Hidrológiai Közlöny 1967 (47. évfolyam)
7. szám - A „Szervesanyag meghatározási problémák édesvizekben” című 1966. szeptember 25–28. között Tihanyben rendezett Szimpózium előadásai - Procházková, Lidmilá: A Kjeldahl- és a protein nitrogén (Folin–Lowry eljárás) közötti különbség különböző típusú édesvizekben
Hidrológiai Közlöny 1967. 7. sz. 314 The Relationship of Protein Nitrogén Determined by the Folin Reaction and Organic Nitrogén according to Kjeldahl in Various Types of Freshwaters 1IDMUA P R O C H A Z K O V A* The Folin reaction, that is the action of phosphowolframate and phosphomolybdate in an alkaline médium on certain type of redueing substances accompanied by the development of an intensive blue color, was originally used for the determination of phenols. Later it was emploved, particularly in clinical practice, for the determination of proteins. It is commonly known that the tyrosine and trvptophan groups of the peptidic chain are responsible for the reaction of proteins with this reagent. Both these aminoacids give an intensive color even if much diluted (Folin and Cioralteu 1927). Lowry (1951), using this reaction, investigated the influence of pH, reaction time and particularly the added bivalent copper. Povoledo and Gerletti (1962) modified this method for the protein determination in water and alsó defined the optimál conditions for this purpose. Folin's reaction is very sensitive, however its specifity is rather poor. The course of this reaction is not clear up to now. At the present time, Billman, Borders et al (1965) and Billman and Seiling (1965) have dealt with the reduction of heteropolvacids. They studied before all the reaction of the molybdosilicic acid, which they recommend as a relatively specific reagent for carbonvl compounds having at least one hvdrogen atom in alpha position. They assume that these compounds are not directlv oxidized but rather their products occurring in an alkaline médium, namelv the /5-hydroxy carbonvl compounds. As concerns the phosphowolframic acid they onlv mention that it is much less specific giving a positive test with somé noncarbonyl compounds. The aimjof our investigation has been to outline the main interferences in determining the proteins by the use of the Folin reagent and find the ratio of the Folin nitrogén to the Kjeldahl organic nitrogén in various types of freshwaters. During our studv on interferences, we used mainly substances of a redueing character. From the literature (Folin, Denis 1912) it is known that Folin's reagent reacts before all with phenols and structurally related compounds. Besides somé simple phenols, we alsó used tanúin, calciumhumate and lignin, which all give a positive reaction. Tyrosine, responsible for the protein reaction, alsó belongs to phenolic substances. Trvptophan, which gives a somewhat slighter reaction than the latter, does not possess any phenolic group, but the imino-group of indole may be considered as a related one. Folin and Looney (1922) report that from seventeen aminoacids studied, only tyrosine and tryptophan have given a positive reaction. We * Hydrobiological Laboratory of the Czechoslovak Aeademy of Sciences. Praha 5, Vltavská 17. have alsó found a positive reaction. yet somewhat smaller in the presence of cysteine. Histidine has given a slight blue color only at very high concentrations, while under the same conditions no reaction was found with other aminoacids. A positive test has been alsó observed in the presence of the following organic substances: uric acid (alsó reported by Folin and Denis 1912), ascorbic acid, thiourea, triethanolamine, aniline. Urea reacts negatively. Alsó the easily oxidizable inorganic substances react inasimilar wav:* Ferrous sulphate, sodiumbisulphite and manganous sulphate. A positive reaction of cyanides has been alreadv inentioned by Folin and Cioralteu (1927). No positive reaction has been observed with sulphocyanides. An intense coloration is caused by hydrazine and hydroxylamine. Benzaldehvde reacts positively, but acetaldehyde only at very high concentration. This is apparently caused by a rapid aldolic or the Canizzarro reaction in an alkaline médium. In presence of sugars we found no positive reaction. Table 1 presents the main reacting substances, arranged according to their absorbance. Table 1. Substances reacling positively with the Folin reagent arranged according io (he value of their absorbance A Substance A Substance 1,16 Tyrosine 0,24 MnS0 4 1,06 Phenol 0,19 NaCN 0,95 Hydrochinone 0,18 Uric Acid 0,90 Hydrazine. H 2S0 4 0,16 Thiourea 0.80 Hydroxylamine. . HC1 0,16 FeS0 4 0,80 Tryptophan 0,14 Ca-Humate 0,60 Tahin 0.14 Cvsteine 0,35 Aniline 0,09 Na 2S0 3 0,30 Lignin 0,09 Ascorbic Acid 0.28 Triethanolamine 0,03 Benzaldehvde 20 mg/l; 1 cm path length From this it follows that the main source of interference in natural waters are substances of liumic character, the positive reaction of which has been made use of bv Povoledo and Gerletti (1964) during their studv of organic substances in sediments. Onlv in an anaerobic médium the interference of inorganic redueing substances might alsó come into account. For a time period of one and a half year parallel determinations were made on protein nitrogén according to Povoledo and Gerletti (1962) and the * In easily oxidizable substances distilled water was used for dilution from which oxvgen was removed by bubbling with nitrogén.
