TRANSGLUTAMINASE 15 Glued ends ß •Hai r sample Figure 1. Hair sample configuration for isotope experiments. An example of a stainless steel hair clip and a plastic microvial are shown. Hair samples and clips were incubated in individual microvials and removed for rinsing in a larger container. Hair samples were removed from clips after rinsing and put into scintil- lation vials for analysis. containing 2 mM DTT and 2 mM EDTA. One unit of enzyme activity was equivalent to 1 •mole substrate converted/min at 25øC. Separate sample and reference cuvettes were used for the optical assay. 1.0 ml assay solution, 10 •1 NADH solution, 10 •1 amine solution, and 40 •i GiDHase were added to each cuvette. The solutions were mixed and equilibrated for approximately five minutes and the absorbance read at 340 nm in a spectrophotometer. A 10-•1 aliquot of transglutaminase preparation was added to the sample cuvette and 10 •i of distilled water to the reference cuvette, and both cuvettes were incubated for at least one hour. Absorbance was read at 340 nm at specific time points. Enzyme activity was calculated based on •moles amine/min, using the difference in absorbance between the sample and reference cuvettes (i.e., AA sample vs AA reference). An NADH mM extinction coef- ficient of 6.22 i/mmol/cm was used for the calculation. RADIOISOTOPE ASSAY A filter paper assay based on incorporation of [14C]-putrescine into DMC was adapted with minimal change from the method described by Lorand et al. (38). Lyophilized transglutaminase was reconstituted in Tris buffer (10 mM Tris hydrochloride, 2 mM EDTA, 2 mM DTT, pH 7.5) and stored on ice until use. The assay buffer (50 mM Tris hydrochloride, 5 mM CaCi 2, 20 mM DTT, 5 mg/ml DMC, pH 7.5) was made up in bulk and stored frozen until use. The DMC was omitted from incubations with hair, analogous to the procedure used for the optical assay. The labeled putrescine stock solution was prepared with 50 •Ci of [14C]-putrescine (@ 115 mCi/mmol in 1.0 ml of 2% ethanol/water) and was diluted to 2.0 ml with cold putrescine hydrochloride in 50 mM Tris hydrochloride (pH 7.5) to provide final stock solution of 1.2 mM putrescine. For all studies, the stock solution was diluted 1:10 to provide a final assay concentration of 0.12 mM. The solution was stored frozen until use.

16 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS Assays were performed at 37øC unless indicated otherwise. For hair studies, a variety of rinsing protocols, described below, were utilized to ensure removal of non-covalently bound [•4C]-putrescine following enzyme incubation. Sample activity was assessed in counts per minute (cpm) using 4 ml of Cytoscint (Fisher) cocktail and counting with a Beckman liquid scintillation counter with a wide open (0-1000) window to 1.0% error. HYDROXAMATE OPTICAL ASSAY Transglutaminase activity was evaluated before use with a colorimetric assay based on the formation of a colored ferric-hydroxamate complex (39). Hydroxamates are formed enzymatically by reaction of hydroxylamine with glutamine-containing peptides. This assay was not applicable for evaluation of transglutaminase activity with hair, but was used to assess activity of transglutaminase preparations and stocks as received. The activity of the transglutaminase supplied by Sigma was tested using the hydroxamate assay because the enzyme was sold on the basis of units derived from this assay. Figure 2 shows that the specific activity obtained with the hydroxamate assay was in excellent agreement with product specifications. The enzyme was also found to be highly active with the radioisotope assay (not shown). However, the results of the two assays were not compared on a "unit" basis because the assays utilized different substrates. The isotope assay was therefore used only to provide information on relative activities. RESULTS OPTICAL ASSAY The approach first conceived for evaluation of transglutaminase activity was a continuous optical assay based on coupling the primary reaction to a secondary enzymatic reaction (37). Free ammonia released by transamidation was enzymatically converted in the presence of ot-keto glutarate to glutamic acid by glutamate dehydrogenase. Since this oxidizes reduced nicotinamide adenine dinucleotide (NADH) to NAD +, the rate of transamidation was monitored by loss of absorbance at 340 nm: TRANSAMIDATION REACTION L•340 { • NH 3 NADH )• tz-Ket)Glut NAD + Glutamate The fixed-time assay described above was first optimized by monitoring transglutami- nase activity continuously with dimethyl casein as the glutamine donor, and histamine, putrescine, or hydroxylamine as the amine (Figure 3). The assay was found to be linear, both with time and enzyme concentration, and considered suitable for evaluation with hair in either a fixed-time or continuous format.