphoglycerate binding by this hemoglobin was in good agreement with that expected from the dimer-tetramer dissociation constant over a wide range of hemoglobin concentrations. In contrast to the liganded hemoglobins, both deoxyhemoglobin and aquomethemoglobin bind the two polyanions as tetramers.

The interaction of folylpolyglutamates with deoxyhemoglobin. Identification of the binding site.

Previous studies have shown that pteroylheptaglutamate (PteGlu7) can form a 1:1 complex with deoxyhemoglobin. The solution and crystallographic studies reported in this paper delineate the nature of the PteGlu7 binding site. We find that the three structural elements of PteGlu7 (the pteridine moiety, the p-aminobenzoyl portion, and the glutamate groups) each contribute to the binding energy by interacting with residues in the central cavity between the beta subunits and with residues at the alpha 1 beta 1 interface. Identification of the 2,3-diphosphoglycerate (DPG) binding site as part of the PteGlu7 binding site was accomplished in two ways; first by the demonstration of reduced PteGlu7 binding to hemoglobin selectively modified by pyridoxylation at this site, and second by the finding that DPG and PteGlu7 bind to deoxyhemoglobin in a competitive manner. In addition, since analogs of PteGlu7 in which the pteridine moiety is modified display reduced binding, it can be concluded that the pteridine group also contributes significantly to the binding energy. The crystallographic studies are completely consistent with the results determined in solution. A difference electron density image at 4.3 A resolution shows that the pteridine and p-aminobenzoyl groups are nestled against an interior edge of the alpha 1 beta 1 interface with the pteridine ring interacting with Phe 36 alpha 1 and the p-aminobenzoyl group positioned against a portion of the H helix between residues Lys 132 beta 1 and Ala 135 beta 1. The difference density for the glutamate residues is less well resolved (for reasons described in the text), but it is clear that some of the carboxylate side chains must interact with residues at the DPG binding site.

The binding of folyl- and antifolylpolyglutamates to hemoglobin.

A binding method that detects only the strongest binding site for a ligand on a protein has been used to show that folates and folate analogs, conjugated with poly-gamma-glutamates, are bound to hemoglobin. When the concentration of hemoglobin is much larger than that of the polyglutamate, as is the case in the red cell, the fraction bound is a direct function of the hemoglobin concentration and is independent of the total polyglutamate concentration. Binding to deoxyhemoglobin tetramers is competitive with 2,3-diphosphoglycerate. In oxyhemoglobin the folyl and methotrexate polyglutamates are bound preferentially by free alpha beta dimers, but removal of the pteridine moiety leads to tetramer binding even in oxyhemoglobin. Changes in the length of the polyglutamate side chain and alterations of the pteridine structure such as reduction and/or methylation have a much larger effect on the constant for binding to deoxyhemoglobin tetramers than on that for oxyhemoglobin dimers. The implications of these results for the storage of pteroylpolyglutamates in the erythrocyte and their release from the red cell under the influence of the degree of oxygenation and variations in the 2,3-diphosphoglycerate level are discussed.

Changes in the Bohr effect due to pyridoxylation of the alpha-chain terminal amino groups of hemoglobin.

Deoxyhemoglobins substituted with pyridoxal 5'-phosphate or pyridoxal 5'-deoxymethylenephosphonate at the N-terminal amino groups of the alpha-chains were investigated by 31P-NMR spectroscopy. Titration curves of the 5'-side-chains show a substantial increase in acid strength in alpha-pyridoxylated deoxyhemoglobins when compared to the corresponding CO-liganded hemoglobins. These derivatives therefore contain a new oxygenation-linked acid group which opposes the normal Bohr effect. The loss in stabilization of the monoanion of the phosphate or phosphonate group derived from the three-dimensional structure can account for the lower pK of this ionization in deoxyhemoglobin as compared to CO-liganded hemoglobin. The reduction in the Bohr effect caused by modification of the alpha-chains with pyridoxal 5'-deoxymethyl-enephosphonate is quantitatively equal to the expected contribution of alpha-chain N-terminal amino groups.

Enhanced oxygen unloading by an interdimerically crosslinked hemoglobin in an isolated perfused rabbit heart.

Coronary perfusion has shown that an intramolecularly crosslinked hemoglobin (Hb) with a very low affinity for O2 (Hb crosslinked covalently between the beta chains with 2-nor-2-formylpyridoxal 5'-phosphate, HbXL) has several advantages over ordinary Hb. As predicted from in vitro oxygenation curves, much more O2 was unloaded to the heart at three different heart rates, at two perfusion rates, and when the perfusate was equilibrated with 25% as well as 95% O2. In all cases, the improved O2 unloading occurred at higher tissue O2 pressures than with normal Hb. The greater O2 consumption with HbXL was accompanied by better mechanical performance because, after 90 min of perfusion, the HbXL-perfused hearts maintained two-thirds of their original contractility (dp/dt), while that of the Hb-perfused hearts had declined to one-fifth. A special advantage of HbXL is its ability to unload significant amounts of O2 even at low temperature (10 degrees C), in contrast to whole blood. This should make it useful for supporting aerobic metabolism during low-temperature cardioplegia in cardiac surgery and for organ preservation.

A pteroylpolyglutamate binds to tetramers in deoxyhemoglobin but to dimers in oxyhemoglobin.

The binding of a physiological concentration of pteroylhepta(glutamate) to oxy- and deoxyhemoglobin in large excess was measured by ultrafiltration. The variation of free to bound folate with hemoglobin concentration showed that a single molecule of the pteroylpolyglutamate is bound by deoxyhemoglobin tetramers and by alpha beta dimers in oxyhemoglobin. Although the binding sites are different, the affinity constants are the same and very similar to the 2,3-bisphosphoglycerate binding energy. Nevertheless, in view of the small proportion of dimers in oxyhemoglobin much more pteroylhepta(glutamate) is bound by deoxyhemoglobin over a wide range of hemoglobin concentrations. Because even 2% deoxyhemoglobin is enough to bind all of the erythrocyte folate as polyglutamate, the bulk of it will be bound at physiological oxygen pressures. Free folate could only be expected in fully oxygenated erythrocytes. Therefore, the reaction of pteroylpolyglutamates with hemoglobin represents an oxygenation-dependent storage mechanism that can account for the 40-fold excess of the vitamin in the erythrocyte over the amounts in the serum. Because methotrexate is also converted to polyglutamate derivatives in the erythrocyte, this drug is likely to be concentrated and stored there by the same mechanism.

Specific receptor sites for pyridoxal 5'-phosphate and pyridoxal 5'-deoxymethylenephosphonate at the alpha and beta NH2-terminal regions of hemoglobin.

hemoglobins have been prepared which are substituted with pyridoxal 5'-phosphate or pyridoxal 5'-deoxymethylenephosphonate at both NH2-terminal amino groups of either the alpha or the beta chains. 31P NMR titration curves of the 5' side chain show a substantial decrease in acid strength in hemoglobins pyridoxylated at the alpha chains and the reverse effect in the hemoglobins labeled in the beta chains. These changes can readily be explained by the interaction of the 5' side chains with surrounding residues which provide specific binding sites for the monoanion at the alpha chain termini and for the dianion in the case of the beta chains. Since the proportion of monoanion is at lest ten times greater for pyridoxal 5'-deoxymethylenephosphonate than for pyridoxal 5'-phosphate at neutral pH, the phosphonate reacts preferentially with the alpha chain NH2-terminal amino groups and the phosphate with the beta chain ones.