Serum albumin beads: an injectable, biodegradable system for the sustained release of drugs.

Biologically active compounds were entrapped in cross-linked serum albumin microbeads. Injection of these drug-impregnated beads into rabbits produced no adverse immunological reactions. Sustained release (20 days) of progesterone was demonstrated in vivo.

The effect of calcium ion on the urea denaturation of immobilized bovine trypsin.

The effect of calcium ion on the urea denaturation of trypsin has been investigated. By using trypsin immobilized on glass beads, all possibilities of autolysis occurring during the denaturation process are eliminated. It was found that in 8 M urea calcium ion markedly decreases the denaturation rate of the immobilized trypsin. Conversely, the presence of calcium ion markedly accelerates the rate of renaturation of denatured immobilized trypsin. Calcium may exert its stabilizing effect on the tertiary structure of the protein by coordination to the side chains of Asp 194, Ser 190 and the carbonyl group of Ser 139 (using the chymotryptic numbering system).

Use of immobilized carboxypeptidase Y (I-CPY) as a catalyst for deblocking in peptide synthesis.

CPY is a metal-free carboxypeptidase from yeast with broad specificity [1]. In addition to exopeptidase activity at acid pH, the enzyme is an effective esterase at alkaline pH. N-alpha-acetyl-L-tyrosine ethyl ester is hydrolyzed faster by CPY than by chymotrypsin. These observations suggested that the immobilized form of the enzyme would be of value in removing ester groups from the C-terminal ends of peptides. In this report we describe sequential synthesis using I-CPY and alpha-COOH deblocking of peptides made by conventional methods.

Stabilization of kappa-carrageenan gel with polymeric amines: Use of immobilized cells as biocatalysts at elevated temperatures.

Spherical beads of kappa-carrageenan containing entrapped cells were prepared in a two-step process. First, the beads were formed by dispersing a warm carrageenan cell suspension into stirring oil. After cooling (gelation) the beads were cured by treatment with amines. Ten amines of various sizes and structures were tested. We evaluated the mechanical strength and the applicability of aminetreated gels as immobilization matrices. The results of critical compression tests indicate that linear and branched polyethylenimines (PEI) are both good curing agents. PEI treated carrageenan beads also exhibited superior resistance to heat and abrasion. Furthermore, PEI polymers were demonstrated to be effective in stabilizing the lactase activity of the free and immobilized Bacillus stearothermophilus cells. The immobilized cell preparations of Saccharomyces cerevisiae, B. stearothermophilus, and Flavobacterium sp. were treated with branched PEI and evaluated for the activity of invertase (EC 3.2.1.26), lactase (EC 3.2.1.23), and glucose isomerase (EC 5.3.1.18), respectively, in a packed bed reactor at 60 degrees C. The apparent half-lives were 108, 39, and 64 days, respectively.

Synthetic derivatives of polyethyleneimine with enzyme-like catalytic activity (synzymes).

A synthetic polymer has been prepared which contains dodecyl groups (to bind small substrate molecules) and methyleneimidazole side chains (as nucleophilic catalytic sites) linked to a polyethyleneimine framework. This macromolecule, with a high local concentration of binding and catalytic groups, catalyzes the hydrolysis of uncharged nitrophenyl esters in water at pH 7 with rates markedly greater than previously observed with any other synthetic substances under similar conditions.

Substrate-induced conformational changes in lactate dehydrogenase. Proteolysis of the immobilized enzyme in the presence of specific substrates.

We report here a new approach to the study of the conformation of enzymes in the presence of specific substrates. Rabbit muscle lactate dehydrogenase was attached to CL-Sepharose via a cleavable spacer arm (-NH-(CH2)6NHCO(CH2)2SS(CH2)2CO-). The bound lactate dehydrogenase was digested with subtilisin BPN' in the presence of substrates of lactate dehydrogenase. The use of a flow system permits the maintenance of saturating levels of substrates. Proteolysis was followed by loss of activity of the enzyme column. The time course of proteolysis in the presence of either NADH, NAD+, or pyruvate alone did not differ from the control. However, when NADH and pyruvate were present simultaneously, the enzyme became more susceptible to proteolysis. The initial rate of proteolysis was increased by 40%. The abortive ternary complex (lactate dehydrogenase - NAD+ - pyruvate) also showed an increase in susceptibility to proteolysis. These findings clearly show that the productive ternary complex (lactate dehydrogenase - NADH - pyruvate) is conformationally different from the apoenzyme and binary complexes under optimal catalytic conditions.