Mario A. Marini

Peroxidative activities of hemoglobin and hemoglobin derivatives.

Publisher Summary This chapter discusses the peroxidative activities of hemoglobin and hemoglobin derivatives. Methemoglobin and metmyoglobin can react with preexisting lipid hydroperoxides to yield lipid-associated free radicals, which resulted in the propagation of free radical-mediated reactions that produced extensive lipid peroxidation. Methemoglobin and metmyoglobin can initiate the peroxidation of polyunsaturated fatty acids. Substantial membrane lipid peroxidation for model cells composed of hemoglobin entrapped in chemically defined liposomes. Human erythrocytes are also shown to be susceptible to hemoglobin-catalyzed, H 2 O 2 -dependent lipid peroxidation. Additionally, lipids, proteins, and carbohydrates are also susceptible to oxidative degradation by hemoglobin. Thus, the addition of H 2 O 2 to erythrocytes or erythrocyte membranes produces high molecular weight membrane protein aggregates, whereas deoxyribose is readily degraded by H 2 O 2 in the presence of hemoglobin. The mechanism by which hemoglobin exerts its peroxidative activity is similar to that of the peroxidases, in which hydrogen peroxide first reacts with the native enzyme and withdraws two electrons.

Preparation of human hemoglobin ao for possible use as a blood substitute

A procedure is presented for the preparation of a purified fraction of adult human hemoglobin (HbAo) from one unit of outdated blood. The entire process requires less than 16 h and gives a sterile, endotoxin-free solution of HbAo (approximately 30 g) in a yield of 50%. The solutions are isoionic with a conductivity of less than 15 mu mhos and less than 2 mmol total phosphorus per mol heme. The methemoglobin content is less than 1% and on storage at 4 degrees C rises less than 1% per month.

Reduction of extracellular methemoglobin by erythrocytes.

Erythrocytes, suspended in a glucose-containing buffer, catalyzed the partial reduction of extracellular methemoglobin. Physiological concentrations of ascorbic acid or dehydroascorbic acid greatly enhanced the rate of reaction and the ultimate extent of reduction. The relationship between erythrocyte concentration and initial reaction rate was nonlinear, which suggested that the rate limiting factor was not an erythrocyte membrane enzyme. Also, significant dehydroascorbate-stimulated reduction occurred even when the erythrocytes and methemoglobin were separated by a dialysis membrane. The above observations indicate that the transfer of reducing equivalents across the erythrocyte membrane and reduction of extracellular methemoglobin can be accomplished by release and recycling of ascorbic acid.

Thermal titrimetric evaluation of the heats of ionization of the commonly occurring amino acids and their derivatives.

Abstract By using instrumentation of relatively simple design it has been shown that calorimetric data of good precision can be obtained with rapidity and ease of operation. The heats of ionization of all the commonly occurring amino acids have been determined and, depending on the differences in ionization constants, heats of ionization of individual groups in a mixture can also be obtained.

Heats of precipitation of calcium phytate

Abstract The heat of precipitation has been determined for the reaction, in aqueous solution, of calcium ion with the phytate anion. The reaction is endothermic. From the value of the equilibrium constant associated with the reaction, the entropy change has been calculated. The large, positive entropy change is consistent with the view that when calcium ion reacts with the phytate anion there is a decrease in the hydrophilic character of the phytate anion. The quantitative aspects of the interaction of phytic acid with calcium provides a rational basis for the understanding of the mechanism of metal deprivation by phytic acid.

Heats of precipitation of zinc phytate

Abstract The heat of precipitation for the reaction of zinc ion with the phytate ion in aqueous solution has been determined. The reaction is endothermic. From the value of the equilibrium constant associated with the reaction, the entropy change has been calculated. The entropy change is large and positive. This is consistent with the view that when zinc reacts with phytate to form solid zinc phytate there is an increase in the number of particles in the system.

Thermal and Potentiometric Titrations of Phytic Acid

Abstract Values for the ionization constants and the heats of ionization of phytic acid (inositol hexaphosphate) have been determined by simultaneous potentiometric and thermal titration in a titration microcalorimeter. The potentiometric results are in substantial agreement with those found by discontinuous titration; the heat liberated from pH 10.4 to 6.8 is essentially that found using a batch microcalorimeter. Ionization of phytic acid may be generally characterized as having five groups similar to pK of phosphoric acid, two groups similar to pK2 of phosphoric acid and 4 groups similar to pK4 of pyrophosphoric acid. A single group has pK ∼ 5 with ΔHi ∼ -1.1 kcal/mol.

Microcalorimetry in Biochemical Analysis

I. INTRODUCTION Recent years have seen the development of methodology transcending mere application of older techniques that could be applied to the rapid, precise determination of specific components in complex mixtures — usually of biologic origin. The development of ion-selective,1 other than for protons, and enzyme-specific electrodes2 are examples in point. Although the latter has its specific limitations of application, the generality of approach is limited only by miniaturization technology and the sophistication of the chemist/biochemist to attach enzyme molecules to insoluble matrices with retention and stability of biological activity. Solid surface fluorescence methods as developed by Guilbault and co-workers3 are also of recent origin and offer great potential for routine and inexpensive assays in the clinical laboratory.

Modification of the amino group of isoleucine-16 in chymotrypsin with retention of activity

Abstract The N-terminal isoleucine-16 residue of δ-chymotrypsin has been modified to the corresponding amidine by reaction with either ethyl acetimidate or methyl picolinimidate. The modified δ-enzymes show no change in specific activity or in the active site concentration but have one less isoleucine amino group as determined by both end-group analysis and potentiometric titration. Despite the fact that the amidinated amino group has a pK′ about 12, k cat K′ m versus pH profiles (acetyl-L-tryptophan ethyl ester as substrate) are bell-shaped with the upper pK′ 9.3. Thus, the Ile-16 amino group is neither essential for chymotrypsin activity nor responsible for the kinetically seen group with pK′ 9.

Formol titration of α-chymotrypsin, chymotrypsinogen, and model compounds in 4.8 M guanidine hydrochloride

Titration of model compounds in 4.8 M guanidine0.15 M KCl at 20° show that pK′ values of imidazoyl, phenolic, and carboxyl groups are shifted only slightly. Amino group ionizations are affected most, varying ±0.3 unit. The useful range of this system is from pH 1.5 to 12.0, in which all the normally ionizing groups of proteins can be titrated. Form-aldehyde titrations may also be performed to obtain additional information about the amino groups of proteins. In guanidine/formaldehyde, the pK′ of the hydroxymethylamino derivatives are shifted to lower values than in water. When chymotrypsinogen and α-chymotrypsin titrations were performed, the data indicated 36 ionizing groups in chymotrypsinogen, 15 of which are amino groups. Analysis of the difference between the ionic behavior of these proteins shows that α-chymotrypsin has two additional groups with pK′ 3 and two with pK′ 7.5 in guanidine. The groups with pK′ 7.5 are shifted to pK′ 4.2 in 3.1 M formaldehyde, which is indicative of the behavior of α-amino groups. The comparison of the Sorensen titration of α-chymotrypsin in water or guanidine indicates that all the amino groups of the protein react with formaldehyde in the native or denatured form.