J. Seric. Sci. Jpn. 65 (2), 109-113 (1996)

Possible involvement of and in the

yellowing of acid hydrolyzed silk

CHEN KAILI, ZUGLUL RAIDER AYUB and KIYOSHI HIRABAYASHI Department of Biotechnology, Faculty of Technology, Tokyo University of Agriculture and Technology (Received June 6, 1995)

Tryptophan and are known to be involved in the yellowing of silk hydrolysate. The present study confirmed that serine and glycine become yellow on hydrolysis with hydrochloric acid like and tyrosine. Decomposition of glycine was not fast in comparison with that of serine due to the absence of hydroxy group. Serine and glycine required water for yellowing. However, yellowing of these 2 amino acids was not influenced by the presence of oxygen during hydrolysis. The degree of yellowness (Y.I.) was high in serine and glycine, but not in when they were treated with acid. The proportion of carbon in serine and glycine increased during hydrolysis with acid. The formation of double bond in the hydrolyzing process might be responsible for the yellowing effect.

Key words: serine, glycine, yellowing, acid hydrolysis, silk

Acid hydrolysis always makes fibroin solu- , cystine, and tion yellow. Chemical changes in amino tryptophan (HIRs, 1964). , glutamic acids, such as tryptophan and tyrosine, is acid and glycine are highly fragile during heat generally considered as the main reason for treatment (SETOYAMA,1982). In this report, free yellowing (OKAMOTOand KIMURA, 1964). How- amino acids as model compounds were ever, the yellowing of fibroin solution was treated with acid to detect the yellowing proc- observed again even after the decolourization ess, and the yellowing of silk fibroin due to the of acid hydrolysate with activated charcoal. presence of amino acids, especially serine and This phenomenon indicates that other amino glycine, is discussed. acids than tryptophan and tyrosine made the Materials and Methods decolourized fibroin solution yellow. Serine and threonine break down easily during hy- Analysis of the yellowing of amino acids: drolysis. Other amino acids in fibroin decom- Amino acids were purchased from Kokusan pose in the following order: tyrosine, Chemical Company. These were each treated with 6 N hydrochloric acid or neutral water at Koganei, Tokyo 184, Japan. 110°C for 24 hr and the solutions were observed

-109- 110 Journal of Sericultural Science of Japan Vol. 65 No. 2 for the colouring. Then, the amino acids that phenylalanine, , and exhibited extensive yellowing were each alanine were treated with 6 N hydrochloric treated with 6 N hydrochloric acid or neutral acid or neutral water. Only serine and glycine water at 110°C for 72 hr. The solutions (after remarkably turned into yellow (Table 1). neutralized with sodium hydroxide if neces- Then, further description will be restricted sary) were dialyzed using a Microacelyzer-G3 mainly to serine and glycine. Deep yellow

(Asahi Kasei Corporation) and freeze-dried. colour was observed with the increase of time. The resulting powder was dissolved to prepare Yellowing of serine was faster than that of 2% aqueous solutions, which were measured glycine. The deep yellow colour of serine for the degree of yellowness using Colour and finally became brown.

Colour Difference Meter TC-1500MC (Tokyo Table 2 shows Y.I.s in serine and glycine.

Denshoku Co. Ltd.). The yellowness index Serine turned into yellow more rapidly than

(Y.I.) was determined applying the equation glycine even though the treatment time was the 100 (1.28X -1.06Z) / Y, where X, Y, Z are same. Moreover, Y.I.s were higher in acid tristimulus values (YABE, 1982). treated amino acids than water treated ones,

Analysis of the proportions of carbon, indicating that the yellowness was accelerated hydrogen and nitrogen: The yellow powders by acid treatment. of amino acids prepared as described above were dried by heating in vacuo at 60°C for 72 hr to eliminate moisture and a 2 mg aliquot of each sample was subjected to the analysis Table 1. Change in colour of amino acids treated with 6 N hydrochloric with an Elementary Analyzer MT-5 (Yamato acid or neutral water at 100•Ž for 24 hr. Kagaku Corporation).

Examination of the effect of oxygen and water on the yellowing of amino acids:

Deionized water was boiled in a stream of nitrogen for 48 hr and used to prepare deoxi-

dized 6 N hydrochloric acid. With this acid

solution, amino acids were hydrolyzed at

110•Ž for 72 hr in a stream of nitrogen. As a

control, usually prepared aqueous solutions - , no change. of amino acids were irradiated at 265 nm with

an ultraviolet lamp (Black Ray Lamp, UVG- Table 2. Yellowness indices (Y.I.s) of D15, San Gabriel California 91778 U.S.A.). To serine and glycine treated with 6 N examine the effect of water for the yellowing, hydrochloric acid or neutral water.

solutions were individually heated

for 72 hr at 100°C with water, and Y.I. of the

solutions was determined.

Results and Discussion

Yellowing of amino acids

First, 6 amino acids serine, glycine, Chen et al.: Yellowing of serine and glycine 111

Yellowing mechanism of serine and glycine This n -electron type double bonded com- On the neutralization of acid-treated serine pound is considered to be responsible for the solution with sodium hydroxide, ammonia yellowing effect. During hydrolysis of this gas was generated as confirmed by the occur- imino acid, CH3COCOOH (pyruvic acid) and rence of white fume in contact with fuming ammonium chloride were formed. Possibly, hydrochloric acid and by the Nessler's reagent the double bond in pyruvic acid is also in- test. Ammonia gas produced during the acid volved in the yellowing. The anticipated reac- treatment may react with hydrochloric acid to tion process is shown in Fig. 1. produce ammonium chloride. Generation of ammonia gas on the neutrali- Elementary analyses (Table 3) showed that, zation of acid treated glycine solution sug- when glycine was treated with neutral water, gests that glycine is transformed into the proportions of carbon and nitrogen in- CH(=NH)COOH like serine during the heat creased instead of decreased hydrogen, treatment. However, in the case of glycine the whereas the proportions of hydrogen and reaction was slower in comparison with nitrogen decreased instead of increased car- bon when glycine was treated with hydrochlo- ric acid. As to serine, similar results were obtained to those of glycine when treated with water, but the proportion of carbon markedly increased when reacted with hydrochloric acid. The proportions of nitrogen and hydro- gen of serine decreased when it was treated with acid. Dehydration occurring between the hydrogen atom attached to alpha carbon and hydroxy group of serine molecule may result in the formation of a double bonded com- pound CH2=C(NH2)COOH. Under acidic conditions dehydration process is accelerated to produce CH3C(=NH)COOH (imino acid).

Table 3. Proportions (w/w%) of H, C and N in serine and glycine treated with 6 N hydrochloric acid or neutral water.

Fig. 1. Decomposition process of serine when treated with 6 N hydro- chloric acid or neutral water. 112 Journal of Sericultural Science of Japan Vol. 65 No. 2

Fig. 3. Change in yellowness index (Y.I.) of serine treated with 6 N hydro- chloric acid in air (solid circles) or in vacuo. (open circles).

Table 4. Yellowness indices (Y.I.s) of Fig. 2. Decomposition process of heat treated serine and glycine. glycine when treated with 6 N hydro- chloric or neutral water. serine, because there was no hydroxy group in glycine. However, partial transformation of CH(=NH)COOH finally produced ammonia and HCOCOOH when the reaction was acceler- ated by the presence of acid. This phenomenon is in agreement with the extent of the genera- for 72 hr in the absence of water (Table 4), no tion of ammonia gas and the colour of the yellowing effect was found. solution. The proposed decomposition proc- ess of glycine is illustrated in Fig. 2. Possible contribution of glycine and serine in the yellowing of silk Effects of oxygen and water on the yellowing We infer that the yellowing of silk is largely of amino acids contributed by the presence of glycine and Fig. 3 shows that Y.I.s of serine hydrolyzed serine, whose contents in fibroin are as high in the presence and absence of oxygen were as 46% and 12%, respectively. Above results almost the same. This suggested that there indicate that the yellowing of acid hydrolyzed was no influence of oxygen on the yellowing silk is stimulated by the presence of water. effect during the hydrolysis process. When Further study to confirm the actual contribu- glycine and serine were dehydrated at 110°C tion of these amino acids in the yellowing of Chen et al.: Yellowing of serine and glycine 113 silk is under way in our laboratory. fibroin. Sen-i Gakkaishi, 10, 629-631. SETOYAMA,K. (1982): Effect of water on the References heat-yellowing of silk fabric and the changes in amino acid composition in silk HI s, C.H.W. (1964): The Chemistry of pep- fibroin in the sealed tubes by heat treatment. tides and proteins. Ann. Rev. Biochem., 33, J. Seric. Sci., 51, 365-369. 597-632. YABE, A. (1982) : "Senshoku Jiten", p. 130, OKAMOTO,S. and KIMURA, T.(1964): The de- Asakura Shoten, Tokyo. composition of silk fibroin by sunlight (II) on the decomposition of tryptophan in

陳 開 利 ・ZUGLUL RAIDER AYUB・ 平 林 潔:酸 加 水 分 解 絹 の 黄 変 にお ける セ リン と グ リ シ ン の寄 与 の可 能 性

酸 加 水 分 解 絹 の黄 変 に トリプ トフア ン とチ ロ シ ン以外 に も他 の ア ミノ酸 の 影 響 が あ る可 能 性 を 明 らか に した。 グ リシ ン とセ リン は ト リプ トフ ア ンと チ ロ シ ンと 同様に 黄 変 した。 グ リ シ ン に は 水 酸 基 が な い の で,セ リ ンに比 べ て グ リシ ンの分 解 速 度 は さ ほ ど早 くな か った。 ア ミノ酸 の 黄 変 は水 分 が存 在 す る と促 進 さ れ た。 しか し,こ れ らの 黄 変 に は酸 素 の 影 響 は認 あ られ な か っ た。 酸 で処 理 した ア ミノ酸 の黄 変 度 は高 く,セ リ ン とグ リ シ ンを酸 で処 理 した と き炭素 の 割 合 が 増 加 し た。黄 変 に は加 水 分 解 過 程 に お い て二 重 結 合(π 電 子)の 形 成 が 関与 す る もの と推 察 され る。