Alfred C. Olson

Properties and enzymatic activities of papain insolubilized with glutaraldehyde.

Abstract Glutaraldehyde at a concentration of 2.3% and at 0 ° reacted in aqueous solutions with papain to form a water-insoluble product with enzymatic activity after activation by reducing agents. A rapid reaction of glutaraldehyde with the essential sulfhydryl of papain was not involved in the reaction since after activation the insoluble enzyme retained esterolytic and proteolytic activity. The amount of enzymatic activity retained by the insoluble material depended on the conditions of the reaction. The insolubilizatiou of papain with glutaraldehyde was pH dependent; the higher the pH the more rapid the reaction. Complete precipitation occurred in 24 hr over the range pH 5.2–7.2. The lower the pH of the reaction, the greater was the retention of esterase activities. Similar results were obtained when the reaction was carried out in the presence of added cysteine. However, the insoluble enzyme prepared in the absence of cysteine was fibrous while that prepared in its presence was crystalline. The reaction of mercuripapain with glutaraldehyde resulted in an insoluble enzyme that possessed, after treatment with cysteine, approximately twice as much esterase and proteinase activities as did the insoluble enzyme prepared from papain. Compared with soluble papain, preparations from glutaraldehyde mercuri-papain retained 48% of the ester, 12% of the casein, and 16% of the hemoglobin hydrolyzing ability. The reaction of the reduced soluble and insoluble enzymes with iodoacetamide-1- 14 C demonstrated that insoluble mercuripapain retained all of its essential sulfhydryl groups whereas those in insoluble papain had been reduced to two thirds. Amino acid analysis on hydrolyzed insoluble enzyme showed that only the lysine had been significantly altered by the glutaraldehyde reaction, having been reduced to approximately one-half that of soluble papain under all the conditions studied.

Force extension analysis of Avena coleoptile cell walls

SummaryA method is described for measuring the cell wall mechanical properties of Avena coleoptiles in the absence of turgor stress or influences of a living protoplast. Forceextension curves obtained with a constant-rate-of-extension instrument and standard fiber-testing techniques demonstrate the permanence of cell wall loosening effects of prior indoleacetic acid (IAA) treatment of living tissue and provide evidence that these changes involve interactions between cell wall polymers. By this method various chemical and enzymatic modifications of cell walls can be evaluated in terms of altered mechanical properties. Thus, it was possible to remove over 97% of the cell nitrogen (including some hydroxyproline-containing protein) by hot methanol followed by enzymatic treatment and not change the extensibility properties of the tissue. In contrast, coleoptile mechanical properties were markedly influenced by chemical acetylation procedures or cellulase treatment.

Cross-linking of α-chymotrypsin and other proteins by reaction with glutaraldehyde☆

Abstract The rate of inactivation of α-chymotrypsin by reaction with glutaraldehyde increased with increasing pH, but the formation of insoluble and active α-chymotrypsin was most rapid at pH 6.2. The pH values for the most rapid glutaraldehyde insolubilization (pH optima) of other proteins were chymotrypsinogen-A, pH 8.2; bovine serum albumin, pH 4.8; soybean trypsin inhibitor, pH 4.8; lysozyme, pH 10.5; and papain, pH 8.6. With the exceptions of α-chymotrypsin and chymotrypsinogen-A, the pH for the most rapid glutaraldehyde insolubilization of these proteins was the same as their isoelectric points. The proteins, which varied in their initial total lysine content, showed the same percentage decrease in lysine upon insolubilization. Insolubilization of α-chymotrypsin was more rapid the lower the ionic strength of the reaction mixture. When the insolubilization was conducted in the presence of the inhibitor, β-phenylpropionate, or with the monoacetyl derivative of α-chymotrypsin, the insolubilized product was more enzymatically active. Concurrent glutaraldehyde insolubilization of bovine serum albumin and α-chymotrypsin was possible only at a pH between the optimum for each protein. An insolubilized cross-linked mercuripapain-α-chymotrypsin was prepared which possessed the activities of each enzyme.

Physical chemical observations on the α-chymotrypsin glutaraldehyde system during formation of an insoluble derivative

Abstract Physical chemical observations have been made on the α-chymotrypsin-glutaraldehyde system during formation of an insoluble, enzymically active product. Lightscattering measurements indicate a two-step reaction, the second step being a linear condensation polymerization reaction. The effects of pH and ionic strength on the rate of the second-step reaction are best explained in terms of an acid shift in the p K a of the ϵ-amino groups of glutaraldehyde-modified lysine residues and a decrease in attractive forces between enzyme particles with increasing ionic strength, respectively. The large cross-linked particles formed appear to be branched flexible coils. Time-dependent ultraviolet spectra demonstrate an apparent hyperchromism due to increased scattering with no new absorption bands. Optical rotatory dispersion (ORD) and circular dichroism (CD) measurements show no gross unfolding of the α-chymotrypsin molecule upon cross-linking, although small local changes in conformation could be indicated by changes in the CD spectrum at 255 and 229 nm. Measurements on small soluble polymers show that there is a large loss in activity (60%–70%) during formation of these relatively small derivatives, suggesting that reaction of glutaraldehyde with primary amino groups and not intermolecular cross-link formation could be the main reason for the loss in activity.

Activation of glutaraldehyde-crosslinked chymotrypsinogen-A. Enzymatic activity and circular dichroism studies

Abstract Glutaraldehyde insolubilized chymotrypsin has been prepared by trypsin activation of glutaraldehyde insolubilized chymotrypsinogen-A and found to have esterase activities of 28 and 22% (TEE, ATEE) and proteinase activities of 4.4 and 3.8% (hemoglobin, casein) relative to α-chymotrypsin. A soluble glutaraldehyde crosslinked chymotrypsin, similarly prepared, had a weight average molecular weight of 130,000 and had esterase activity of 90% (ATEE) and proteinase activity of 42% (casein) relative to α-chymotrypsin. These activities were appreciably greater than those shown by glutaraldehyde crosslinked α-chymotrypsin prepared by direct reaction of glutaraldehyde and α-chymotrypsin. Amino acid analyses on the crosslinked enzymes prepared by activation of insoluble and soluble crosslinked zymogens suggest that the insoluble crosslinked enzyme is entirely in the π form and the soluble crosslinked enzyme partly in the π form. Full esterase activity is retained by the trypsin-activated glutaraldehyde insolubilized chymotrypsinogen-A at 25 °C for 14 days. The circular dichroism (CD) changes associated with activation in the native system were also found to occur in the soluble crosslinked system, suggesting that certain conformational changes are necessary for development of enzymatic activity. A CD change at 255 nm toward less negative values was observed when glutaraldehyde was reacted with either native chymotrypsin or crosslinked chymotrypsin generated by activation of glutaraldehyde crosslinked zymogen but not when glutaraldehyde reacted with chymotrypsinogen-A.

Scintillation counting of the ninhydrin-amino acid-C14 reaction products from an automatic amino acid analyzer

Abstract The effluents of an amino acid analyzer which contain C14-labeled amino acids may be collected and aliquots of the ninhydrin colored solution counted in a scintillation counter. The colored ninhydrin reaction product does not quench the counting when as much as 0.4 ml containing 0.2 μmole amino acids is added to 20 ml of counting solution. Since there is loss of the carboxyl group in the reaction, only those amino acids with C14 in positions other than the carboxyl will be detected by this method. An analysis of commercially available uniformly labeled amino acids from protein hydrolyzates by this procedure suggests that each carbon in the amino acids may not have the same specific activity and that the carboxyl carbon may have a disproportionate share of the activity. The technique has been applied to studies of proline metabolism.

The Use of Tannic Acid and Phenol-Formaldehyde Resins with Glutaraldehyde to Immobilize Enzymes

Our investigations with immobilized enzymes have been guided by how they may assist in finding out more about enzymic phenomena and how they could ultimately be utilized in processes and analyses, particularly those related to agriculture and food. In the latter area economic as well as scientific factors play a very important role. A complete evaluation of all of these factors is beyond the scope of this paper. However, we have considered many of these factors in both the choice of the systems for immobilization and the applications we have elected to study. The criteria we used in selecting methods for enzyme immobilization included: successful achievement of stable enzyme immobilization; an acceptable degree of retention of enzyme activity; good pH and temperature characteristics; simplicity of the method; use of impure or inexpensive enzyme preparations; a potential for large volume application; and low cost.

Electrophoretic migration and redox behavior of malate dehydrogenases from cell suspension cultures of tobacco.

1. 1. The presence of one distinct mitochondrial malate dehydrogenase (l-malate: NAD+ oxidoreductase, EC 1.1.1.37), at least three cytosol malate (NAD+) dehydrogenase isoenzymes and only one cytosol malic enzyme component (l-malate:NADP+ oxidoreductase (decarboxylating), EC 1.1.1.40) were demonstrated in preparations from plant suspension cells cultivated at 25 °C. 2. 2. Bulk separation of the cytosol malate (NAD+) dehydrogenase isoenzymes was achieved by serial fractionation with (NH4)2SO4. Each of the three enzyme forms possessed different kinetic properties and one of the isoenzymes did not require phenazine methosulfate to reduce tetrazolium after gel electrophoresis. 3. 3. Cultivating the plant suspension cells at temperatures at least 10 °C above or below 25 °C prevented expression of one or the other of three cytosol malate (NAD+) dehydrogenase isoenzymes. 4. 4. Treatment of the particulate preparation with deoxycholate increased the electrophoretic mobility of mitochondrial malate (NAD+) dehydrogenase. Depending on the concentration or assay method used, deoxycholate either inhibited or reversed the reaction catalyzed by soluble as well as particulate forms of malate (NAD+) dehydrogenase.