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K W Olsen

Publications and source records attributed to K W Olsen.

At least 19 recordsLinked to original sources
PubMed2000

Crystal structure of Lysbeta(1)82-Lysbeta(2)82 crosslinked hemoglobin: a possible allosteric intermediate.

The crystal structure of human hemoglobin crosslinked between the Lysbeta82 residues has been determined at 2.30 A resolution. The crosslinking reaction was performed under oxy conditions using bis(3, 5-dibromosalicyl) fumarate; the modified hemoglobin has increased oxygen affinity and lacks cooperativity. Since the crystallization occurred under deoxy conditions, the resulting structure displays conformational characteristics of both the (oxy) R and the (deoxy) T-states. beta82XLHbA does not fully reach its T-state conformation due to the presence of the crosslink. The R-state-like characteristics of deoxy beta82XLHbA include the position of the distal Hisbeta63 (E7) residue, indicating a possible reason for the high oxygen affinity of this derivative. Other areas of the molecule, particularly those thought to be important in the allosteric transition, such as Tyrbeta145 (HC2) and the switch region involving Proalpha(1)44 (CD2), Thralpha(1)41 (C6) and Hisbeta(2)97 (FG4), are in intermediate positions between the R and T-states. Thus, the structure may represent a stabilized intermediate in the allosteric transition of hemoglobin.

Allosteric Regulation↗
PubMed1999

Nuclear magnetic resonance and oxygen affinity study of cesium binding in human erythrocytes.

We investigated the interaction of the cesium ion (Cs(+)) with the anionic intracellular components of human red blood cells (RBCs); the components studied included 2,3-bisphosphoglycerate (BPG), ADP, ATP, inorganic phosphate (P(i)), carbonmonoxy hemoglobin (COHb), and RBC membranes. We used spin-lattice (T(1)) and spin-spin (T(2)) (133)Cs NMR relaxation measurements to probe Cs(+) binding, and we found that Cs(+) bound more strongly to binding sites in BPG and in RBC membranes than in any other intracellular component in RBCs at physiologic concentrations. By using James-Noggle plots, we obtained Cs(+) binding constants per binding site in BPG (66 +/- 8 M(-1)), ADP (19 +/- 1 M(-1)), ATP (25 +/- 3 M(-1)), and RBC membranes (55 +/- 2 M(-1)) from the observed T(1) values. We also studied the effect of Cs(+) on the oxygen (O(2)) affinity of purified Hb and of Hb in intact RBCs in the absence and in the presence of BPG. In the absence of BPG, the O(2) affinity of Hb decreased upon addition of Cs(+). However, in the presence of BPG, the O(2) affinity of Hb increased upon addition of Cs(+). The O(2) affinity of Cs(+)-loaded human RBCs was larger than that of Cs(+)-free cells at the same BPG level. (31)P NMR studies on the pH dependence of the interaction between BPG and Hb indicated that the presence of Cs(+) resulted in a smaller fraction of BPG available to bind to the cleft of deoxyHb. Our NMR and O(2) affinity data indicate that a strong binding site for Cs(+) in human RBCs is BPG. A partial mechanism for Cs(+) toxicity might arise from competition between Cs(+) and deoxyHb for BPG, thereby increasing oxygenation of Hb in RBCs, and thus decreasing the ability of RBCs to give up oxygen in tissues. The presence of Cs(+) at 12.5 mM in intact human RBCs containing BPG at normal concentrations did not, however, alter significantly the O(2) affinity of Hb, thus ruling out the possibility of Cs(+)-BPG interactions accounting for Cs(+) toxicity in this cell type.

2,3-Diphosphoglycerate↗
PubMed1996

Tris(3,5-dibromosalicyl) tricarballylate crosslinked hemoglobin: functional evaluation.

Both oxy and deoxy human hemoglobin A were crosslinked with tris(3,5-dibromosalicyl) tricarballylate. The major species from both reactions contained an inter-subunit crosslink. The denaturation transition (Tm) of the oxy crosslinked hemoglobin increased 14.5 degrees C and that of deoxy crosslinked hemoglobin, 13.0 degrees C. The apparent rate constant (kapp) of autoxidation for oxy crosslinked hemoglobin remained the same as native hemoglobin but that of the deoxy crosslinked hemoglobin increased by 34%. The higher oxygen affinity and lower cooperativity of the crosslinked proteins compared with native hemoglobin indicated that the crosslink shifted the conformation to the R state.

Allosteric Regulation↗
PubMed1994

The modification of hemoglobin by a long crosslinking reagent: bis(3,5-dibromosalicyl) sebacate.

Bis(3,5-dibromosalicyl) sebacate is a bifunctional protein crosslinking reagent. It reacts with oxy form of human hemoglobin A to produce a crosslinked hemoglobin between two beta chains in the 2,3-bisphosphoglycerate binding cleft, decreasing the oxygen affinity. The oxygen binding curve of crosslinked hemoglobin had a P50 of 18.5 mmHg compared to a P50 of 11.0 mmHg for native hemoglobin and remains highly cooperative, n = 2.2. Crosslinking hemoglobin between the two beta chains also results in a 12.5 degrees C increase in the thermal denaturation temperature. The crosslinked hemoglobin is oxidized more rapidly to methemoglobin. Its autoxidation rate in 0.01 M MOPS, pH 7.4, at 37 degrees C was 1.4 times as fast as that of Hb A.

2,3-Diphosphoglycerate↗
PubMed1994

Enzymatic protection from autoxidation for crosslinked hemoglobins.

The autoxidation rates of hemoglobins crosslinked between the alpha subunits (alpha 99XLHb A) and between the beta subunits (beta 82XLHb A) were reduced in the presence of catalase and/or superoxide dismutase. In the presence of catalase the rate for alpha 99XLHb A decreased 2.3 fold and for beta 82XLHb A, 1.9 fold. Superoxide dismutase reduced the rate 1.6 fold for alpha 99XLHb A and 1.8 fold for beta 82XLHb A. In the presence of both catalase and superoxide dismutase the rate of autoxidation decreased by 3.0 fold in alpha 99XLHb A and 4.0 fold in beta 82XLHb A. The presence of catalase and superoxide dismutase or both in the crosslinked hemoglobin samples increases the autoxidation half-life of oxyhemoglobins. This suggests that crosslinked hemoglobins to be used as blood substitutes could be protected from oxidation in storage by these enzymes.

Aspirin↗
PubMed1992

Stabilities and properties of multilinked hemoglobins.

Crosslinking of both human and dog hemoglobin has been done with a variety of reagents to produce singly, doubly and multiply crosslinked hemoglobins. Succinate and glutarate diaspirins did not crosslink deoxy human hemoglobin in good yield, in contrast to the fumarate analog (DBSF). Deoxy dog Hb did not react well with DBSF, but oxy dog Hb did react, giving crosslinked tetramers as well as dimers on SDS electrophoresis. Crosslinking with a short, rigid reagent (difluorodinitrobenzene) resulted in a similar product for both oxy and deoxy hemoglobin that had high stability and oxygen affinity. The trilinker, tris-chloroethylamine, produced a more stable product than the corresponding crosslinker, bis-chloroethylamine. Double crosslinking oxy Hb with DBSF and dimethylpimelimidate or with DBSF followed by deoxygenation and recrosslinking with DBSF gave products with higher denaturation temperatures. The diaspirin double crosslinked product had high oxygen affinity.

Animals↗
PubMed1991

Crystallization and preliminary X-ray analysis of peanut agglutinin-N6-benzylaminopurine complex.

Preliminary diffraction data collected on peanut agglutinin (PNA) crystals grown in the presence of N6-benzylaminopurine (BAP) indicate a monoclinic cell (P2) with a = 67.0 A, b = 35.2 A, c = 65.8 A and beta = 68.6 degrees. This is the first example of a legume lectin crystallized with a bound phytohormone. Crystals of PNA grown previously in the presence of lactose had an orthorhombic space group (P2(1)2(1)2) with a = 128.8 A, b = 126.0 A and c = 76.1 A and one tetramer per asymmetric unit. The Vm value for the PNA-BAP crystals is 2.62 A3/Da, assuming one monomer of PNA per asymmetric unit. Thus, while the PNA-lactose complex crystallized as tetramers, the PNA-BAP complex has, at most, dimers in the crystal. These results indicate that BAP, a naturally occurring phytohormone, can modify the quaternary structure of PNA by dissociation and change its carbohydrate valence.

Adenine↗
PubMed1991

Thermal stability of hemoglobin crosslinked in the T-state by bis(3,5-dibromosalicyl) fumarate.

Bis(3,5-dibromosalicyl) fumarate was used to crosslink oxyhemoglobin between Lys 82 beta 1 and Lys 82 beta 2 (Walder, J. A., et al. (1979) Biochemistry 18, 4265) and deoxyhemoglobin between Lys 99 alpha 1 and Lys 99 alpha 2 (Chatterjee R.Y., et al. (1986) J. Biol. Chem. 261, 9929). Thermal denaturations demonstrated that alpha crosslinked hemoglobin (alpha 99XLHb A) has the same stability as the beta crosslinked one (beta 82XLHb A). Both alpha and beta crosslinked methemoglobins have a denaturation temperature in 0.9 M guanidine of 57 degrees C compared to 41 degrees C of Hb A. The second product from the T-state crosslinking reaction was found to be crosslinked between the beta chains by chain separation and amino acid analysis. The possible positions for this crosslink are limited to the bisphosphoglycerate binding site in the three-dimensional structure. Its stability is comparable to that of the alpha 99XLHb A or beta 82XLHb A. These modified hemoglobins are potential blood substitutes.

Amino Acids↗
PubMed1989

The effect of crosslinking by bis(3,5-dibromosalicyl) fumarate on the autoxidation of hemoglobin.

Bis(3,5-dibromosalicyl) fumarate was used to crosslink hemoglobin both in the oxy and deoxy states. This double headed diaspirin was known to crosslink oxy Hb A selectively between Lys 82 beta 1 and Lys 82 beta 2 (Walder, J. A., et al. (1979) Biochemistry 18, 4265) and deoxy Hb A between Lys 99 alpha 1 and Lys 99 alpha 2 (Chatterjee R. Y., et al. (1986) J. Biol. Chem. 261, 9929). The autoxidation at 37 degrees C of oxy alpha 99 crosslinked hemoglobin was found to be 1.8 times as fast as that of Hb A while that of the oxy beta 82 crosslinked hemoglobin was only 1.2 times as fast. After 5 hours the formation of methemoglobin in the alpha crosslinked Hb A is 21.3% compared to 10.8% in beta crosslinked Hb A and 6.4% in Hb A. These results may effect the proposed use of alpha 99 crosslinked hemoglobin as a blood substitute by demonstrating the need for protection from autoxidation during storage.

Aspirin↗
PubMed1989

Thermal stability and cross-linking of Hb New York [beta 113(G15)Val----Glu].

Hb New York [beta 113(G15)Val----Glu] has been cross-linked with bis (3,5-dibromosalicyl) fumarate, a reagent known to cross-link Lys 82 beta 1 and Lys 82 beta 2. Thermal denaturations of met Hb New York and its derivative have been compared to those of the corresponding Hb A samples. The structural transitions, observed as absorbance changes at 418 nm, were at 40.2 degrees C for Hb New York, 42.2 degrees C for Hb A, 53.7 degrees C for cross-linked Hb New York, and 56.2 degrees C for cross-linked Hb A. Transitions observed at 280 nm were approximately 2 degrees C higher. Thus, a single inter-subunit cross-link can stabilize an abnormal hemoglobin. A model of Hb New York in which Glu beta 113 forms a salt bridge to His beta 117 can explain the small changes in both the stability and the electrophoretic mobility of this protein.

Aspirin↗
PubMed1988

Hemoglobin Brockton [beta 138 (H16) Ala----Pro]: an unstable variant near the C-terminus of the beta-subunits with normal oxygen-binding properties.

Hemoglobin Brockton [beta 138 (H16) Ala----Pro] is an unstable variant associated with a mild anemia. It has the same electrophoretic mobility as and cannot be resolved from Hb A. Oxygen affinity measurements of blood and hemolysate do not indicate biphasic oxygen saturation, showing that the functional properties of the variant are very similar to those of Hb A. This implies that the introduction of proline into the H-helix at position 138 does not disrupt the critical inter- and intrasubunit hydrogen bonds and salt bridges at the beta carboxyl-terminal dipeptide, since these polar interactions are essential for the normal oxygen-binding properties of hemoglobin. X-ray crystallographic data are consistent with these findings and show that the consequences of the beta 138 Ala----Pro substitution are almost entirely confined to the immediate vicinity of the mutation site. Instability probably results from the inability of a buried hydrogen bond to form between Pro 138 beta and Val 134 beta.

Amino Acid Sequence↗
PubMed1988

Effects of crosslinking on the thermal stability of hemoglobins. II. The stabilization of met-, cyanomet-, and carbonmonoxyhemoglobins A and S with bis(3,5-dibromosalicyl) fumarate.

Hemoglobins A and S were crosslinked between Lys 82 beta 1 and Lys 82 beta 2 using bis (3,5-dibromosalicyl) fumarate (J. A. Walder et al. (1979) Biochemistry 18, 4265). Thermal denaturation experiments were used to compare the stabilities of the met, cyanomet, and carbonmonoxy forms of these crosslinked hemoglobins to the corresponding uncrosslinked proteins. Uncrosslinked carbonmonoxy- and cyanomethemoglobins had transition temperatures about 11 degrees C higher than the corresponding met samples. The increase in denaturation temperature (Tm) due to crosslinking was 15 degrees C for the methemoglobins, 10 degrees C for the cyanomethemoglobins, and 4 degrees C for the carbonmonoxy ones. There was no significant difference in stability between the met and carbonmonoxy crosslinked proteins. In order of increasing stability the samples were: met Hb S less than met Hb A less than CO Hb S less than CO Hb A = CN-met Hb A less than met XL-Hb S = CO XL-Hb S less than met XL-Hb A = CO XL-Hb A less than CN-met XL-Hb A. The slight decrease in the stability of Hb S (beta 6 Glu----Val) compared to Hb A can be explained by the replacement of an external ionic group by a hydrophobic residue in Hb S. In mixtures of crosslinked and normal Hb A, the Tm of the uncrosslinked material was slightly increased by the presence of the more stable crosslinked hemoglobin. The effects of both crosslinking and cyanide or carbon monoxide binding can be explained by Le Chatelier's principle since both would favor the native form of the protein.

Aspirin↗
PubMed1988

A spectrophotometric assay for the degradation of the siderophore deferrioxamine B.

A spectrophotometric assay using ferric perchlorate in a perchloric acid solution has been developed to monitor the degradation of the trihydroxamate siderophore deferrioxamine B to monohydroxamates. Using the ferric perchlorate solution and employing various concentrations of acetohydroxamic acid (as the model monohydroxamic acid) while maintaining a constant amount of deferrioxamine B resulted in the shifting of the absorption maximum from that of ferrioxamine B to longer wavelengths and toward that of a pure ferri-acetomonohydroxamic acid solution. A similar result was noted when a cell-free extract, from a bacterium capable of using deferrioxamine B as its sole carbon source, was given the siderophore in a phosphate buffer and aliquots of the enzyme-deferrioxamine B solution were removed for analysis. The assay may thus be used to monitor the formation of the monohydroxamic acid degradation products of the siderophore by the enzyme(s) in the cell-free extract.

Deferoxamine↗
PubMed1987

Effects of crosslinking on the thermal stability of hemoglobin. I. The use of bis(3,5-dibromosalicyl) fumarate.

The double-headed aspirin, bis(3,5-dibromosalicyl) fumarate, has been used to crosslink hemoglobin A between Lys 82 beta 1 and Lys 82 beta 2 (J. A. Walder et al. (1979) Biochemistry 18,4265). Denaturation experiments were used to compare the stability of this crosslinked protein to that of hemoglobin A. Thermal denaturations, done in 0.01 M 4-morpholine-propanesulfonic acid, pH 7, containing 0.9 M guanidine to prevent precipitation at high temperatures, were monitored by changes in absorbance between 190 and 650 nm using a diode array spectrophotometer. The sample was heated from 25 to 70 degrees C at 0.3 degrees C/min. The data were analyzed by using both a two-state model and a novel first derivative method. As expected, methemoglobin A had a single, broad transition with a midpoint of 40.7 degrees C. The crosslinked methemoglobin showed a transition at 57.1 degrees C. Two minor transitions, one of which was apparently due to residual unmodified hemoglobin, were also observed in the crosslinked sample. Thus, a single crosslink between only two of the four subunits can lead to a significantly more stable molecule. These results can be explained by Le Chatelier's principle, since crosslinking prevents dissociation of the beta-subunits and, thereby, holds the entire tetramer together.

Aspirin↗