TY - JOUR
T1 - Water-Insoluble Enzymes. Synthesis of a New Carrier and Its Utilization for Preparation of Insoluble Derivatives of Papain, Trypsin, and Subtilopeptidase A
AU - Goldstein, L.
AU - Pecht, M.
AU - Blumberg, S.
AU - Atlas, D.
AU - Levin, Y.
PY - 1970/5/1
Y1 - 1970/5/1
N2 - A new type of highly insoluble polyfunctional, diazotizable resins, (dialdehyde)-starch-methylenedianiline (S-MDA), could be prepared by the condensation of dialdehyde starch (a commercially available periodate oxidation product of starch) with p,p′-diaminodiphenylmethane (MDA) and the subsequent reduction of the Schiff's base-type polymeric product. The S-MDA resins following diazotization were coupled with papain and mercuripapain, subtilopeptidase A (subtilisin Carlsberg), and polytyrosyl trypsin, giving highly active water-insoluble derivatives of these enzymes. The S-MDA resins had diazotization capacities of 0.26–0.33 mequiv/g and a protein binding capacity of about 100 mg of protein/g. The water-insoluble S-MDA–protein conjugates had particulate form, were easily filtered and could be conveniently used in columns. Amino acid analysis of acid hydrolysates of S-MDA derivatives of papain, subtilopeptidase A, and polytyrosyl trypsin showed that the tyrosine, arginine, and lysine contents of the insoluble derivatives were considerably lower than the respective values obtained for the native enzymes. It was assumed that the missing amino acids represented the amino acid residues through which the covalent bonds between the protein and the diazotized carrier were formed. The pH–activity profiles of S-MDA–papain, S-MDA–subtilopeptidase A, and S-MDA–polytyrosyl trypsin, acting on benzoylglycine ethyl ester, acetyl-L-tyrosine ethyl ester, and benzoyl-L-arginine ethyl ester, respectively, were displaced toward more alkaline pH values by one to two pH units, as compared with the native enzymes. This effect was found to be essentially independent of the ionic strength of the medium.
AB - A new type of highly insoluble polyfunctional, diazotizable resins, (dialdehyde)-starch-methylenedianiline (S-MDA), could be prepared by the condensation of dialdehyde starch (a commercially available periodate oxidation product of starch) with p,p′-diaminodiphenylmethane (MDA) and the subsequent reduction of the Schiff's base-type polymeric product. The S-MDA resins following diazotization were coupled with papain and mercuripapain, subtilopeptidase A (subtilisin Carlsberg), and polytyrosyl trypsin, giving highly active water-insoluble derivatives of these enzymes. The S-MDA resins had diazotization capacities of 0.26–0.33 mequiv/g and a protein binding capacity of about 100 mg of protein/g. The water-insoluble S-MDA–protein conjugates had particulate form, were easily filtered and could be conveniently used in columns. Amino acid analysis of acid hydrolysates of S-MDA derivatives of papain, subtilopeptidase A, and polytyrosyl trypsin showed that the tyrosine, arginine, and lysine contents of the insoluble derivatives were considerably lower than the respective values obtained for the native enzymes. It was assumed that the missing amino acids represented the amino acid residues through which the covalent bonds between the protein and the diazotized carrier were formed. The pH–activity profiles of S-MDA–papain, S-MDA–subtilopeptidase A, and S-MDA–polytyrosyl trypsin, acting on benzoylglycine ethyl ester, acetyl-L-tyrosine ethyl ester, and benzoyl-L-arginine ethyl ester, respectively, were displaced toward more alkaline pH values by one to two pH units, as compared with the native enzymes. This effect was found to be essentially independent of the ionic strength of the medium.
UR - http://www.scopus.com/inward/record.url?scp=0014963341&partnerID=8YFLogxK
U2 - 10.1021/bi00813a016
DO - 10.1021/bi00813a016
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C2 - 5430716
AN - SCOPUS:0014963341
SN - 0006-2960
VL - 9
SP - 2322
EP - 2334
JO - Biochemistry
JF - Biochemistry
IS - 11
ER -