High‐performance liquid chromatography (HPLC) is still, today, the technique of choice for the analysis of natural dyes in artistic objects and historical textiles, particularly in association with photodiode array (PDA) detection. In the last two decades, surface‐enhanced Raman spectroscopy (SERS) gained also increasing importance for these investigations thanks to its sensitivity and limited requirements in terms of sample quantity. In favor of SERS, its high specificity in molecular recognition typical of vibrational spectroscopy should be mentioned, whereas this non‐separative technique is obviously disadvantaged in the analyses of mixed chromophores, as is often the case of many natural dyes and also of tints obtained by the combined use of different colorants. An optimized experimental setup combining the two techniques, HPLC‐PDA and SERS, is proposed in the present work, allowing online SERS detection of different dyeing compounds eluted from the HPLC column. Examples are presented concerning some of the colorants most widely used in history, such as morin and luteolin for yellow dyes, alizarin, purpurin, laccaic, kermesic, and carminic acids for red ones, and indigotin for blue tints. Copyright © 2016 John Wiley & Sons, Ltd.

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Diethyl pyrocarbonate inactivated the tetrameric 3 alpha,20 beta-hydroxysteroid dehydrogenase with second-order rate constants of 1.63 M-1 s-1 at pH 6 and 25 degrees C or 190 M-1 s-1 at pH 9.4 and 25 degrees C. The activity was slowly and partially restored by incubation with hydroxylamine (81% reactivation after 28 h with 0.1 M hydroxylamine, pH 9, 25 degrees C). NADH protected the enzyme against inactivation with a Kd (10 microM) very close to the Km (7 microM) for the coenzyme. The ultraviolet difference spectrum of inactivated vs. native enzyme indicated that a single histidyl residue per enzyme subunit was modified by diethyl pyrocarbonate, with a second-order rate constant of 1.8 M-1 s-1 at pH 6 and 25 degrees C. The histidyl residue, however, was not essential for activity because in the presence of NADH it was modified without enzyme inactivation and modification of inactivated enzyme was rapidly reversed by hydroxylamine without concomitant reactivation. Progesterone, in the presence of NAD+, protected the histidyl residue against modification, and this suggests that the residue is located in or near the steroid binding site of the enzyme. Diethyl pyrocarbonate also modified, with unusually high reaction rate, one lysyl residue per enzyme subunit, as demonstrated by dinitrophenylation experiments carried out on the treated enzyme. The correlation between inactivation and modification of lysyl residues at different pHs and the protection by NADH against both inactivation and modification of lysyl residues indicate that this residue is essential for activity and is located in or near the NADH binding site of the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

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