Carlyle B. Storm

A rapid and convenient preparation of apocarbonic anhydrase

Abstract A method for the rapid removal of Zn(II) from carbonic anhydrase using pyridine-2,6-dicarboxylic acid is reported. Either flow dialysis or direct equilibration with the chelating agent followed by gel filtration permits removal of >97% of the Zn(II) in less than 2 hr. This represents an improvement of about two orders of magnitude over currently used methods in the time required for the preparation of apocarbonic anhydrase.

A possible approach to the investigation of the structures of copper proteins: 1H n.m.r. spectra of azurin

The 1H n.m.r. spectra of apo-, Cu(I) and Cu(II) azurins from Pseudomonas aeruginosa have been measured. Three of the four histidines have been assigned. The effect of the copper(II) ion acting as an intrinsic paramagnetic perturbant leads to the proposal that one of the histidines is far from the metal and another is closer, but not bound, to the copper. The possibility that the remaining two histidines are ligands to the copper is considered. The relationship to the sequence is discussed.

The proton magnetic resonance spectra of a cobalt (II) azurin.

Abstract The proton magnetic resonance spectrum of a cobalt(II) derivative of Pseudomonas aeruginosa azurin is reported. The temperature dependence of 26 resonances is described together with a study of the pH∗ titration behaviour over a range 4.7 to 9.3. A few resonances are observed shifted by more than 30 ppm from their diamagnetic positions. Of the remainder most extrapolate to the aliphatic region at T = ∞. Two lines are assigned to the C2 and C4 protons of a freely titrating histidine residue far from, and only slightly affected by, the Co(II) centre. A further two lines are assigned to the C2 hydrogen of protonated and deprotonated forms of a histidine residue in slow exchange with bulk aquaeous protons and closer to, but not bound to, the cobalt. The structure of the protein in the vicinity of the paramagnetic centre is found to be essentially insensitive to pH∗ over the range 4.7 to 9.3.

Cobalt (III) carboxypeptidase A: Preparation and esterase activity

Abstract Co(II) carboxypeptidase A has been oxidized to Co(III) carboxypeptidase A with hydrogen peroxide. The resultant metalloprotein has an absorption spectrum different from that of the Co(II) enzyme and the metal is no longer removable by dialysis. The Co(III) carboxypeptidase A retains esterase activity comparable to that of the Co(II) enzyme and has very low peptidase activity. This demonstrates that scission of a bond to the first coordination sphere of the metal is not necessary for the hydrolysis of ester substrates.

The structure of abelsonite

Abelsonite, a C31 nickel-porphyrin of the deoxophylloerythroetioporphyrin type, is shown to have methyl groups in the 2, 3, 7, 12, and 18 positions and ethyl groups in the 8 and 17 positions by high-resolution, high-field 1H nuclear magnetic resonance and nuclear Overhauser effect studies. Removal of the nickel by treatment with methanesulfonic acid permitted confirmation of the structure on the free base porphyrin and demonstrated structural integrity under the conditions required for demetallation. The structure is best accounted for geochemically by the hypothesis that abelsonite is derived from a chlorophyll.

The preparation of stroma-free hemoglobin by selective DEAE-cellulose absorption

The preparation of stroma-free hemoglobin by selective DEAE-cellulose absorption is reported. The stroma-free hemoglobin prepared by this method is compared to the product obtained by crystallization from sodium phosphate. Both show normal serum potassium, sodium, and pH values, and no coagulant activity or blood type activity by blood type test. PAA gradient gel electrophoresis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and isoelectric focusing in polyacrylamide gel all show the same well-defined bands in both preparations. The DEAE procedure requires 11 h as compared to 4 days for the crystallization method. The recovery of hemoglobin is 77% (less than 1% methemoglobin) in the DEAE preparation compared to 34% (greater than 3% methemoglobin) by the crystallization method. In addition, far fewer expensive materials are required.

Cobalt(III) carboxypeptidase A: an activity depending on the method of metal ion replacement.

Abstract There are two different methods available for replacing the Zn(II) in carboxypeptidase A with Co(II). One involves direct metal ion exchange using a large molar excess of Co(II) while the other requires the preparation of the metal-free apoenzyme and reconstitution with Co(II). Oxidation of the product obtained by the first route gives Co(III) carboxypeptidase A which is inactive towards synthetic peptide substrates but active towards synthetic ester substrates. In contrast, oxidation of the Co(II) carboxypeptidase obtained by the second strategy gives a Co(III) carboxypeptidase A having neither peptidase nor esterase activity.

Nephrotoxic Effects of Oxygen Transport Media in the Isolated Rat Kidney

Abstract Perfusion of isolated kidneys from rats demonstrated the following nephrotoxic effects of Fluosol-DA: decreased glomerular filtration rate (GFR), urine flow rate (UFR), and fractional reabsorption of potassium (FrK+) (P < 0.01). Fluosol-DA perfusions were at flow rates about equal to the physiologically normal rodent renal plasma flow rate of 4 ml/min. Stroma-free hemoglobin (SFH) perfusions, also at 4 ml/min, were associated with physiologically normal renal functions, as were those of control Krebs-Ringer bicarbonate (KRB) perfusions at 32 ml/min.