Aminotransferase activities in mouse, Mus domesticus, erythrocytes separated according to age.

Erythrocyte aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities are often used as indices of vitamin B-6 nutritional status; however, results using a mixed population of erythrocytes can be quite variable. Erythrocytes from two strains of mice (Mus domesticus), A/Ibg and DBA/Ibg, were separated according to age by centrifugation through discontinuous Percoll density gradients into three fractions: top (least dense, youngest), middle and bottom (most dense, oldest). A sufficient yield of age-fractionated erythrocytes was obtained from a single mouse for all of the enzyme measurements. The activities of AST, ALT and three age-marker enzymes, pyruvate kinase, acetylcholinesterase and hexokinase, were found to be significantly higher in the youngest cell fractions, and declined in the older, more dense fractions. A mice had significantly lower AST and ALT activities in the age separated fractions than did DBA mice. The measurement of enzyme activities in low density, young cells may be especially useful in studies involving conditions in which the proportion of young erythrocytes may be elevated with respect to the entire erythrocyte mass.

Hydrogen exchange measurement of the free energy of structural and allosteric change in hemoglobin.

The inability to localize and measure the free energy of protein structure and structure change severely limits protein structure-function investigations. The local unfolding model for protein hydrogen exchange quantitatively related the free energy of local structural stability with the hydrogen exchange rate of concerted sets of structurally related protons. In tests with a number of modified hemoglobin forms, the loss in structural free energy obtained locally from hydrogen exchange results matches the loss in allosteric free energy measured globally by oxygen-binding and subunit dissociation experiments.

A dodecamer of globin chains is the principal functional subunit of the extracellular hemoglobin of Lumbricus terrestris.

Repeated dissociation of the approximately 3600-kDa hexagonal bilayer extracellular hemoglobin of Lumbricus terrestris in 4 M urea followed by gel filtration at neutral pH produces a subunit that retains the oxygen affinity of the native molecule (approximately 12 torr), but only two-thirds of the cooperativity (nmax = 2.1 +/- 0.2 versus 3.3 +/- 0.3). The mass of this subunit was estimated to be 202 +/- 15 kDa by gel filtration and 202 +/- 26 kDa from mass measurements of unstained freeze-dried specimens by scanning transmission electron microscopy. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of this subunit showed that it consists predominantly of the heme-containing subunits M (chain I, 17 kDa) and T (disulfide-bonded chains II-IV, 50 kDa). Mixing of subunits M and T isolated concurrently with the 200-kDa subunit resulted in partial association into particles that had a mass of 191 +/- 13 kDa determined by gel filtration and 200 +/- 38 kDa determined by scanning transmission electron microscopy and whose oxygen affinity and cooperativity were the same as those of the 200-kDa subunit. The results imply that the 200-kDa subunit is a dodecamer of globin chains, consisting of three copies each of subunits M and T (3 x chains (I + II + III + IV], in good agreement with the mass of 209 kDa calculated from the amino acid sequences of the four chains, and represents the largest functional subunit of Lumbricus hemoglobin. Twelve copies of this subunit would account for two-thirds of the total mass of the molecule, as suggested earlier (Vinogradov, S. N., Lugo, S. L., Mainwaring, M. G., Kapp, O. H., and Crewe, A. V. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 8034-8038). The retention of only partial cooperativity by the 200-kDa subunit implies that full cooperativity is dependent on the presence of a complete hexagonal bilayer structure, wherein 12 200-kDa subunits are linked together by approximately 30-kDa heme-deficient chains.

A photoacoustic calorimetric study of horse myoglobin.

The dynamics of the enthalpy and volume changes for the photodissociation of CO from horse myoglobin has been studied by time-resolved photoacoustic calorimetry which measures the dynamics of enthalpy and volume changes on the nanosecond time scale. The role of the Lys 45 salt bridge in the ligand dissociation is discussed.

Role of the arginine-45 salt bridge in ligand dissociation from sperm whale carboxymyoglobin as probed by photoacoustic calorimetry.

The dynamics of the enthalpy and volume changes found in the photodissociation of CO from sperm whale carboxymyoglobin and two site-directed mutants in which arginine-45 is replaced by glycine and asparagine are examined by photoacoustic calorimetry. An intermediate is observed whose lifetime at 20 degrees C is 700 ns. The enthalpy of the intermediate increases by approximately 7 kcal/mol upon replacing arginine-45 with either asparagine or glycine. These observations support recent proposals that an arginine-45 salt bridge is broken upon ligand dissociation.

A time-resolved photoacoustic calorimetry study of the dynamics of enthalpy and volume changes produced in the photodissociation of carbon monoxide from sperm whale carboxymyoglobin.

The dynamics of the enthalpy and volume changes produced in the photodissociation of carbon monoxide from sperm whale myoglobin is investigated by time-resolved photoacoustic calorimetry. The enthalpy and volume changes for the formation of the geminate pair, which occurs within 50 ns of photolysis, are delta H = -2.2 +/- 2.8 kcal/mol and delta V = -10.0 +/- 1.0 mL/mol relative to carboxymyoglobin. The enthalpy and volume changes associated with formation of deoxymyoglobin and solvated carbon monoxide, formed with a half-life of 702 +/- 31 ns at 20 degrees C, are delta H = 14.6 +/- 3.4 kcal/mol and delta V = 5.8 +/- 1.0 mL/mol relative to carboxymyoglobin.