Peter Bernfeld

Reversible dissociation of enzymes at high dilutions and their inhibition by polyanions

Abstract Crystalline preparations of aldolase and lactic dehydrogenase from rabbit muscle, of porcine pancreatic α-amylase and of sweet potato β-amylase exhibit a decrease of their specific activities upon dilution. This phenomenon can best be explained by a dissociation of the enzymes into enzymically inactive products. The enzyme concentrations at which specific activities drop to one half of their maximum values are 2, 1.5, 0.005 and 1 μg per milliliter of digest, respectively. Various macromolecular polycations and diaminodecane, as well as proteins acting in this capacity, reverse the dissociation and, hence, are activators of the diluted enzymes. They have no effect on the activity of concentrated enzyme solutions. Certain polycations which thus activate one enzyme do not necessarily have the same effect on another enzyme. Many macromolecular polyanions, notably polysaccharide sulfate esters, polyvinyl sulfate and polystyrene sulfonate, inhibit the enzymes. This effect depends not only on the presence of electronegative groups in these substances but also on the chemical nature of their negative groups, as well as on the chemical nature and, to a much lesser degree, on the molecular weight of the polyaniona. Different enzymes are inhibited to widely varying extents by polyanions of a different nature. Polyanionic enzyme inhibition may be due either to a promoting effect of polyanions on the dissociation of enzymes or to some other mechanism. Polycations, including many proteins, counteract the enzyme inhibition by polyanions.

Water-insoluble enzymes: Arrangement of aldolase within an insoluble carrier☆

Abstract Crystalline, radioactive muscle aldolase was prepared from a rabbit injected subcutaneously with a mixture of fifteen 14 C-labeled amino acids. The radioactive enzyme exhibiting 1.4 × 10 −4 μC per mg protein was transformed into a water-insoluble state by embedding it into a highly cross-linked, insoluble synthetic polyacrylamide carrier, according to the procedure previously described by us ( Science 142 : 678, 1963). The distribution of enzyme activity and of total enzyme protein, as measured by the radioactivity, between insoluble carrier and remaining aqueous phase was determined. While only 44.5% of the total enzymic activity could be recovered, all of the radioactivity was accounted for. The ratio of enzyme activity to radioactivity in the aqueous phase, which remained after termination of the polymerization, was not much lower than the corresponding ratio of the original soluble aldolase (25% reduction). This suggests that the contact of enzyme with the reagents for polymerization caused no more than 25% enzyme inactivation. However, the respective ratio in the insoluble phase was four times smaller, indicating that four times more enzyme protein than enzymic activity was associated with the carrier. The most plausible explanation of these findings is the assumption that only that portion of the embedded aldolase deploys enzymic activity, which is located at or near the surface of the carrier particle. Once the aldolase is embedded in the synthetic polymer, the enzyme can no longer be separated from its insoluble carrier.

Factors influencing the activity of testicular hyaluronidase

Abstract 1. 1. The influence of dilution and of addition of polyelectrolytes, mainly of macromolecular nature, on the activity of testicular hyaluronidase was studied. 2. 2. Two samples of hyaluronidase, i.e., a commercial preparation and a product purified fivefold by zone electrophoresis, served for this study. Enzyme activity was measured by two independent procedures, namely by the viscosity-reducing and by the turbidity-reducing methods. The latter technique has been modified and adapted for use at high enzyme dilutions. 3. 3. Upon dilution, hyaluronidase is transformed into an enzymically inactive form, recognizable by the progressive loss of specific activity. This phenomenon is due to a reversible dissociation of the active enzyme into inactive components. 4. 4. Dissociation of hyaluronidase can be reversed by the addition of polycations, in particular by poly- l -lysine, protamine, chitin, and 1,10-diaminodecane, in the order of decreasing potency. These substances are, therefore, activators of dilute hyaluronidase. 5. 5. Macromolecular polyanions, such as polystyrene sulfonate, heparin, sulfated pectic acid, polymethacrylate, amylopectin sulfate, and deoxyribonucleate are inhibitors of hyaluronidase. They act by sequestrating the activator and, hence, they promote dissociation of the enzyme into inactive components. The inhibition is of the “activator-competitive” type, since it can be attenuated and even completely overcome by increasing the activator concentration.

“Activator-competitive” enzyme inhibition. Interrelation of dissociation, activation, inhibition and surface inactivation of β-glucuronidase☆

Abstract 1. 1. Dilution of β-glucuronidase causes reversible dissociation of the enzyme into inactive polypeptides. 2. 2. Many polycations are able to prevent the dissociation and, thus, act as activators of the diluted enzyme. 3. 3. Polymethacrylic acid and polystyrene sulfonate reversibly inhibit dilute β-glucuronidase by sequestering its activator with which they form complexes. Enzyme and inhibitor compete with each other for the activator. The potency of the inhibition depends on the chemical nature of the activator and on its concentration, a mechanism which results in an antagonism between inhibitor and activator. 4. 4. The products of dissociation of β-glucuronidase are irreversibly altered upon contact with rough surfaces, such as scratched glass or quartz sand, inasmuch as the addition of activators no longer restores the original enzyme activity. Thus, handling of diluted β-glucuronidase in ordinary glassware causes an irreversible inactivation. 5. 5. The inactivation can be prevented by the presence of activators during the contact with glass, or it can be boosted by the presence of polymethacrylate or polystyrene sulfonate. Concentrated enzyme solutions are stable in glass, and diluted enzyme solutions are not inactivated in polyethylene containers.

Kinetics of water-insoluble phosphoglycerate mutase

Abstract 1. 1.|An insoluble form of phosphoglycerate mutase (2,3-diphospho- d -glycerate: 2-phospho- d -glycerate phosphotransferase, EC 2.7.5.3) from rabbit muscle was prepared by mechanically immobilizing the crystalline enzyme within a water-insoluble carrier of highly cross-linked polyacrylamide. This procedure has previously been devised in our laboratory and is known to involve no covalent bonds between enzyme and carrier. 2. 2.|The kinetic behavior of the soluble and insoluble forms of this enzyme were identical in many respects, i.e. upon variation of the enzyme concentration, substrate concentration and temperature. Thus, their Michaelis constants, activation energies and susceptibilities to substrate inhibition were the same. 3. 3.|In contrast, the pH optimum of activity of the insoluble form of mutase was one whole unit lower than that of the water-soluble form, although the carrier of the insoluble enzyme was electrostatically neutral. 4. 4.|Insoluble mutase was also more sensitive to activation by 2,3-diphosphoglycerate than the soluble form. 5. 5.|A comparison between the insoluble forms of phosphoglycerate mutase and enolase, prepared by the same procedure, shows that differences between soluble and insoluble forms of these enzymes depend more on the nature of the enzyme than on that of the insoluble carrier.

Characteristic individual electrophoretic patterns in humans.

Summary 1. The detailed examination of the contours and fine architecture of the elec-trophoretic patterns of human plasma in different individuals may reveal considerable dissimilarities between persons, even in those cases where the relative amounts of the plasma protein components, calculated from the elec-trophoretic patterns, are similar. The careful examination of the electrophoretic patterns, of the shape, symmetry or asymmetry, and width of each protein peak and of the tendency of adjacent protein peaks to separate from or to approach each other is considered to be of great importance to characterize a given pattern. 2. The general features and the appearance of the electrophoretic patterns in one and the same person usually remain constant over long periods of time, although the plasma protein composition may vary considerably during this period. 3. Characteristic individual electrophoretic patterns of the plasma proteins have been observed in numerous cases of both normal individuals and patients with various diseases. The distinguishing characteristics encountered in individual electrophoretic patterns are independent of sex and age; in healthy persons they are confined mainly to the a-globulins; with only a few exceptions they are not related to the disease state in patients.

Water-insoluble enzymes: Kinetic; of rabbit muscle enclose embedded within on insoluble carrier☆

Abstract Crystalline rabbit muscle enolase was transformed into a water-insoluble form by embedding the enzyme in a highly cross-linked, synthetic polymer, without formation of covalent bonds between enzyme and insoluble carrier, according to the procedure previously published by us (Science142, 678 (1963); Arch. Biochem. Biophys.127, 779 (1968)). Enzyme kineties of the insoluble form of enolasc were compared to those of the soluble form. There were no differences between the two forms of enolasc with regard to the influence of variations in pH, enzyme concentration or substrate concentration on enzyme activity. Michaelis constants, maximum rates of substrate turnover and degrees of substrate inhibition were the same for both forms of the enzyme. This behavior of insoluble enolase is consistent with that of an enzyme immobilized in an electrostatically neutral, insoluble carrier. Considerable differences existed between the two forms of enolase in their behavior toward temperature and magnesium. Between 1 and 24 °, the activity of the insoluble form increased much more slowly than that of the soluble form; optimum activity was reached at 44 and 50 ° for the soluble and insoluble forms, respectively; the former was no longer active at 50 °, but insoluble enolase still retained 23% of its activity at 56 °. Activation energies were similar for both forms of the enzyme in the upper temperature range (above 24 °); i.e., 11,800 and 15,300 cal/mole for the soluble and insoluble forms, respectively. But they differed widely at lower temperatures (below 8 °); i.e., 29,600 and 4600 cal/mole, respectively. Both forms of enolase required magnesium for maximum activity (0.68 × 10−3 m ), but the insoluble form, in contrast to soluble muscle enolase, was not inhibited by an excess of magnesium. Zinc inhibited both forms of the enzyme to about the same extent, except at low magnesium concentrations where the insoluble form was some-what less affected by Zn2+ than soluble enolase.

Characteristic Electrophoretic Patterns in Hemophilia and Idiopathic Thrombocytopenia.

Summary Characteristic electrophoretic patterns have been observed in all 5 cases of hemophilia and in all 8 cases of idiopathic thrombocytopenic purpura. These patterns show the following characteristics: (a) appearance of an unusual peak in the group of the α-globulins (αx), and (b) disappearance of the usual α2- and α3-globulin peaks. The same anomaly has been observed in some cases of secondary thrombocytopenia, “anaphylactoid” type of vascular purpura, and hemolytic anemia with evidence of platelet dysfunction.

Electrophoretic Plasma Protein Patterns in Families with Hemophilia.

Summary 1. An electrophoretic anomaly, called αx-globulin, has been found in the plasma of 14 hemophiliacs, i.e., in all of the active bleeders studied. 2. The same anomaly also has been observed in 8 members of hemophilic families who are not active bleeders (over 50% of this category), and who are not afflicted with any of the other diseases in which this anomaly has been found. 3. The electrophoretic anomaly is seen in female as well as in male subjects unlike the bleeding manifestation of hemophilia, found only in males. 4. There is a definite relationship between the prothrombin utilization and the appearance of the αx-globulin anomaly in the plasma of members of hemophilic families.

Further Studies on Abnormal Serum Proteins in Tumor-Bearing Hosts.

Summary The presence of an abnormal serum protein in C3H mice bearing spontaneous mammary adenocarcinoma was confirmed. A rabbit antiserum specific for this abnormal murine serum protein was prepared, and the specific antiserum obtained did not react at all with the serum of normal male C3H littermates. The procedure for its preparation was based on 3 essential features: 1. Immunization of the rabbits with purified antigen; 2. development of a schedule of “cross immunization” consisting of alternate injections with 2 slightly different murine serum protein fractions; and 3. quantitative absorption of all antibodies against normal murine serum proteins by the use of water-insoluble antigens. The abnormal serum component is present in all tumor-bearing animals but only in minute amounts; it was also found in pregnant females and in about 20% of normal females, but never in males; it is assumed to be a forerunner of tumor growth. It migrates on electrophoresis as an a-globulin and only insignificant amounts of it were found in extracts of washed tumor or liver cells of the hosts. It is not known whether it is related to Bittners Milk Agent.