1

Sci.Rep_Fukushima Univ.,'No.64 (2000)

Adsorption of Volatile Organic Compounds onProteins and Protein Models 1.

Hitoshi KANAzAwA and Nobuko SATe

Faculty of Education, Fukushima UniversityMatsukawa-machi, Fukushima 960-1296, Japan

(、Recet11ed Apri l .10, 2000)

27

ABSTRACT

Adsorption of volatile organic compounds such as alcohols NN-dimethyi formamide,o- dich]orobenzene,decane,etc. on silk fibroin and synthetic polypeptides was investigated. Each of polypeptides gave its own adsorption property,which seemed convenient for the identification of amino acid residues in the polymer. Methanol and ethanol gave high adsorptivity among the examined compounds because of the high vapor pressure. Acetronitrile adsorbed much on both poly(BLA)and poly(BLG)wh通e methanol adsorbed extensively on the other polypeptides. The vapor pressure of the compounds and molecular interaction between the compounds and the polymer are considerd to affect the adsorption property.

l . INTRODUCTION

The adsorption of organic compounds to materials is a very important phenomenon in science or even in our life but the mechanism does not seem to be revealed well.

When usual fibers were exposed to saturated vapor of organic compounds,each fiber gave its in- herent adsorption behavior of the compounds. The authors proposed a novel method to identify the kind of fibers by using the adsorption property.l'2 Protein fibers gave high adsorbability of some organic compounds. It is also known empirically that protein fibers adsorb we11someodororganic compounds. The authors have been studying the adsorption of organic compounds on proteins such as silk fibroin,silk sericin and wool keratin.3-5 Systematic experiments should be carried out in order to reveal the mechanism of the adsorption. Protein fibers such as silk fibroin and wool keratin were dissolved in appropriate solvents and recrystanized from the solution in order to avoid the effect of fiber structure. In this article,the adsorptior1of volat通eorgarlic compounds on silk fibroin was mainly in- vestigated. The adsorption property of s通k sericinf cotton fiber and synthetic polypeptides such as poly(glycine)and poly(L-alanine)was studied for comparison,considering that the molar contents of main amino acid residues in fibroin are glycine (42.8% ), L-alanine (28.3%), L-serine (14.7% ) and L-tyrosine (13.8% ).

2

28 Kanazawa and Sate:Adsorption of volat通e organic compounds on proteins

2 . EXPERIMENTAL

2_l Reagents and materialsReagents:Guaranteed grade reagents of methane](MeOH),ethanol (EtcH), 1-propanol (1- PrOH), 2-propanol (2-PrOH), 1-butanol (1-BuOH),NN-dimethylformamide (DMF),decane, o-dichlorobenzene (DCB),dioxane,acetonitrile(ACN),p-xylene,anisole,and ethyl acetate were pur- chased from Wake Pure Chemical Industries,Ltd. and used as received.Fibroin fiber:A buffer solution,0.2M boric acid/hydrochloric acid was prepared;50m1of 0.025mo1/1 Na2B407solution was mixed with 4.6m1of 0。lmoi/1 HCl solution and the whole volume was in- creased to 100m1 with water. Cocoon of Bombyxmori was mixed with the buffer solution of 50 times of the volume,and the mixture was boiled for one hour. The separated fibroin fiber was washed sufficiently with water and dried m t1acuo.Fibroin powder:Degummed fibroin fiber was dissolved in a solution of calcium chloride in water and ethanol (weight ratio, 8:2:1). After removing contaminations by the centrifugation,the fibroin solution was dialyzed overnight in water and it was concentrated unt通the volume became 30% of the original one by a fun. The concentration of fibroin solution (39.8g/1)was determined from the ab- sorbance at 275.7nm by the spectroscopy with a Shimazu spectrophotomer UV2200. The obtained

fibroin solution was 12.6g/1. The fibroin solution was gelated at PH= 3.5 by adding 0.12 N hydrochloric acid. The gel was freeze-dried and fibroin powder was obtained。Sericin powder:The remaining liquor obtained by washing cocoon Bombyxmori with a buffer so-

hltion, 0.2M boric acid/hydrochloric acid was dialyzed overnight in water and it was concentrated untnthe volume became 30% of the original one by a fun. The concentrated solution of sericin (conc 12.6g/1)was freeze-dried and sericin powder was obtained.Polypeptides:7,-Benzyl-L-glutamate(BLG)andβ一benzyl-L-aspartate (BLA)were prepared by the method reported by Yuki et at. with a slight modiiication.6 N-Carboxy amino acid anhydrides (amino

acid NCAs)were synthesized by the reaction between amino acids or substituted amino acids and triphosgen,and recrystallized in ethyl acetate and hexane. Polypeptides were prepared by the poly-merjzatjonof amino acid NCAs in acetonitile or in hexane (solid state)with butylamine as an initiator at 40℃ for 14 days. The molar ratio of NCA to butylamine was 200.

2.2 Adsorption of volat通e organic compoundsA given amount of proteins or synthetic polypeptides were hung in a glass bottle filled with satu-

rated vapor of organic compounds at 25℃ for a given time. The adsorbed compounds on polymers were extracted twice in ethyl acetate at 50℃. The extracts were analyzed by the gas chromatography (GC). Undecane was used as a standard material.

Analytical GC was performed on a Shimadzu 14A GC using a nonpolar capillary cohlmn,CBP l of Shjmadzu Co. Ltd.(i.d. 0_25ｵm, length 25 m). Column temperature for the analysis of alcohols, 35 ℃ for 1 min and 35-145℃ at 25℃/min and that for the other compounds, 70℃ for 1 min and 70-145℃ at 15℃/min. Temperatures of column injector and a detector,FID were 180℃ and 200 ℃, respectively.

3

Sci.Rep.Fukushima Univ.,No64 (2000) 29

3.RESULTS AND DISCUSSION

Adsorption experiments were carried out by two methods;the adsorption from the mixture of several kinds of volatile organic compounds and that from a single compound. These methods are described as the mixture method and the single compound method,respectively. Each compound in the mixture should be influenced to each other in the mixture method. But,it is easy and convenient te sco the whole adsorption property or adsorption selectivity of polymers. The single compound method gives the essential interaction between the compound and polymers.

First,the time change in the adsorption of volatile organic compounds on polymer materials was examined. Figs.1; 2 and 3 give the time change in the adsorption amount on fibroin,sericin,and poly(L-alanine)from the mixture of compounds at 25℃ (the mixture method). Each figure suggests that the adsorption amount gave a little or no change after around 25h. Thus,main experiments of adsorption were carried out at 25℃for 25h. The three figures suggest the adsorption property of the adsorbent (polymer)to the compounds:alcohols adsorb on these substances more than the other compounds. The vapor pressure of the compound should play an important role in the adsorption.

The values for the compounds except DMF,decane,acetonitrile,jrxylene and anisole at 25℃ were given in the literature,7 and the values for DMF and decane were calculated from the Kanazawa's method.8 Vapor pressure values of acetonitrile,・ xylene and anisole were plotted against temperature,9 and the regression analysis was carried out in order to estimate the vapor pressure at 25℃. Fig.4 gives the result and equations. The vapor pressure values of acetonitrile and anisole calculated from the equations are given in Table 1. The value of p-xylene given in Table 1 was calculated as 8 76 from the Antoine's equation.9 The value was estimated as 8.72 from the equation given in Fig.4. Although the vapor pressure value of DCB, 2_69 is much smaller than that of dioxane, 39.6,its adsorption amount seems more than that of dioxane in Figs.2 and 3. Thus,both of vapor pressure and molecular interaction between the compounds and polymers are considered to influence

20

10

5

/-o一一o-)一V

10 20 30 40 50 60

Time/h

Fig.l Adsorption of methanol or ethanol from their mixture on fibroin powder at 25℃

4

30 Kanazawa and Sate: Adsorption of volatile organic compounds on proteins

3

2

1

」amf

一ed‘3/-oulu一/Punodu-o.)Poq」osPV

l8

o Ethanol ・DMFi DCB

a D i e n n e

口Becaae

Time/h

Fig.2 Amount of adsorbed organic compounds on sericin powder at 25℃

.SO

.25

e

0

0

u-P-suP--)一一eu一要-一eu一、一eu一、Punodu-ooPa一(-espy

0 10 20 30Tiln lh

Fig.3 Amount of adsorbed organic compounds on poly(L-Ala)at 25℃

the adsorption property.Fig.5 gives the adsorption of alcohols to fibroin powder,sericin powder,fibroin fiber and cotton

fiber by the single compound method. The adsorption on the powder is much more than that on fibers, which should be mainly influenced by the difference between the surface area of the powder and that of fiber. Fibroin powder gives larger adsorptivity than sericin fiber,which suggests the difference in the amino acid residues in these proteins is important. In addition,protein seems adsorb compounds more than cellulose from the difference between fibroin fiber and cotton fiber. The order of the ad- sorption amount of these alcohols seems mainly in the order of higher vapor pressure of the alcohols,

5

Sci。Rep,Fukushima Univ_,№64 (2000)

Table 1 Vapor pressure of used volatile organic compounds at 25℃

31

Organic compolmd

MeOH EtcH 1-PrOH 2-PrOH 1-BuOH ACN DMF DCB Dioxane Anisole p-Xyierle Decaae

Vapor pressure (mmHg)

122.2 59_3 20.3 44.3 6.44 91,5 3.82 2.69 39.6 3.22 8_76 1.95

VPAc N _ 26 5 54 + l.41a5t + 3.52 15e-2f 2 + 4。5880e-4t 3_l 4 6 44 9e-7 4 + 3.09l4 e 8t 5, R 2 = 1 000

VPx y - _0。85025 + 0 .35904 _ 3 6037e_3t2 + 1_7 560e_4t3_1 1.2479e_7t 4+ 5.3l55e_9t 5. R2= 1,000

VPAnl _ _l4.210 -1 13320 _39360e-2t2 +6.4915e-4t3 4 0 l16e 61 4 _1 1 5657e-8t 5 R 2 _ 1 000

Fig.4 Plot between vapor pressure and temperature,and the fitted curves andequations for the data of acetonitrile,p-xylene and anisole.

but 1-butanol was adsorbed more or similarly as compared with 1-propanol.In order to consider the adsorption property of fibroin,it is significant to see the property of poly

(Gly)and poly(L-Ala)because the molar fraction of Gly residue (0.475)and L-Ala residue (0.265) in fibroin is very high. Fig.6 gives the adsorption of five compounds on poly(Gly)by the mixture method and the single compound method. Adsorption amount in the single compound method is more than the corresponding value in the mixture method. This should be caused by the occupation of ad_ sorption sites by the compounds. In the single compound method,it is remarkable that DCB and DMF adsorbed more than dioxane although the vapor pressure values of DCB and DMF are 2.69 and 3.82mmHg,that of dioxane, 39.6EmHg,respectively. It is also remarkable that decane considerably adsorbed on fibroin in spite of the low vapor pressure. Poly(L-Ala)gave the similar result to Fig.7 The adsorption amount of DMF is also more than that of dioxane.

Fig.8 gives the adsorption of the eight compounds on poly(Gly),poly(L-Vat),poly(L-Leu), poly(L-Phe),poly(BLA),poly(BLG)and fibroin powder. The amount of adsorbed compound was estimated per lmolaminoacid residue in each polypeptide. The formula weight of amino acid residue in fibroin was estimated averagely as 76_02 from the molar fraction of each amino acid residue in fibroin。jo lt is remarkable that each polypeptide has its inherent adsorption property of the compounds.

Acetronitrile adsorbed much on both poly(BLA)and poly(BLG)although methanol adsorbed much

6

32 Kanazawa and Sate:Adsorption of volat通e organic compounds on proteins

iu-od‘a-eu一u一/一o一一o-)-epoq」espy

30

20

10

Fibroin Soricine Fibroin Fiber on

r

〇ttjbe

CF

Fig。5 Adsorption of alcohols on fibroin,sericin and cotton by single compound method

1s

t2

的

部

的

0

0'

0'

8・

e

8

a.p-s=p言

ou.Ea・一ou-1-om/punodmopo(一」espy

F「Om l, i ・ h i r e

Fig.6 Adsorption of organic compounds on poly(Gly)at 25℃ for 25h.

on the other polypeptides. These results gave a good reproducibility. The present result gives a convenient method for the characterization of amino acid residues in polypeptides.

In allot the adsorption experiments,methanol and ethanol gave high adsorptivity. This iS mainly due to the extremely high vapor pressure. In order to consider the interaction between alcohols and polypeptides,the adsorption of a series of alcohols on seven polypeptides was examined by the Single adsorption method. Fig.9 gives the result. Methanol gives the high adsorptivity in the results fo「

each polymer. Vapor pressure of methanol is about twice of that of ethanol (see Tab. 1) . But the

7

Sci.Rep.Fukushima Univ_,№.64 (2000)

富 Ethanol ・ D M F

翻 DCB 口 Dio n n e

■ Dm ae

From From eachmix tur e eOmPOm d

Fig.7Adsorptionof organic compounds on poly(Gly)at 25℃ for 25h

一,-

1

0

0

MP-a.P-o.-oue齒d一一・一om、一eu-/Punodmo-)PaqJosPv

Gly Ala Val Leu Phe BLA BLG FibroinPolypeptide

Fig.8 Adsorption of organic compoundsonpolypeptidesandfibroinbythemixture method at 25℃

33

for 25h

adsorption amount of ethanol is more than half of the adsorption amount of methanol. The amount of adsorption of 2-propanol and 1-butanol seem rather high in spite of the low vapor pressure. This indicates that the interaction between the hydrophobic stn1cture in the volatile organic compounds and polypeptides should be important. The effect of vapor pressure and the role of hydrophobic groups in the volatile organic compounds will be examined in more detail.

8

34 Kanazawa and Sate: Adsorption of volatile organic compounds on proteins

3

2

1

0

0

0

0

aiP--M

P-.-eou-u一e,-om/一om/Punodmo-)Paq」osPV

Gly Ala Val Leu Phe BLG BLAPolypeptide

Fig.9 Adsorption of alcohols on polypeptides by the single compound method at 25℃ for 25h

References

1.H.Kanazawa and Y.0nami,Science Report,.Fukushima Univ.,48, 37 (1991).2.H.Kanazawa and Y.0nami,Sen-i-Gakkai Preprints, 1993 (S), p.136.3.H.Kanazawa and Y.Mats11shima,Science Reports of Fukushima University, 59, 13-17 (1996). 4.H.Kanazawa and T.0nami,Science Report,Fukushima Univ.,60, 25-31 (1997).5.H.Kanazawa,Nihon Kasei Gakkai,Tohoku-Hokkaido,.Preprints;p.32 (1998).6.H.Yuki,S.Saskakibara,and H.Tar1i,Bu通.Chem.Soc,Jpn,29, 654 (1965).7. Chem.Soc.Jpn,“Kagaku Binran,Kiso-hen I[ ,3rd.Ed.,Maruzen,Tokyo,pp.1l8-132(1975) 8.H.Kanazawa,Science Reports of Fukushima University, 60, 25 (1997).9.Chem.Soc.Jpn,“Kagaku Binran,Kiso-hen I[', 2nd. Ed.,Maruzen,Tokyo,pp.710-731(1975).

10.Senl Gakkai,“Sen-i Binran,Kako-hen”,Maruzen,Tokyo,p.874 (1969).