Eugene F. Jansen

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.

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.

Recovery of 14C-labeled sugar and alcohol derivatives in gas chromatography

Abstract The recovery of trimethylsilyl (TMS) derivatives in gas chromatography was determined by using 14 C-labeled compounds. Under the conditions used approximately one-half of the cholesterol derivative was recovered. Four-fifths of the glycerol derivative and one-fourth of the stearyl alcohol derivative were recovered. Approximately one-fourth of the TMS derivatives of glucose, fructose and sucrose were recovered. Rechromatography of the TMS derivative of glucose resulted in the same loss as found initially. With the TMS derivative of glocuse- 14 C, radioactivity was found throughout the length of the column. Thus decomposition and/or deposition of the derivative occurred throughout the column. The recovery of the triflouroacetyl derivative of glucose was less than of the TMS derivative.

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.

Cysteine and glutathione in orange juice

Abstract The major part of the sulfhydryl compounds of both Valencia and Navel orange juice was found to exist as cysteine and glutathione. These two compounds were isolated and analyzed as their crystalline S -benzyl derivatives from concentrates prepared by preferential mercuric ion precipitation. Cysteine analysis before and after hydrolysis showed that all of the sulfhydryl of the concentrates was cysteine and glutathione. The glutathione analysis was confirmed by Woodwards glyoxalase method.