Garfield P. Royer

A new cleavable reagent for cross-linking and reversible immobilization of proteins☆

Abstract We have prepared a new bifunctional reagent for the cross-linking and reversible immobilization of proteins through their amine groups. This compound, ethylene glycolyl bis(succinimidyl succinate), reacts rapidly with proteins, at pH 7 and at high dilution. The resulting protein cross-links are readily cleaved at pH 8.5 using hydroxylamine for 3–6 hr. at 37°C. Substantial enzymatic activity was observed with lactic dehydrogenase after such reversible cross-linking. Trypsin immobilized on agarose using this reagent retains full specific activity, is stable for weeks in the cold, and may be released with hydroxylamine at 25°C. This compound appears suitable for studies involving proteins with essential disulfide linkages.

Immobilization of glycoenzymes through carbohydrate side chains.

Abstract Glucoamylase, peroxidase, glucose oxidase, and carboxypeptidase Y were covalently bound to water-insoluble supports through their carbohydrate side chains. Two approaches were used. First, the carbohydrate portions of the enzymes were oxidized with periodate to generate aldehyde groups. Treatment with amines (ethylenediamine or glycyltyrosine) and borohydride provided groups through which the protein could be immobilized. Ethylenediamine was attached to glucoamylase, peroxidase, glucose oxidase, and carboxypeptidase Y to the extent of 24, 20, 30, and 15 mol/mol of enzyme, respectively. These derivatives were coupled to an aminocaproate adduct of CL-Sepharose via an N -hydroxysuccinimide ester or to CNBr-activated Sepharose. Coupling yields were in the range of 37–50%. Retained activities of the bound aminoalkyl-enzymes were 41% (glucoamylase), 79% (peroxidase), 71% (glucose oxidase), 83% (carboxypeptidase Y). A glycyltyrosine derivative of carboxypeptidase Y was bound to diazotized arylamine-glass. Coupling yield was 42% and retained esterase activity was 84%. In the second approach, the enzyme was adsorbed to immobilized concanavalin A and the complex was crosslinked. Adsorption of carboxypeptidase Y on immobilized concanavalin A followed by crosslinking with glutaraldehyde was also effective. The bound enzyme retained 96% of the native esterase activity and showed very good operational stability.

Immobilization of enzymes on aldehydic matrices by reductive alkylation.

Abstract We report here a convenient and inexpensive method of attaching enzymes to solid supports which contain diols. Dextran coated porous glass, Sepharose and glass coated with a glyceryl silane were oxidized with NaIO4. Trypsin, carboxypeptidase A, and carboxypeptidase B were bound to the oxidized supports by treatment with NaBH4. The pH dependence of the coupling reaction and loss of lysine in bound trypsin indicate that the immobilization occurs via reductive alkylation. The bound enzymes display good catalytic activity against synthetic substrates and proteins.

Stable attachment of redox groups for modified electrodes via cyanuric chloride

Abstract Methylaminopropylviologen (MAV) was covalently attached to glassy carbon electrodes via cyuranic chloride. X-ray photoelectron spectroscopy (XPS) analysis confirmed the presence of bound MAV on the electrode surfaces. Electrochemical experiments of these electrodes indicated that the bound MAV was stable and electrochemically active for extended periods of time in aqueous media. The surface coverage of MAV was in the range, of 2.0–3.0×10−10 mol cm−2.

N-(β-iodoethy)trifluoroacetamide: A new reagent for the aminoethylation of thiol groups in proteins

Abstract β-Iodoethyltrifluoroacetamide was prepared and evaluated for use in the aminoethylation of sulfhydryl groups in proteins. Lysozyme, ribonuclease A, chymotrypsinogen A, and soybean trypsin inhibitor were reduced using dithiothreitol in 6 m guanidine hydrochloride. The reduced proteins were then treated with the reagent. Conversion of cysteinyl to aminoethylcysteinyl residues averaged 97%; no destruction of nontarget residues was observed.

Immobilized carboxypeptidase Y. applications in protein chemistry

Carboxypeptidase Y (CPY), a metal-free exopeptidase found in yeast [l-S] , catalyzes the hydrolysis of both peptide and ester substrates. The enzyme contains serine and histidine at the active site and exhibits burst kinetics with p-nitrophenyl trimethylacetate [6-81. CPY is of interest to protein chemists because of its broad specificity. Proline is readily released from peptide linkages. Glycine and aspartic acid are released very slowly. We have demonstrated total hydrolysis of proline-free peptides with mixtures of immobilized enzymes [9]. The bound CPY described in this report has no endopeptidase activity and should be of considerable worth when combined with other enzymes in total hydrolysis of polypeptides which contain proline. We demonstrate herein the utility of the bound enzyme in the sequencing of a polypeptide containing proline. After many unsuccessful attempts to prepare an immobilized CPY derivative exhibiting good operational stability over a wide pH range, we have found that hexamethylenediamine-Cl-Sepharose 4B (prepared by periodate axidation and reductive alkylation) is the support of choice. The enzyme conjugate was made with water-soluble carbodiimide. The modified Sepharose prepared by the method described here should be useful as a support for other enzymes and affinity ligands.

Cross-linking of reversibly immobilized enzymes

There are at least two reasons for the introduction of covalent, intramolecular cross-links into proteins. Valuable information concerning the solution structure of the protein may be obtained [l] . In addition stabilization of the protein may be achieved. One problem inherent in the reaction of proteins with bifunctional reagents is the production of aggregates as a result of the formation of intermolecular crosslinks. We report here a new method of cross-linking which yields monomeric protein exclusively (fig.1). First, the protein is attached to a solid support via a cleavable linkage. Cross-linking is the second step. The last step is the release of the cross-linked monomer. Since the protein molecules are fixed and widely spaced on the matrix, intermolecular cross-links are prevented. Trypsin was attached to CL-Sepharose-NH(CH2)6 NHCO(CH2)2 S-S(CH2)2 CO-(N-hydroxy-succinimide) (I), cross-linked with diimidates, and released by reduction of the disultide linkage between the enzyme and support. In a second approach the protein was attached to the support through free thiols rather than through amino groups. To illustrate this method papain was reacted with CL-Sepharose-(glutathione-2,2’dipyridyl disulfide) (II). The bound enzyme was crosslinked with glutaraldehyde and released by treatment with DTT*.

Evidence for the Induction of a Conformational Change of Bovine Trypsin by a Specific Substrate at pH 8.0

A method is described that permits the covalent attachment of enzymes to solid matrices in the presence of saturating levels of a specific substrate under conditions where the enzyme is optimally active. Bovine trypsin immobilized on glass and agarose in the presence of saturating levels of N-α-benzoyl-L-arginine ethyl ester differs in a number of respects from the enzyme bound in the absence of substrate. Trypsin bound in the presence of substrate (ES form) is attached to porous glass through three more side chains than the enzyme coupled in the absence of substrate (E form). The rate of inactivation by iodoacetamide for the ES form is much greater than that for the E form. The two enzyme forms differ also in their reactivity with TLCK and DFP, active site-directed inhibitors. The pH dependences of the ES and the E forms bound to agarose differ; the pH optima are 9.0 and 8.5 for the ES and E forms, respectively. The thermal denaturation of the agarose-trypsins occurs in two distinct steps. The inactivation rate of the ES form is significantly slower than the E form in both stages. Also, for the hydrolysis of both N-α-benzoyl-L-arginine ethyl ester and N-α-benzoyl-Larginine amide, the Km values for the ES form were lower than the Km values for the E form. Our data suggest that a substrate-induced conformatonal change occurs with trypsin, and that this conformation is stabilized when the enzyme is bound by multiple attachments to a solid support. These findings support the “induced-fit” theory in general and lend support to the interpretation of the activation of trypsin by methyl guanidine that is part of a specific substrate (Inagami, T., and Hatano, H. [1969] J. Biol. Chem. 244, 1176).

Column chromatography on polyethylenimine-silica: Rapid resolution of nucleotides and proteins with short columns and low pressures

Abstract Silica gel (100–200 mesh) can be coated with a stable layer of crosslinked polyethylenimine (PEI). The resulting material is useful in column chromatography for quantitative separation of adenine nucleotides. For example, 3′:5′-cyclic AMP can be separated from a mixture containing AMP, ADP, and ATP. A mixture of adenosine, AMP, ADP, and ATP can be resolved with quantitative recovery of components. Convenient separation of cytochrome c from albumin illustrates the applicability of this system to protein purification. PEI-Xama silica gel and PEI-glutaraldehyde silica gel have ion-exchange capacities of 0.27 and 0.21 meq/g, respectively. The materials are dimensionally rigid and chemically stable except in alkaline solutions (>pH 10) for prolonged periods.

An ESR study of the active-site conformations of free and immobilized trypsin.

Abstract Trypsin covalently bound to porous glass free trypsin have been spin-labeled with 1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl methylphosphonofluoridate. The active-site conformations are identical whether the enzyme is labeled before or after immobilization. The active-site normality may be determined for immobilized trypsin by the spin-count technique.