Mitochondrial malate dehydrogenase (L-ma-1ate:NAD' oxidoreductase, EC 1.1.1.37) has been purified from both rat liver and rat heart by using Sepharose-Blue Dextran pseudoaffinity chromatography. Under the conditions employed most of the mitochondrial malate dehydrogenase and some cytosolic malate dehydrogenase are adsorbed, whereas many proteins simply pass through. The blue column is then subjected to several treatments which elute a variety of other proteins (notably residual cytosolic malate dehydrogenase and lactate dehydrogenase) which also have an affinity for the bound dye Cibacron Blue F3GA. Mitochondrial malate dehydrogenase is then eluted by forming an abortive ternary complex with reduced nicotinamide adenine dinucleotide-sodium D( +)-malate. Pooled active fractions are passed through a preequilibrated diethylaminoethyl-Sephadex column which retains contaminating proteins. The active fractions are concentrated, and residual contaminants are removed by very small stepwise pH changes on a CM-52 cellulose column. The procedure is mild and rapid and yields enzyme which is homogeneous by the applied criteria. In M i t o c h o n d r i a l malate dehydrogenase (m-MDH) has been isolated and partially characterized from various sources (Ochoa

Alpha-Chymotrypsin was light sensitized by acylating with cis-cinnamoyl ester, a substrate interconvertible to the trans form by ultraviolet (UV) light. The degree of acylation by this method was complete leaving no residual activity of the enzyme. Upon UV irradiation the inhibited enzyme regained about 70% of its original activity, thereby adding light-sensitiveness to the proteolytic enzyme. In seeking a photographic application of the light-sensitized enzyme, a pigmenting enzyme was incorporated with it. The coupled enzyme system was shown to exhibit a light signal in the form of dark pigment slurry.

SummaryA unique silver-free and ultraviolet (UV)-sensitive photographic process using enzymes has been developed. It utilizes a-chymotrypsin acylated to a light-sensitive stereoisomeric ester as the basic photographic material. When U V light is exposed to the film, the signal is registered by the appearance of melanin pigment through the chymotrypsin-mediated activation of pre-tyrosinase.

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