Trichloroacetic acid tyrosine

Application and Principle This procedure is used to determine the proteolytic activity, expressed as hemoglobin units on the tyrosine basis (HUT), of preparations derived from Aspergillus oryzae var. and Aspergillus niger var., and it may be used to determine the activity of other proteases at pH 4.7. The test is based on the 30-min enzymatic hydrolysis of a hemoglobin substrate at pH 4.7 and 40°. Unhydrolyzed substrate is precipitated with trichloroacetic acid and removed by filtration. The quantity of solubilized hemoglobin in the filtrate is determined spectrophotometrically. 

The properties of protein fragments can Ik also used for the fluorometric assay of proteolytic activity. Curoffproposed that the increase in trichloroacetic acid-soluble tyrosine (both free and bound in small peptides) can be fluorometrically assayed after the condensation with nitrosonaphthol. 

When 1.5 volumes acetone is added to the trichloroacetic acid filtrate of the gastric juice, an abundant flocculent precipitate forms, which contains all the components of dissolved mucin with the exception of soluble mucus. If this precipitate is taken up in dilute alkali and then acidified with dilute HCl down to pH 3.5, a fine flocculent precipitate forms, which we named dissolved mucoprotein (G27, G36). It was later renamed glandular mucoprotein (G9, G38) because of its close relationship to the fundic glands of the stomach. This material contained much protein its nitrogen content was 12.61 0.44% and its tyrosine content 7.50 0.65% by the Folin-Giocalteu reaction. The reducing substance content was 6.38 1.48% before and 12.5% after hydrolysis (G9, G27, G36) (see Table 4). Werner (W9) determined the composition of this mucoprotein fraction and found that it contained 11.2% N by Kjeldahl, 8.8% hexosamine, 4.8% uronic acid, and 2.0% sialic acid. 

The iodometric method was then modified (G23, G24) by changing the final concentration of trichloroacetic acid from 8% to 3.3%, leaving more proteose-like substances in the filtrate, and quantitating the mucosubstances in the filtrate colorimetrically by the Folin-Ciocalteu reaction with the phenol reagent, instead of iodometric titration. This colorimetric reaction determines the tyrosine and tryptophan content after alkaline hydrolysis of the total dissolved mucin. 

Protease activity was measured using 1% (w/v) casein as substrate dissolved in 0.1 M phosphate buffer (pH 3.0). Aliquots (2 ml) of diluted supernatant were mixed with 2 ml of 1% (w/v) casein and the reaction mixtures incubated at 40 °C for 10 min. An equal volume of trichloroacetic acid (0.4 1) was added to the reaction mixture, after filtration, the tyrosine released was quantified by the absorbance of the filtrate at 275 nm (A275). 

Autolytic assays were undertaken according to the method described by Morrissey et al. (1993) with the following changes. A 3 g surimi gel sample was finely chopped with a razor blade and incubated at 55°C for 1 hr. Autolysis was stopped by adding 27 mL 5% cold trichloroacetic acid (TCA), incubating the mixture at 4" C and centrifuging at 6100 x g for 15 min. The supernatant was analyzed for oligopeptide content by the Lowry assay (Lowry et al, 1951) and expressed as mmoles of tyrosine released. 

Suppose that sequence analysis of a protein has provided the data that the given protein contains 7 residues of t5Toslne. React the protein with radioactive iodine - 131 in a sultabk reaction mixture so that tyrosine residues pick up the iodine. After this precipitate the protein with the help of trichloroacetic acid. Count the precipitate. Suppose that the count given Is 4000 cpm. Now denature amother aliquot of the protein and subject this too to iodination reaction in the presence of radioactive iodine 131. After iodination is over, precipitate the protein and count for radioactivity. Suppose now that the count is 9000 cpm. 

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