John W. Donovan

Spectrophotometric observation of the alkaline hydrolysis of protein disulfide bonds

Abstract In the spectrophotometric titration of the phenolic groups of ovomucoid, a time-dependent increase in ultraviolet absorption is observed at high pH ( Donovan, 1967 ). The present communication shows that the pseudo-first order rate constant for the absorption increase is proportinal to the hydroxide ion concentration. The absorption change resembles the absorption of sulfhydryl ion, which is produced at the same rate as the absorption spectrum changes. The stoichiometry is consistent with S-S splitting of the disulfide bonds.

Biological activity and conformational stability of the domains of plasma fibronectin

Abstract As measured by two assays of biological activity, fibronectin was readily denatured by heat. Both by the rat liver slice assay and by gelatin-latex agglutination, 90% of the activity disappeared in about 10 min at 60 °C. In contrast, immunological activity, as measured by microcomplement fixation, showed little change at 10 min and was at least 60% as great as unheated fibronectin after 20–50 min at 60 °C. Binding of heparin was unaffected by heating up to 52 min, but at very long times (48 hr at 60 °C), it also was lost. Differential scanning calorimetry of native fibronectin showed three endothermal denaturing transitions, at 68, 82, and 119 °C. Enthalpies of denaturation for the three transitions are approximately 2.6, 0.4, and 0.7 cal/g of flbronectin. These results are consistent with a three-domain structure for fibronectin. The domain which unfolds at 68 °C is associated with gelatin binding and cell. binding. The 82 °C domain appears to be associated with much of the immunological activity, and the 119 °C domain with heparin binding, as well as with some immunological activity. Residual immunological activity after loss of heparin binding may reside in nonordered portions of the molecule.

Scanning calorimetry of complex biological structures

Abstract Scanning calorimetry, a method long used in polymer chemistry and more recently in membrane studies, is now gaining increased use in biochemical studies on the stability, conformations and interactions of complex structures of proteins, nucleic acids, carbohydrates and lipids.

Sugar nucleotides of chicken egg white

The sugar nucleotides GDP-mannose and UDP-N-acetylgalactosamine sulfate have been found in about equal amounts in chicken egg white, and account for nearly 90% of the nucleotides observed. UDP-N-Acetylglucosamine and UDP-N-acetylgalactosamine are also observed, as well as small amounts of GDP and UMP. There are approximately 0.2 μmole of combined nucleotides per milliliter of egg white. The ease of separation of GDP-mannose and UDP-N-acetylgalactosamine sulfate makes egg white appear to be a convenient source of these nucleotides.

Spectroscopic evidence for perturbation of tryptophan in Al(III) and Ga(III) binding to ovotransferrin and human serum transferrin

Ultraviolet spectroscopy has been widely used to study metal binding to transferrins [l-8] . The observed difference spectra are usually interpreted in terms of changes in ionization of tyrosyl residue, although earlier work suggested perturbation [5] or direct involvement [3,9] of tryptophan in Fe(II1) and Cu(I1) binding to transferrins. But Feand Cutransferrins have broad charge-transfer absorption bands in the near ultraviolet [5,10,1 l] which extend into the wavelength region in which the tyrosine and tryptophan chromophores absorb. This makes interpretation difficult. Difference spectra obtained at pH 11 .O have been used to correct for this background absorption of Fe-transferrins [2,4]. But, ionization of tyrosines may not be complete at pH 1 I .O [l] and time-dependent exposure of ‘buried chromophores at alkaline pH [ 121 and alkaline hydrolysis of disulfide bonds [ 131 may further complicate determination of background absorption by this method. Colorless transferrin metal complexes appear to be more suitable for difference spectral studies. Zn(I1) [4,7] and trivalent lanthanides [6] give difference spectra which are similar to that for tyrosine ionization, with no apparent interfering absorption due to the metal ion. Ga(II1) binding to ovotransferrin has been reported [8,14] to give a difference spectrum characteristic of tyrosine ionization. A difference was noted between the spectrum of the first Ga bound and that of the second’ [8] . We have measured, (i) the difference spectra produced by Zn, Ga and Al(II1) binding to ovotransferrin and human serum

Calorimetric studies on the binding of iron and aluminium to the amino- and carboxyl-terminal fragments of hen ovotransferrin.

The binding of iron(II1) and other metal ions to ovotransferrin confers thermal stability on the protein [ 11. This observation prompted the use of differential scanning calorimetry (DSC) for a more detailed study of the binding of Cu(II), Al(III), Fe(III), Zn(I1) and Ga(II1) to ovotransferrin [2-41. The disappearance and appearance of the various liganded forms of a protein titrated with metal ions can be followed by DSC, provided that each species has a characteristic denaturation temperature. Saturation of iron-free ovotransferrin with Fe(II1) at pH 7.5 raises its denaturation temperature from 63-84’C. On partial saturation of the protein, intermediate endotherms (peaks of heat absorption) at 68’C and 77’C are observed, tentatively assigned to two monoferric species [3] . When AI(II1) is added to the apoprotein to 50% saturation, the denaturation temperature is raised to 68°C. Further addition of Al has no effect on the thermal stability. Here, we have examined the effects of Fe(II1) and Al(II1) on the thermal stability of the aminoand carboxyl-terminal fragments of ovotransferrin which can be isolated by proteolytic digestion [5,6] . Our results show that there is preferential binding to Fe(II1) to the amino-terminal fragment, and that this fragment is more stabilized by Al(II1).

Heat stabilization dependence on redox state of cytochrome cd1 oxidase from Pseudomonasaeruginosa

Abstract The irreversible thermal denaturation of cytochrome cd1 oxidase from P. aeruginosa as a function of the oxidation-reduction states of its hemes was observed with a differential scanning calorimeter. Upon full reduction of the four hemes, the apparent denaturation temperature decreases by about 10° and the denaturation enthalpy decreases slightly: oxidized, 5.9 cal/gm; reduced, 5.4 cal/gm. At pH 7.5, the first order rate constants for denaturation at 90°C are: reduced, 33 × 10−3s−1; oxidized, 3 × 10−3s−1. Thus, oxidation of the hemes reuults in heat stabilization of the cytochrome oxidase. The activation energy for denaturation of fully reduced oxidase, 53 kcal/mol, is less than that for fully oxidized protein (73 kcal/mol).