Comp. Biochem. Physiol. Vol. 78B, No. 2, pp. 351-353, 1984 0305-0491/8453.00+ 0.00 Printed in Great Britain © 1984 Pergamon Press Ltd A MODIFIED LOWRY ASSAY TO MEASURE LEVELS OF PROTEINS LACKING AROMATIC AMINO ACID RESIDUES (e.g. METALLOTHIONEINS) V. W. T. WONG and P. S. RAINBOW School of Biological Sciences, Queen Mary College, Mile End Road, London E1 4NS, UK (Tel: 01-980-4811) (Received 25 November 1983) Abstract--1. A combination of two modifications to the Lowry protein assay is recommended for estimation of metallothionein (lacking aromatic amino acid residues and requiring reducing conditions for isolation). 2. Serum thymic factor (STF), a decapeptide lacking aromatic amino acids, is recommended for use as a standard after allowance for the effects of short chain length on colour intensity development. INTRODUCTION Metallothioneins are a group of low mol. wt metal- binding proteins which are thought to occupy a central position in the metabolism of particular trace metals such as copper, cadmium, zinc and mercury (Vallee, 1978). They act as ligands for these toxic metals absorbed by animals exposed to metal pollu- tion (Noel-Lambot, 1976) and they may play a role in the regulation of levels of these trace metals in the organism. Metallothioneins are widespread among vertebrates and invertebrates, differing only slightly between species in their amino acid sequences. Char- acteristically, metallothioneins have a low mol. wt (about 12,000), are very high in cysteine residues (about 30~o) and lack the aromatic amino acids tyrosine and tryptophan (VaUee, 1978). Under ox- idising conditions, the isolated proteins tend to form aggregates via disulphide bridges on the cysteine residues (Minkel et al., 1980), but can be protected by the use of a reducing agent such as DTT* at concen- trations of 1 mM (Cleland, 1964). The present authors are working to isolate and study metallothionein in the crab Carcinus maenas, and in the course of this study, we needed to obtain absolute protein values to relate to metal values. It is difficult to obtain sufficient amounts of isolated crab metallothionein for gravimetric or amino acid anal- ysis and purified metallothionein standards from other sources are as yet unavailable commercially. A routine procedure for measuring metallothionein was therefore required using a common protein as a standard. A simple, direct and sensitive procedure for ana- lysing metallothionein in crude homogenates has not yet been devised (Olafson and Sim, 1979). Con- sequently, workers in this field have relied on a variety of crude and cumbersome techniques: ultra- *Abbreviations: BSA, bovine serum albumin; CHM, chymotrypsinogen-A; DTT, dithiothreitol; OVA, oval- bumin; STF, serum thymic factor. violet absorption at 250nm based on the molar extinction coefficient of 6.8ml/mg/cm for metal- lothionein (Kagi and Vallee, 1961) is too non-specific to be sufficiently accurate: a modified biuret method (Nordberg et al. 1972; Piscator, 1962) was found to be too insensitive for our purposes; dye-binding methods with reagents such as Amido Black (Pis- cator, 1962) and Coomassie Blue (Overnell and Grant, 1981) were unsuitable because of the variable nature of their responses to different proteins (Pierce and Suelter, 1977; Van Kley and Hale, 1977); staining with Schiff's reagent (Overnell and Grant, 1981) requires further developments; and finally, elaborate electrochemical techniques (Olafson and Sim, 1979), fluorescence techniques (Bohlen et al., 1973; Naka- mura and Pisano, 1976) and determinations based on amino acid analysis (Hoch and Vallee, 1953; Buhler and Kagi 1974; Kagi et al., 1974; Overnell, 1982a,b) were considered to be too time-consuming for routine application. Lowry's method for protein assay (Lowry et al., 1951) is a widely used procedure in routine analysis. The Lowry assay can be interpreted in terms of two reactions: (1) the biuret reaction--an initial reaction whereby copper ions are thought to bind to the protein backbone, and (2) the Folin phenol addition--whereby tyrosine and tryptophan residues enhance the final colour development. Protein mea- surements using the Lowry method are often made against a common "standard" protein such as bovine serum albumin (BSA) and protein values are given in "BSA units" rather than exact values. The lack of aromatic amino acid residues in metallothionein will clearly affect further colour development in com- parison with BSA. The unmodified Lowry method was therefore considered unsuitable for our purposes. Modifications have since been made to the Lowry method to render it more suitable for our require- ments. Dorsey et al. (1977) devised a simple yet effective modification of Lowry's method which ap- pears to bring different proteins to a similar final colour development (by the inclusion of a heating 351 C.B,P. 78/2B~D