Instructions for use Title GENERATION OF

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GENERATION OF CARBOXIMETHYL-CHITIN-METAL ION COMPLEX AND ITS PROPERTY

Uraki, Yasumitsu

Citation

Issue Date

Doc URL

1989-12-25

http://hdl.handle.net/2115/32568

Right

Type

theses (doctoral)

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Instructions for use

Hokkaido University Collection of Scholarly and Academic Papers : HUSCAP

GENERAT I ON OF CARBOXYMETHYLCHI TIN -

METAL

AND

ION COMPLEX

I TS PROPERTY

YASUMITSU

URAKI

A DOCTRAL DISSERTATION

FACULTY OF SCIENCE, HOKKAIDO UNIVERSITY, SAPPORO, JAPAN

CONTENT Page ABSTRACT

INTRODUCTION

I.

II.

1

Preparation of various CM-chitins and its identification

19

EXPERIMENTAL

20

RESULTS AND DISCUSSION

25

CONCLUSION

42

Adsorption of metal ions to CM-chitin and surface property of the complex.

43

EXPERIMENTAL

44

RESULTS AND DISCUSSION

50

CONCLUSION

103

III. Preparation of peptide and its adsorption to CM-chitin

104

EXPERIMENTAL

105

RESULTS AND DISCUSSION

108

CONCLUSION

115

CONCLUDING REMARKS

116

ACKNOWLEDGEMENT

119

REFERENCES

120

ABSTRACT

Variously ch i tin)

substituted

6-0-carboxymethyl-chitins(O-CM-

and 3,6 -O-CM-ch i tin were prepared from a- and

{:J-

chitins, and their characterization were performed with 13CNMR, ties

FT-IR and others. At first, of

these

materials

were

metal

ion-binding proper-

investigated

exhaustively.

Either 6-0-CM-chitins, prepared from a- or {:J-chitin adsorbed calcium ion specifically and tightly among various divalent metal

ion.

The adsorption was

found

to be contributed not

only by the electrostatic interaction of carboxyl group but also

by

secondary

acetamide

group,

hydroxyl

as

groups,

specific adsorption site, binding).

The

specific

enhancement ions.

the

the the

primary

and

existence

of

of

6-0-CM-chi tin

toward

by the stretching process in fiber

adsorption

This phenomena

by

constructed with multiple way of

the CM-chitin of

as

suggesting

adsorption

calcium ion disappeared spinning of

(

well

owing

to

capacities

the for

the

appreciable other

metal

suggested the importance of geometri-

cal arrangement of those functional groups for the conformation of the metal-binding sites. Nevertheless 6-0-CM-chitin from {:J-chitin forms gel by the addition of transition metals owing to the wide distributed carboxyl

group,

the

specific

adsorption

for

calcium

ion

still remained, so suggested the geometrical arrangement of binding site could not be destroyed. On the other hand, Nonspecific

binding

for

calcium

chitin ,neither from a- or

ion

{:J-,

-1-

was

found

in

3, 6-0-CM-

even though it formed gel by

addition of

some metal

ions

owing

to high negative charge

density. The second, chitin

the adsorption of amino acids to the Ca 2 + -CM-

complex

was

examined

configuration aspects ion.

in

order

to

identify

the

of specific binding sites of calcium

Adsorption of amino acids to the complex was found to

be proportional to the hydrophobicity of amino acid. Enormously

high

quantities

their

chiral

of

specific property was

containing Phe was also CM-chitin

phenylalanine

from

found

was

adsorbed

observed.

to be

and

The peptide

adsorbed

to

Ca-6-0-

a-chitin complex tightly and the adsorbed

peptide in the complex could not be hydrolyzed by peptidase. It

was

noted

complex when

that it

the peptide fragment

was

treated

splitted out

sequentially by

from

lysozyme

and

peptidase remaining only the Phe residue in the complex. These results suggested the existence of specific adsorption site for aromatic ring of Phe and in calcium specific adsorption si te, group is

that

is

to

say,

the side chain of phenyl

incorporated to generate tight complex constructed

with CM-chitin, Ca 2 +

ion and Phe residue.

Since 6-0-CM-chitin was found to protect the peptide from peptidase and stabilize it in animal body, 6-0-CM-chitin was suggested

to

be

a

useful

material

as

a

polymeric

drug,

especially, as a drug carrier of sustained release system. The present the

mechanism

study wi 11 of

the

also proposed a model

interaction

protein.

-2-

of

CM-chi tin

to

explain

wi th

blood

INTRODUCTION

Chitin

is

a

polysaccharide

consisting

of

N-acetyl-

glucosamine(2-acetamido-2-deoxy-D-glucopyranose) by

residueslinked

(3-1,4-glycoside

the

bonds.

The

deacetylated derivative of chitin is called as "chitosan". Chitin

is

known

organism,

to

exist

including

in

the

bacteria,

large

number

p I an t s

and

of

living

an i rna Is.

In

bacteria and plants, chitin exists in a large amount in the cell wall of bacterium, mainly in mold, yeast and mushroom, and

is also

found

in some algae groups.

components of cell wall

Polysaccharide as

is not chitin but peptideglycan in

the more primitive living organisms, while is cellulose in higher order plants. In the attaching bacteria, on the other hand,

the cell

ch it i n (1)

wall

contains chitosan also

in addition to

•

In animal, chitin distributes in the various animals from Protozoa

to

Pogonophora.

Ability

to

synthesize

chitin,

which was established in the beginning of the evolution, has been

kept

in

Triproblastica, of

evolution

organisms(2,3).

number

a

but to

Diproblastica

of

and

most

the ability disappeared on the process Deuteromia

except

a

Namely chitin exists

part

of

in Crustacea

I i vi ng such as

crab or shrimp, in Arthropoda such as spider or tick, and in Nematoda.

However,

it

does

higher order.

These

physiological

"ecdysis".

saccharide

chain

as

seems

not to

As

exist

be

the

animals

of

ser i ous I y concerned wi th

chitin

described

in

has

below,

it

very is

rigid used

polyas

the

protection

cloth

Arthropoda,

in

supporting material

for stigma,

and

also

used

sensitive organ,

as

the

vessel

of

secret glandura and both end of digestive tube in addition to the cuticle. The cuticle is consisted of, chitin,

proteins,

lipids,

protect

effectively from severe natural

the cuticle prevents and

invasion

80% of

of

total

in addition to

inorganic salts and catechols environment.

to

Thus,

the evapolation of water from surface

foreign

animal

Arthropoda

enemies(4,5).

species

and has

occupies

been prospered

for

a

long period by this way. Since the rigid and less expensive cuticle dose not match well for the growth of animals, an i mal s

need

animals,

the ecdys is.

chitin seems

In

the case

to be converted

0

f

these

the higher order

to proteins such as

collagen to obtain the ability of more rapid growth. Considering aspects

of

the

evolution

chemical

of

structure,

animal

cuticle

peptideglycan

from

in

the

the very

primitive bacteria seems to be converted to cellulose simply to

form the plant cuticle. On the other hand,

chitin which

has a skeleton such as cellulose and amide bonds similar to protein

is

an

intermediate

material

on

the

process

of

evolution to proteins in higher animals. Chitin belongs glycan) "

toge ther

heparin,

heparitin

acid,

so-called "mucopolysaccharides(proteo-

to

wi th

0

ther

sulfuric

mucopo 1 ys acchar ides acid,

chondroitin

such

as

4-sulfuric

chondroitin 6-sulfuric acid, hyauronic acid and etc ..

These polysaccharides except heparin have repeating unit of mono-

or di-

repeating

saccharide.

unit

of

Ce 11 u lose and mannan havi ng

monosaccharide

have

supporting

the and

storaging

functions,

whi Ie

most

of

acidic

mucopoly-

saccharides have physiological activity to keep the function of living body. For example, hyauronic acid contributes as lubricants in the corpus vitreum liquid and joint parts. It binds to protein and protects tissues from bacterial attack. According to the recent report,

it plays important roles in

the cell construction such as angiogenesis (6). known

to

bind

to

fibronectine,

which

has

Heparin is

a

function

to

regulate a cell attachment by its conformational change(5l). It inhibits of serine protease activity working thrombin chitin

III is

to prevent

useless

for

hydrolyse chitin and

the blood coagulation(7).

animals,

wall

as

animals

chitosan as

body protection since chi tin cell

described

with anti-

one of

is a

above.

the

enzymes

to

functions

of

component of bacterial

So

the animal body.

have

Though

most

of

the

invading

bacteria die

in

When chitin invades

into

animal body,

it is digested slowly by enzymes in the body

and nothing else occurs. This digestive property in animal body is very important for the utilization of chitin. l09_l011t of chitin is produced biologically similarly to lOl1t of of

environmental

pollutions

attached proteins. one

example

of

functionalization project,

also

cellulose(9). owing

per year(S),

It has been the cause to

its

abandon

wi th

Use of chitosan as a coagulant is only

its

large

scale

of

chitin

has

from

the

utilization. become

standpoint

of

to this

the

Thus,

the

attractive

environmental

cleaning up. Studies

on

the

functionalization

3

of

chitin

have

been

developing

on

converted

the

to

chemical

basis

the material

modifications,

lation,

since

chemical

the chitin molecule

they

position

is

position

is

are

Hydroxyl

to

chi tin

for

reactive

can

be

activity by or

carboxy-

N-acetylglucosamine

in

that of acidic mucopoly-

groups at C-l and C-4 position

residue

protected

that

sulfonation

as

structure of

used

not

ideas

having physiological

such

N-acetylglucosamine

because

the

is similar

saccharides(lO-12). in

of

of

chitin

glycoside since

by

the

acetyl

is

not

reactive

linkage. amino

Also

group

group_

The

C-2

of

C-2

secondary

hydroxyl group at C-3 position and primary hydroxyl group at C-6 position are on

the chemical

the only functional modification.

group

Thus

to be regulated

chitin

is

regarded

as

a valuable starting material for the chemical modification. Three kinds of crystalline structure are known for chitin. Chitin

of

the

shrimp

shell)

orientation termed

as

largest is

of

termed

as

molecular

~-chitin

mixture of

existence

chains. has

these two forms

~-chitin

reported

by

X-ray

d iff erence crystal

0

f

the

lattice.

has

Chitin

in

parallel

crab

or

anti-parallel Loligo

Pen

orientation.

is The and

a-Chitin forms rigid crystalline

forms

soft

one,

crystallography number

Figure

It

(e.g.

is in stomach wall of squid,

is called as y-chitin(19). and

nature

a-chitin.

which

structure

in

0

of

f

its

was

come

from

the

bond i ng

ina

un it

to

hydrogen

difference

crystalline

structure

and

the

unit crystal lattice constant are shown in Fig.l(20,21). a-Chitin P21 21 21.

An

is

rhombic

uni t

system

lattice

of

contains

which two

space

group

is

N-acetylgl ucosamine

(a)

-

a=4.75 A

b

b=18.8 6A

c=lO.3 2A (b)

Cryst alline struc ture of a-chi tin

i

(a) bc-sid e (b) ab-sid e

fal

a=4.85 A b=9.26 A

c=lO.J 8J.. b

fbi

Cryst allinE ' structun~

')1

fj-chi tin wirho ut water

(::il a!:'-si de (b) bc-sid e

Fig. l Crys talli ne struc ture of chiti ns from X-ray diffr actio n

5

units

in

two species of chains on each direction of anti-

parallel,

and

the

structure(20).

On

chitin

the

conformation

other

system, space group is P21

hand,

is

/1-chitin

is

21

helix

monoclinic

and an unit lattice contains two

sugar unit in a chain on repeated parallel direction and its conformation

is also 21

are

only

formed

molecular

chain

by

helix.

Hydrogen bonds

in /1-chitin

group

with

hydroxyl

along a

axis.

at

C-6

a-Chitin has

other

more hydrogen

bonds than /1-chitin, because these bonds are formed by the hydroxyl group with the adjacent chain of opposite direction along b axis in addition to that along a axis. Accordingly, a-chitin has rigid structure to form the most packing and stable crystalline structure, while /1-chitin is so soft that it

swells up

in water easily.

a-Chitin makes

rigid shell

together with proteins and calcium carbonate, but /1-chitin makes

soft bone with

amount

of

calcium

large amount

carbonate.

In

of proteins both

chitin

and

small

forms,

the

hydrogen bond within intra residue is between the hydroxyl group at C-3 position and the oxygen atom of hemiacetal at 0-5.

Si nce secondary hydroxyl group at C-3 pos it i on in N-

acetylglucosamine is protected by the hydrogen bond, chitin seems

to be an

analysis

of

ideal

starting material

polysaccharide

and

site

for

the structure

selective

chemical

modification. As one examples, chitin was converted to chitin heparinoid which

has

the

anti-blood

coagulant

property(13-15).

The

heparin has the chemical structure as shown in Fig.2(16). In the

above

mentioned

investigations,

6

the

purpose

was

to

obtain

the

heparin-like

physiological

activity

not

by

constructing the same chemical structure as heparin by the chemical

modification

but

by

regulating

the

position

of

substitution with acidic functional groups. The rigid crystalline structure of chitin interferes the rea c t i vi t yin c hem i c a I mod i f i cat ion.

The g I y cos ide lin k ag e

is destroyed in the acidic condition or acetamide group at C-2 position condition. mild

is hydrolyzed to amino group in Thus

condition

chitin)

was

prepara t i on

the yield of is

always

employed 0

reaction was

needed.

as

a

f hepar i no i d,

the alkaline low since

the

Carboxymethyl-chitinCCM-

starting

material

for

the

and was der i ved by the carboxy-

methylation method which was improved by Tokura et. al.(17). This reaction was carried out in the heterogeneous system to introduce

the

carboxymethyl

acetylglucosaime

group

selectively.

to

C-6

position

Deacetylation

of

at

N-

acetamide

group does not occur even in the high alkaline concentration because

the

Detergent

reaction was

is

used

carried to

out

at

low

increase

temperature. degree

the

of

substitutionCD.S.). Since CM-chitin obtained was a non toxic material

in

the

immunological

property

except

that

it

activated peritoneal macrophages in a short period to induce a little mitogenic activlty(10,52), CM-chitin was suggested to be

the most suitable starting material

for heparinoid.

Heparinoid activities of chitin heparinoids obtained by the sulfonation of CM-chitin under

the several

exhaustively

results

investigated.

OC3,6)-sulfations

of

The

condition were

suggested

partially N-deacetylated

that

N-

6-0-carboxy-

methyl-chitin(70% deacetylation) induced the highest activity. The product had the activity of one-third of heparin and it was non-toxic.

Though sulfated chitosan had

toxicities,

they were found to disappear by the 6-0-carboxymethylation. It suggests that chitin derivatives are converted to be more biocompatible the

by

mechanism

biomaterials blood

of

6-0-carboxymethylation.

interaction

was

proteins

fundamental

the

focused and

between

on.

study

to

6-0-CM-chitin

Adsorption

6-0-CM-chitin evaluate

Accordingly,

was

property

between

investigated

biocompatibility

and

as

and

a the

relationship to blood coagulant system(18). As adsorption of fibrinogen,

a

main

factor

of

blood

coagulant,

to

6-0-CM-

chitin was found to be enhanced in the presence of calcium ion, calcium ion was suggested to play the important role in the interaction between 6-0-CM-chitin and biomaterials. The

purpose

of

the

present

study

is

to

clarify

the

mechanism of interaction between 6-0-CM-chitin and proteins in detail. So the first purpose is to clarify the relationship of 6-0-CM-chitin with calcium ion, especially about the adsorption capacity of calcium ion compared to that of other metal

ions.

As

metal

ions,

alkali-earth

metal

ions

and

transition metal ions, were employed, since their ability of chelation was easily selected owing to pied d orbitals.

As

there is a difference between a-chitin

and ,9-chitin in the crystal rigidity and nic

salts

contained

the variously occu-

in

nature,

it

is

amount of inorgaestimated

that

CM-

chitins derived from both chitins have the different ability in the metal

ions adsorption. Therefore the abi I i ty for the

8

adsorption of metal ions, mainly calcium ion,

to CM-chitins

from both of the a- and P-chitin was examined. The

carboxymethyl at ion

proceed

on

the

predominantly.

described

primary

hydroxyl

above group

Re-carboxymethylation

was

at

found

C-6

under

to

position the

same

condition resulted in the substitution of secondary hydroxyl group at C-3 position

in addition

give

DS>1.0.

the

product

of

to

Effect

the C-6 position of

the

substitution and substituted sites were also

degree

to of

investigated.

CM-chitins used here are 6-0-CM-chitin and 3,6-0-CM-chitin. Adsorptions of metal ions to water insoluble 6-0-CM-chitin (flaky CM-chitin) from a-chitin were measured by the frontal affini ty chromatography(47-49).

The

capaci ty of

flaky CM-

chitin (natural crystalline structure) to calcium ion seems to be higher hand,

than

that of other metal

ions.

On

the other

the specificity of CM-chitin toward calcium ion seems

to have disappeared probably owing to the great enhancement of

the adsorption capacities

for

other metal

ions by

the

stretching process in the spinning of the CM-chitin fiber. These

results

suggested

that

the

specific

adsorption

of

calcium ion is due to the geometrical arrangement of several functional groups. The participations of acetamide group and pr i mary and secondary hydroxyl

groups,

electrostatic

the

suggested by mixture

and

interaction

by

the differential CM-chitin

only.

in add it i on

carboxyl

to

group,

the were

IR spectrum of CM-chitin-Ca The

change

in

pH

to

acidic

condition was not enough for the complete release of calcium ion

from

CM-chitin.

It

needed

9

chelating

reagent

such

as

EDTA,

to release calcium ion. On the other hand,

the release

of other metal

ions was easily performed by the pH shift.

would support

that

due

to

the

It

the strong adsorption of calcium ion is

CM-chitin-Ca

complex

formation.

When

aqueous

solution of calcium ion was added to magnesium ion-adsorbed CM-chitin,

release

observed.

It

CM-chitin

to

calcium

found

toward

specificity

of

relate

which

carbonate, capacity

to

is

to

show

calcium

to in

calcium

for

the

a

selective

mixture.

large

amount

the

The

calcium

ion

amount

CM-chitin

small

show

Moreover,

of

toward

wi th

was

from

of

smaller

of {3-

calcium

adsorption

Water soluble CM-chitins,in which

freedom on molecular motion investigation

a-chitin

a

to

ion.

CM-chitin

Ca 2 +-Mg2+

in

Therefore,

with

expected

from

ability

existence

nature.

exists

the

ion from

its

ion

ion-exchange reaction.

CM-chitin

carbonate

chitin,

magnesium

really an

was

adsorption

seems

is

of

increased,

adsorption

degree of substitution on

were applied

abi 1 i ty.

Effect

to

the

of

the

the adsorption property was

also

investigated with 3,6-0-CM-chitin. In

low concentration

of

metal

ions,

all

CM-chitins

were

found to have the high affinity to calcium ion among several metal

ions.

Especially,

6-0-CM-chitin

from

in the adsorption of calcium ion to

both

of

a-

and

{3-chitin,

negative

cooperativity was not shown in Scatchard plot. But that was shown for the other metal ions. These might suggest that CMchitin calcium

from ion.

{3-chitin

also

obtained

the

The reason might be as

from {3-chi tin forms

the same local

....

.iV

high

follows,

chemical

affinity

for

6-0-CM-chitin

and geometrical

structure

as

that

of

6-0-CM-chitin

from

a-chitin

by

the

carboxymethylation owing to the adsorption mechanism by the all functional groups in N-acetylglucosamine residue. These phenomepa woul d be the func t i onal i za t i on of ch it i n because the specificity to calcium ion was given to carboxymethylation. confirmed

As

to maintain

water-soluble

by the

~-chitin

6-0-CM-chitin

was

the specificity to calcium ion,

adsorption mechanism of calcium ion

the

was investigated by FT-

IR spectra in solution compared to that to non-specific Ni ion.

The

adsorption

contributed

by

of

calcium

carbonyl

ion

groups,

was

suggested

primary

and

to

be

secondary

hydroxyl groups. On the other hand, that of nickel was found to

be

contributed

by

only

carbonyl

nonspecific adsorption of other metal

groups.

Therefore,

ions also seem to be

the simple electrostatic interaction by carbonyl groups. 6O-CM-chitin from to form gel, transition

and all 3,6-0-CM-chitin were found

~-chitin

followed by preCipitation, metal

ions.

As

interchain

in the presence of cross-linking

is

necessary for gel formation, transition metal ions seemed to induce

the

phenomenon carboxyl

cross-linking suggests

groups

in

that the

bundle several chains. to

regulate

crystalline

the

between

transition wide

range

metal of

chains.

bind

to

chains

to

ions

polymer

This

The crystalline structure was found

function

structure

CM-chitin

of

of

CM-chi tin

~-chitin

was

because

maintained

loose in

its

derivatives owing to the distribution of carboxymethyl group in

the

wide

difference

region between

of the

molecules. calcium

11

It ion

clearly binding

showed and

the the

adsorption chitin ions

of

other

tightly

are

region

in

metal

the

supported

of

local

by

polymer

ions.

Calcium

ion

specific area,

only

carboxyl

groups

while adsorbed Fe 3

keeping the soluble state,

to

CM-

and other metal

6-0-CM-chitin

chains.

binds

in

the

wide

adsorbed

Fe 2 +

+

to form gel.

The adsorption site seems to increase by the increase in the charge

of

adsorbed

structure

also

metal

formed

ion.

the

adsorption of barium ion,

gel

ions.

tion

of

the

adsorption

and

which had

among alkali-earth metal on

3,6-0-CM-chitin

Gel

from

precipitate

the largest

both

on

the

ion radius

formation and precipita-

metal

ions

to

3,6-0-CM-chitin

suggest that interchain cross-linking occurred by the adsorption

of

metal

ions

owing

to

the

high

charge

density

of

carboxymethyl group. Especially gel formation proceeds smoothly

in

However,

the presence

of

metal

3,6-0-CM-chitin

calcium ion. adsorption

did

ions not

show

It might be suggested

si te

in

CM-chi tin

with

large the

specificity

that one of

molecule

was

ion radius. to

the calcium

blocked

by

re-

carboxymethylation. Thus the mechanism of calcium ion adsorption

to CM-chitin was elucidated,

chitin was

found

and functionalization of

to be regulated by the crystalline struc-

ture and the degree of carboxymethylation. As CM-chitin has

the specificity to calcium ion,

it would

be important to analyze the surface properties of CM-chitinCa complex

in

order

to

investigate

chitin with biomaterials.

the

interaction

The second purpose of

of CM-

this study

is the investigation on the surface properties of CM-chitinCa complex.

On

this

amino

study,

acids

to

adsorption the

of

surface

of

several the

optically

complex

was

active

analyzed

considering the stereo geometry. The property of the surface can be estimate by analyzing the differences in the affinity for

the various amino acids,

since amino acids have hydro-

ph iIi c a-ami no and a-carboxyl groups in the mai n chai nand hydroph iIi c

or

hydrophob i c

func tiona 1 groups

in

the

side

chain, depending on the kind of amino acid. The interaction of CM-chitin with proteins may be presumed by the profiles of adsorptions of amino acids which are the basic components of proteins. On

the

flaky

adsorption

CM-chitin

capacities

Lys)Phe)Glu,

having amino

of

independently

on

the

natural acids

structure, was

presence

or

the

shown

as

absence

of

calcium ion. The adsorption of D,L-Lys and D,L-Glu reduced to

and 1/5 respectively,

1/10

by the chelation of calcium

the adsorption of D,L-Phe enhanced

ion. On the other hand,

about 50 times. Similar results were obtained also for water soluble neutral

6-0-CM-chitin

from

amino acids having

a-chitin.

the side chain,

adsorption

of

the alkyl side chain was found

to i ncreas e l i near 1y with the i ncremen t city of

The

0

f

the hydropho b i-

while the adsorption of phenylala-

nine having the aromatic side chain increased more strikingly with

the increase of the hydrophobicity. As the contri-

bution of other factor was bigger than that of the hydrophobicity on the Phe adsorption, contribution of benzyl group, of

which

groups,

electron

state

is

different

from

that

of

alkyl

shoul d be very 1 ar ge. When aroma tic ring fo rms the

metal form

ion complex, 1T

electron plays a role as a ligand to

1T

complex. The adsorption of phenylalanine seems to be

contributed phenomena

by seem

the to

similar be

similar

mechanism of calmodulin,

and

with

These

the

adsorption

calcium

binding

a famous calcium binding protein.

The binding sites of metal formed by "E-F hand",

mechanism.

ions in calmodulin molecule are

which has repeating

unit of Asp-Gly

two ex-helix composed of non-polar amino acids and Phe

exists in the adjacent position. of calcium ion has

In this case,

the binding

influence upon the benzyl group of Phe

side chain(51). This mechanism might explain the adsorption of phenylalanine on to CM-chitin-Ca2 + complex. The side chain of Phe was found

to be important for the

adsorption to CM-chitin-Ca complex. But that is not enough to clarify the mechanism of amino acids adsorption. binding of

ex-amino group and especially ex-carboxyl

Ionic group,

which is dissociated in neutral pH, should be examined from the

standpoint

suitable

for

functional

of the

groups

geometric

specificity.

adsorption

experiment

of

main

chain

Though to

it

protect

(ex-position),

is the

non-polar

protective groups are not suitable because they have small solubility in water and such a non-polar material can not be a

model

of

proteins.

To

chain functional group on difference

in

the

investigate

the

effects

of

main

the adsorption of side chain, the

adsorption

capacity

was

examined

by

regulating the distance between main chain and side chain. The adsorption of peptide was investigated using

glycine as

a spacer.

would also

The adsorption studies

for peptides

give us some informations about the interaction between

CM-

chitin and proteins. Since

the

almost

adsorption

same

as

that

capacity for

for

H-Gly-Phe-Gly-OH the

H-Phe-Gly-OH,

is

a- am i no

and

the a-carboxyl side of the Phe residue do not seem to affect on

the

adsorption

adsorption

of

capacity

calcium ion was

Phe

so

for

the

similar

as

This would suggest

much.

The

peptides

increase

in

the

of

the

presence

of

in the case of Phe adsorption.

a contribution of only benzyl

group for

the adsorption of Phe in the peptide. These mechanism seems to

be

explained

justly

using

incorporated

a

in

model the

that

the

adsorption

side site

chain

of

Phe

formed

by

the

calcium adsorption, and main chain of the peptide is exposed on the surface of polymer chain. The function given by the carboxymethylation is thought to be

developed

to

biocompatibility which into of

is

higher

account, drug

biomimetic

material

described

before

than

model

without

side

in

animal

body,

and

metabolism

system

without

planned

to

obtain

fundamental

release

from

the

rate

CM-chitin

of

high

these properties

sustained release system

was

molecule would be released with

the

biodegradability,

Taking

for

effects

utilize

and

chi tin(23).

synthesis

to

planned.

The

adsorbed

the digestion of CM-chitin would

affection

to

disappear the

informations

lysozyme

body. about

hydrolysis,

in

the

I twas sustained rate

of

molecular weight decreasing and the relationship between the adsorption capacity of Phe and chitin.

the molecular weight of CM-

6-0-CM-chitin

was

to

found

be

hydrolyzed

to

1/10

of

original molecular weight in 24 hrs by the lysozyme concentration of the same level as in human tear. It was hydrolyzed finally to 2-3 residue

oligomers in 96 hrs. The adsorption

of Phe decreases by the reduction of the molecular weight. These

results

suggest

that

Phe

adsorbed

to

CM-chitin-Ca

complex is released by the decrease of retaining ability for Phe due to CM-chitin hydrolysis with the glycosidase such as lysozyme.

3,6-0-CM-chitin was not hydrolyzed by lysozyme,

because it seems not to be recognized by lysozyme due to the substitution at C-3 position. Only 6-0-CM-chitin was suggested to be suitable for the carrier of sustained release. 6-0-CM-chitin was reported for

inclusion

type

sustained

to be utilized as a carrier release

by

fer ric ion t.o for m gel (53). The reI e as e

0

the

addition

of

f do x 0 rub i c i n e , a

anti-cancer drug, from the gels was found to increase by the lysozyme digestion results, polymeric

that

in a

CM-chitin-Ca

drugs

specifically,

time-dependent manner.

and

complex

CM-chitin

can

be

All used

adsorbed

these as

metal

a

ions

would indicate the excellent use fullness of

CM-chitin. The results and discussions in detail was described in the following

three

chapters.

Chapter

preparation of various CM-chitins,

I

described

the

their identification and

their digestion by lysozyme. Chapter II described about the adsorption of metal in

order

complex.

to

ions and the adsorption of

analyze

Chapter

III

the

surface

described

16

of

metal

about

the

amino acid

ion-CM-chitin adsorption

of

peptide for cleaning up mechanism of amino acids adsorption and discussed about

the possibility of utilization of CM-

chitin as polymeric drug.

chitin

6-0-CM-chitin

3,6-0-CM-chitin

the active site of heparin

Fig.2. Chemical structures of chitin site of heparin

18

.j'.

~CM-chi tins

and the active

Chapter 1.

Preparation of various CM-chitins and its identification

This chapter will describe

the preparation of variously

substituted 6-0-CM-chitins and 3,6-0-CM-chitins and discuss the

reactivity

of

chitin

crystalline structure.

The

molecule

by

the

susceptibility of

difference

of

CM-chitin

to

lysozyme and its products will also be discussed.

19

EXPERIMENTAL

MATERIALS a-chitin, Shell,

which

is

usually derived

from

Crab

or

Shrimp

was prepared from Queen Crab Shell according to

the

method of Hackman(25) and powdered to 45-60 mesh before use. (:1-chitin was similar

prepared

procedure

as

from Squid BoneCLoligo Pen) those

for

a-chitin

except

by

the

shorter

reaction time, but it took only one-fifth of treatment time in

the

alkaline

and

acidic

condition

because

of

lower

amounts of calcium carbonate and various proteins than those of

a-chitin. Monochloroacetic acid, dichloroacetic acid and other reag-

ents of reagent grade were obtained from Wako Pure Chemical Industries Ltd., and were used without further purification. Hen egg whi te

lysozyme was

purchased

from Seikagaku Kogyo

Co. Ltd. C50000units/mg, 6times recrystallized).

PREPARATION OF 6-0-CM-CHITIN 6-0-CM-chitinsCbelow D.S. 1.0) were prepared by the method reported previously(17) from a or (:1-chitin. In the case of water insoluble 6-0-CM-chitin, 19 of chitin powder was

mercer i zed

in 4mL

0

f

Ccontaining 0.02% sodium dodecyl kept thawed

in

-20·C in

to

be

sulfate)

alkali-chitin.

i -propano 1

neutralization with

20% aqueous NaOH so 1uti 0 n

at

room

The

tempera ture

for

an hour

and

alkali-chitin

was

fa 11 owed

by

the

the addition of monochloroacetic acid.

Crude CM-chitin thus obtained was washed by deionized water

to remove salt completely and dried in air to give pure 6-0CM-chitin. Water

soluble

me thod

as

6-0-CM-chitin

above

wi th

was

performed

Purification

was

prepared

40% CW/W) NaOH by

the

by

aqueous

dialysis

the

same

so I uti on.

of

crude

CM-

chitin against distilled and then deionized water to remove salt followed by concentration of inner solution to a small volume. Pure CM-chitin was obtained by the lyophilization or precipitation with acetone. chitin

was

controlled

by

Degree of substitution of CMalkaline

concentration

on

the

process of mercerization.

PREPARATION OF 3,6-0-CM-CHITIN One gram of 6-0-CM-chitinCD.S. 0.74) was dissolved in 4mL of 50%CW/W)NaOH solution and kept in -20C overnight. It was thawed

in 5mL

i-propanol

at

room

temperature.

Alkal i-CM-

chitin was neutralized by the addition of monochloroacetic acidC4g)

with

chitin.

vigorous

This

crude

stirring

product

to

was

give

crude

dissolved

3,6-0-CM-

in

45mL

dis till ed wa ter and sa tura ted BaCl2

so I uti on was added

precipitate

the

highly

3,6-0-CM-chitin.

precipitate

was

collected

dissolved

in

aqueous

deionized water the

by

the

EDT A solution

centrifugation the EDTA was

wa ter.

Bo th

was

dialyzed

added

and

dialyzed

by

the

against

and dialyzed

inner so I uti on was

obtained

centrifugation,

to

After it

was

against

to remove salts completely. Supernatant of

water,

was

substituted

of

distilled

against

deionized

concen tra ted,

precipitation

21

the

with

then product acetone

or

lyophilization.

Table 1. Yield of 3,6-0-CM-chitin yield yield(%) 3,6-0-CM-chitin precipitated by Ba 2 +

0.55g

CM-chitin not precipitated by Ba 2 +*

Yield(%) was estimated from D.S .. lowly substituted

one,

and

it

was

D.S.

43%

0.30g

1.7

belowI.2

*: It was included by the re-carboxymethylated

to

give 3,6-0-CM-chitin of D.S.I.53.

MEASUREMENT OF INFRA-RED ABSORPTION SPECTRA Infra-red

absorption

spectrometry

of

measured using JASCO A-302 type infra-red

CM-chitin

was

spectrophotometer

to identify the functional groups.

POTENTIOMETRIC TITRATION After 0.05g of CM-chitinCCOOH type) was dissolved in 30mL of

aqueous

O.IM NaCl,

3mL of aqueous

O.IM NaOH was added

and the resulting solution was treated with nitrogen gas for 30 min. solution

Then the solution was using

TSC-IOA

titrated by O.IM HCl aqueous

automatic

titrator(TOA

Electronic

Ltd.) under the nitrogen atmosphere at room temperature.

125MHz- 13 C-NMR MEASUREMENT

13C-NMR measurement D20 at

for

3,6-0-CM-chitin

was

achieved

in

the concentration of 33mg per mL with dioxane as the

outer standard at 60C using JEOL JNM GX-500 spectrometer.

LYSOZYME SUSCEPTIBILITY OF VARIOUS CM-CHITINS The

lysozyme

susceptibi 1 i ty

of

CM-chi tin

was

determined

viscometrically as a standard indicator for biodegradability with an Ubbelhode type viscometer at 30C. One mL of lysozyme solution of

(final

CM-chitin

7.4),

and

concentration,

0.63t,tM)

solution(1.0mg/mL)

the

rate

of

in

hydrolysis

was

applied

10mM Tris-HCl by

to

10mL

buffer(pH

lysozyme was

measured

according to the method of Hamaguchi and Funatsu(26).

PREPARATION OF CM-CHITIN OLIGOMERS CM-chi tin lysozyme

01 igomers

and

the

were prepared by the hydrolysis wi th

rate

of

lysozyme

accessibility

was

also

studied. A

500mL

phosphate

of

6-0-CM-chitin

buffer,

M/15,

units/mL of lysozyme, in which

the

solution(l%

pH6.2)

was

other organs of

human body(27).

CM-chitin

hydrolyzed

Similar level

lysozyme concentration

of

to

that

in

is higher

Moreover,

by

the

in 2500

human tear, than

that

lysozyme of

in the

same concentration was added every day for 9 days at 37C to maintain

constant

physiological

enzymatic

condition.

CM-chitin oligomer, after

7,

acid

was

24,

72

and

added

to

To

activity

hours.

give

to

simulate

the

investigate the compositions of

a part of 96

and

5%

reaction solution was 20% aqueous of

final

taken

trichloroacetic

concentration

to

inactivate and precipi tate was

removed

by

lysozyme.

centrifugation,

After

the precipi tate

trichloroacetic

acid

was

removed from a supernatant by the extraction with ether. The supernatant

was

concentrated

and

slowly

added

to

The precipitate was collected by centrifugation. weight(M.W.)

of

CM-chitin

oligomers

reaction was measured by

the gel

using Sephadex G-50 gel.

As

materials

for

indicating

estimated

bo th

by

end(28)

and

the

reducing end chains total

and

the

method

N-acetylglucosamine

Molecular

M.W.,

of

of

weight

was

in

several

permeation chromatography

molecular of

the

HCI-Indole residues

calculated

weight

titration

HCI-Indole(29).

in saccharides gives the

Molecular

there are no suitable standard

method

method

obtained

acetone.

number gives

of

was

reducing

Titration

of

of molecular the

number

of

in

CM-chitin

oligomer.

by

applying

following

equation

M.W.=

where R.W.

was

number of CM-GlcNAc residues number of molecular chains

residual

molecular

weight

the degree of substitution of CM-chitin.

x R.W.

estimated

from

RESULTS AND DISCUSSION

PREPARATION OF 6-0- AND 3,6-0-CM-CHITIN 6-0-CM-ch it i n was prepared from al kal i -ch it in, derived

by

mercerization

chloroacetic acid the

Williamson

in

wi th

keeping

reaction.

The

sugges ted

0

f

in Fig.3.

tha t

the degree

the

0

-20e,

and

mono-

reaction system by

infra-red

spectra

of

the

Since the new absorption at

due to the stretching of

increase

at

the heterogeneous

products were shown 1735cm- 1

and

wh i ch was

f

carbonyl group appeared

carboxyme thyl at i on,

carboxyme thyl

group

was

it was

introduced

in

chitin molecule. The carboxymethyl group was suggested to be substituted at only primary hydroxyl group of C-6 position in

the

N-acetylglucosamine

residue

by

13C-NMR

investigation(17). The degree of carboxymethylation was found to depend on the concentration of sodium hydroxide used, but not be above 1.0 under this condition, without

removal

of

in which the reaction

excess

alkaline

to

was proceeded

avoid deacetylation

(50). These occurs

results at

condition. between hydroxyl

C-6

would

position

Since

group

at

that

carboxymethylation

predominantly under

chitin

acetamide group

indicate

C-3

molecule

forms

at

position

C-2

position

in

this

reaction

hydrogen and

bonding secondary

N-acetylglucosamine

residue and between its bonding and primary hydroxyl groups at

C-6

position

hydrogen bonding

through

water

between hydroxyl

25

molecule(30),

and

forms

group at C-3 and hemi-

A

B

c

D

t 3600 2800

1735c m

1800

-1

(C=O)

1600 1400_ 1200 1000

Wave numb er (cm 1 ) Fig.3

800

Infra red spec tra of chiti n and CM- chiti ns A;ch i"tin,

B;D. S.

0.08 ,

26

C;D.S .

0.7,

D;D. S.

0.8

acetal

oxygen

al.(31),

atom

secondary

(0-5)

as

hydroxyl

described group

by

at

Sundarajan

C-3

position

et. is

protected by a few hydrogen bonding. As hydroxyl groups at c-3

position

under

is

still

protected

the he t erogeneous

hydroxyl group,

group

is

more

by

chern i ca I

the

hydrogen

mod if i ca t ion

reactive

than

bonding

and pr i mary

secondary

hydroxyl

carboxymethylation of C-6 position occurs more pre-

dominantly than that of C-3 position. 3,6-0-CM-chitin was obtained by the carboxymethylation of mercerized

6-0-CM-chitin

under

the

similar

condition

as

those for 6-0-CM-chitin. 125MHz_13C-NMR spectrum for 3,6-0CM-chitin was shown in Fig.4. Good separation was observed between the chemical shifts at C-3 and at C-5, which were not separated in 6-0-CM-chitin,

and chemical shifts of C-3

itself also separated. Though two kinds of for

carbonyl

group were

magnetic field,

observed

on

chemical shifts

6-0-CM-ch it i n

3,6-0-CM-chitin had several

groups

also

to

C-3

position

in

low

chemical shift

for carbonyl. It would suggest the introduction of methyl

in

carboxy-

addition

to

C-6

position. This reaction was carried out which

6-0-CM-chitin

hydroxide

solution.

was

once

OWing

in homogeneous system,

dissolved to

the

in

aqueous

destruction

in

sodium of

the

crystalline structure, especially by the blocking of hydrogen

bonding

by

carboxymethylation

at

C-6

position,

C-3

position is estimated to become reactive. However, it was impossible to substitute only C-3 position under this reaction condition, because the hydroxyl group at

-O-CH 2

~) C=O(Carbonyl)

H

l\)

00

C-3, C-3'

C=O(Acetamide)

I

C-2

C-l

C-~

~

I

PPM

220

2CX)

Fig.4

125MHz_

18)

13

160

140

120

100

80

C-NMR,spectrurn of 3,6-0-CM-chitin in D2 0 at 60°C

o

position

C-6

should

be

protected

before

carboxy-

methylation. Even if this hydroxyl group can be protected, the

molecular

weight

deprotection

and

would

decrease

complete

on

the

deprotection

process

could

not

of be

carried out due to the steric hindrance of CM-chitin. It was concluded

that

the

substitution

to

only C-3

position was

very difficult so far. Preparation scheme for 3,6-0-CM-chitin was summarized in Fig.5

toge ther

wi th

the

ou t line

0

f

prepara t i on

repor ted

previously by Okimasu(32). The preparation of CM-chitin by Okimasu was carried out in homogeneous

reaction

directly

from

chitin.

Sodium

mono-

chloroacetate was added to alkali-chitin solution which was prepared by dispersing chitin in alkaline ice-water mixture by vigorous stirring. In this reaction condition, CM-chitins of low substitution such as D.S. 0.2-0.25 are obtained, and random substitutions at C-6 and C-3 position occur. Further, the reaction is contaminated with N-carboxymethylation owing to

deacetylation

unsuited

for

the

during

the

purpose

of

reaction.

This

present

study

procedure

is

which

to

is

obtained highly substituted 3,6-CM-chitin. 3,6-0-CM-chitins

derived

from

t1-chitin

easily

by

twice

carboxymethylation from t1-chitin had D.S. 1.5-1.7. 'But these from a-chitin had about D.S. 1.2. This result might be from the difference between the crystalline structures of a- and t1-chitin.

Increase

in

the degree

of

substitution

for

the

preparation of 3,6-0-CM-chitin from a-chitin was achieved by the

following

improvement.

2 ~/'

i)Saturated

sodium

hydroxide

HO

~ H2Gio

c~

c~

~cR ~H20Ho ~'-~OH ~I H

0

0

HO

NH

H

0

CH20H

co

...

HO

n

NH

co

C~

CH20H

C~

Chitin , (A)

1.Na-chitin powder was suspended in i-propanol. 2.ClCH COOH was added into Na-c~itin suspension until neutralization.

~ IAll

C.'d

0

.w

00 n . . U

W

N~

\ \ \ to--

W

cnw '"0

~

\ 0

Ul

\ \~

--- -

0 -...-

\

\ \0

\ \

~

0

c \

~

-

\

0

\

n (J)

\

c \\

'" 3

---3

0

\

\

0..

-

0

-

\

-...-

~\

\

\ \

\

I

~

0

1

N

Fig.16

Plot of

~

2 as a function of the Fe + concentration

at different concentrations of eM-chitin

value

of

Fe 3 +

in

Rapp

is

comparable

to

that

in

oxidized

Fe 2 +, Fe 3 +, reported above. It is concluded that the behavior of scattering profile by the addition of Fe 3 + in which Fe 2 Since

5A

is comparable to that of

the solution

is oxidized as shown in Table IX(42).

+

of

radius

of

gyration

for

cross-section

is

consistence with 4.sA of a unit of crystal lattice ~onstant in

a

axis

estimated

from

X-ray

diffraction,

calcium

ion

seems to adsorb CM-chitin between the inter two chains. 6-0-CM-chitin found

to

presence linking ions

form of is

gel,

followed

transition necessary

seemed

chains

from p-chitin and all

to

metal for

induce

similarly

to

the

by

precipitation,

ions.

gel

3,6-0-CM-chitin were

As

inter

formation,

in

chain

transition

the

crossmetal

cross-linking between CM-chitin

the mechanism of gel

formation by the

addition of Fe 3 +. These phenomenon suggests that transition metal

ions bind with carboxyl

polymer

chains

to

bundle

several

regulate

chains.

structure was

found

because

crystalline structure

loose

to

groups around whole area of

the

function of

maintained in its derivatives owing to carboxymethyl clearly

group

showed

binding and

the

homogeneously difference

The

crystalline of CM-chitin

P-chitin was

still

the distribution of

along

between

to the

molecules.

It

calcium

ion

the adsorption of other metal ions. Calcium ion

binds to CM-chitin tightly in the local specific area which is formed by two chains or residues, which seems to be two residue,

and

other

metal

ions

are

supported

only

carboxyl groups in the wide region of polymer chains.

by

Table IX Radi i of Gyra tion of CM-c hitin Iron Ion Comp lex -Ion Cs(m ol/mo l) Cp(%) Rc(A ) Cons t . of conc . of addi tiona l sa 1 ts Fe 2 + Fe 2 + Fe 2 + Fe 2 + Fe 2 + Fe 2 +

0.15 0.154 0.77 0.77 1.5 1. 54

0.15 0.625 0.03 3.13 0.06 6.25

2.5 2.7 3.3 3.0 4.6 3.9

Cans t. of conc entra tion of CM-c hitin Fe 2 + Fe 2 + Fe 2 + Fe 2 + Fe 2 + Fe 2 + Fe 2 +

0.077 0.3 7.7 0.154 15.4 0.77 3.0

Fe 3 + Fe 3 +

3.0 15.0

I

0.31 0.31 0.31 0.625 0.625 3.13 3.13 3.13 0.63

2.9 3.2 2.9 2.7 6.3 3.0 3.5

9.5

Cs: salt conc entra tion (salt/ CM-G lcNA c) Cp: polym er conc entra tion Rc: radii of gyra tion

A Fe 2 + adsorbed 6-0-CM-chitin adsorbed keeping the soluble state,

site seems charge both

Fe 3 +

while

adsorbed

for

adsorption also

of

metal

forms

the

adsorption of barium ion, alkali-earth

tation

on

suggest

forms

gel.

The

adsorption

to be increased by the increase of the positive

structure

among

one

ion. gel

and

metal

ions.

Gel

inter-chain

from

precipitate by

which had the largest formation

the adsorption of metal

that

3,6-0-CM-chitin

ions

the

ion radius

and precipi-

to 3,6-0-CM-chitin

cross-linking

occurred

by

the

adsorption of metal ions owing to the high charge density of carboxyme thyl

group.

smoothly by metal

Espec i all y

ions

of

ge 1

large

formation

ion radius

proceeds

or multivalent

positive charge. Though the adsorption capacity of calcium ion to 3,6-0-CMchitin shows the highest value among other ions till 5mM of fairly

high

negative

concentration,

cooperativity.

its

3,6-0-CM-chitin

specificity to calcium ion. of

Scatchard

by

calcium

ion

re-carboxymethylation. adsorption

functionalization crystalline

of

did

not

indicates show

the

It might be suggested that one

the calcium adsorption site in

blocked

plot

to

eM-chitin molecular was Thus

CM-chitin

chitin was

the

0

f

elucidated,

and

to be depend on

the

was

found

mechan ism

structure and the degree of carboxymethylation.

SPECIFICITY OF CALCIUM ION TO 6-0-CM-CHITIN All

metal

released

ions

which

thoroughly

by

adsorbed pH

shift,

on but

6-0-CM-chitin calcium

ion

were was

released incompletely by pH shift (88% of total adsorption,

100 +

C\J

80

m

u

OJ

60 40

(J)

m QJ r--i

20

OJ

IT

1

2

3

4

P [He 1] Fig.1 7

Rele ase of Ca 2 + from 6-0-C M-ch itin

O.3g of low subs titut ed 6-0-C M-ch itin was imme rsed in 20mL of O.05M CaC1 aque ous solu tion unde r shak ing for 2 24ho urs at room temp aratu re and the CM- chitin was wash ed by wate r to remo ve free calci um ion. Amou nt of relea sed 2 Ca + was estim ated from the supe rnata nt conc entra tion afte r CM-c hitin -Ca comp lex was imme rsed in seve ral HCl conc entra tion in shak ing for 24h.

7 b',

see

Fig.17).

chelating suggests chitin.

For

the

reagent the

complete

such

tight

as

EDTA

adsorption

Other CM-chitins

release was

of

of

calcium

requested.

calcium

release calcium

ion

ion,

It

to

also

6-0-CM-

ion simply by pH

shift. The adsorptions of calcium

and magnesium ions to 6-0-CM-

chitin were measured by the batch method in the Ca-Mg mixed solution as shown in Table X. The distribution coefficient estimated

from

capacity

magnesium ion, strength.

suggested calcium

calcium

ion

per

was

as

in high

the

ionic

as

4.3.

to

have

the

selective

ion.

When

magnesium

in all

strength

of

adsorbed

was

ability

ion was

found

completely instead of

to

be released

the thus

toward

CM-chitin column

was washed out with aqueous calcium chloride solution, magnesium

for

ionic

0.5,

6-0-CM-chitin

adsorption

ion

which was

capacity

was high value (more than 1.0)

Especially

coefficient

for

for calcium ion,

the

from 6-0-CM-chitin

the adsorption of calcium ion to CM-

chitin as reported previously(33). In

the

high

concentration

chitin-Mg

complex

is

repulsion

between

the

of

unstable magnesium

magnesium probably ions

as

ion,

6-0-CM-

owing

to

the

shown

by

the

negative cooperativity. On the other hand, calcium ion forms a stable complex with CM-chitin. Consequently, ion forced ion

to push out

exchange

mode.

In

the calcium

the magnesium ion from CM-chitin in the

equilibrium

state

also,

only

calcium ion is adsorbed to CM-chitin similarly. The pres en t

study sugges ts

77

that

6-0-CM-ch it i n

ish i gher

Table X

Selective adsorption in Ca 2 +-Mg2+

~ Ion [

Total

j I I

"r: 1 I

O.35(meq/g)

0.56

mixture system

I

50 (me Q / g )

0,48

O.65(meq/g)

i

0.44 -----

-

I !

I

Ca2 +

0.31

0.39

Mg2 +

0.25

D.C

1.2

I

0.28

0.09

I

0.16

4.3

i

I

I

1.8

D.C; Distribution coefficience (Capacity Ca/Capacity Mg) I ; Ionic strength

specific

for

tration,

calcium

ion,

especially

in

the

low

concen-

together with the ion exchange ability.

I tis

very

surface

of

structure

important

CM-chitin-Ca

and

to

to

complex

consider

as biocompatible polymer. clarify from

elucidate

know

estimated

organic

to

the

fundamental

the

The complex surface was

The property was

of

the possibility for utilization

the standpoint

compound

to

property

the

tried

to

of hydrophobic or hydrophilic. from

the adsorption behavior of

6-0-CM-chitin-Ca complex,

which can be

predicted rather simple term of the degree of hydrophilicity or hydrophobicity. Though this method will give the indirect result,

the informations obtained from this experiment would

be significant. Amino acids were selected as the molecules to be adsorbed, as

amino

chain, groups The

acids

and

have

several

a-amino

and

hydrophobic

a-carboxyl and

hydrophilic

in main

functional

in side chain.

investigation

chitin-Ca

complex

on

the

would

adsorption give

of

valuable

amino

calcium

ion,

which

has

high

acids

information

interaction of CM-chitin with a fibrinogen of

groups

affinity

to

CM-

for

the

in the presence to

hydrophobic

materials (18).

ADSORPTION OF AMINO ACIDS ON CM-CHITIN METAL ION COMPLEX Since one of calcium

the

interaction

of

the blood proteins, ion,

the

6-0-CM-chi tin was enhanced in

CM-chitin-Ca

complex

wi th

fibrinogen,

the presence of is

expected

to

interact

tightly with

( 1 8 , 22).

D, L - am i no

used

estimate

to

the hydrophobic domain of

a c ids the

h a vi n g

adsorption

complex(low substi tuted

fibrinogen

d iff ere n t

pI

val u e s

capacity

on

CM-chitin-Ca

6-0-CM-chi tin),

as

1 isted

we r e

in Table

XI. The adsorption of D,L-Phe was enhanced about 50 times in the case of calcium chelation compared to CM-chitin without calcium ion. As

the

effective

adsorption

is

calcium ions, groups

f

0

lower

force

calcium

ion.

reduced

to

of

than

calcium

the

total

ion

number

the geometrical arrangement of

the

driving

number

CM-N- ace ty 1 g 1 uco s ami ne for The

the

Phe

adsorption

almost 1/10 and 1/5

respectively.

in

of

adsorbed

wou 1 d

including

D,L-Lys

of

D,L-Phe

the functional

res i due

adsorption,

for

and

be

the

that

with

D,L-Glu

were

the presence calcium ion

The adsorbed phenylalanine was eluted only by

EDTA, while the other adsorbed amino acids were eluted by a shift

of

the

ionic

calcium effect on 6-0-CM-chi tin, metal

ions,

chitin

strength.

Since

there

was

the adsorption of amino acids

only to

slight fibrous

as expected from its adsorption behavior for

the

molecule

original seems

binding site to

have

been

for

Phe

on

eliminated

the

CM-

by

the

stretching procedure on the preparation of fiber. There is also little selectivity for the optical isomer of phenylalanine The adsorption profiles of D,L-amino acids for high substituted 6-0-CM-chitin-metal

ions complexes from a-

chitin were studied using equilibrium dialysis comparing to that

of

other

CM-chitins,

as

shown

in

Table

XII.

The

adsorption capacities of 6-0-CM-chitin from a-chitin toward

80

TABLE XI Adsorption of Amino Acids to Low Substituted CM-Chitin and Fibrous CM-Chitin in the Presence and Absence of Calcium lon a Capacity, J,Lmol/g Amino acid

Ca2+ chelate

Low substituted (OS 0.1) 0.65

D,L-Phe

+

33

D,L-Lys' HCI

+ D,L-Glu

+

Fibrous 2.0 2.0

2000

73

300

65

0.22

0

0.04

0.2

a Amino acids were applied to the column to equilibrium, and it was then washed with deionized water. Then l),L-Lys and D,L-Glu were eluted with 0.5 M aqueous NaCI solution. D,L-Phe was eluted by 0.1 M aqueous EDTA solution at room temperature.

81

Table XII-a Adsorption of amino acids on water-soluble 6-0-CM-chi tin from a-chitin in the presence of several ions

Ba2 + (fjmol/g)

Mg2+ (fjmol/g)

17 89 110 390

13 21 28 310

24 55 47 43

19 58 54 34

45

48

48

3.2 3.0

60 110

32 28

19 26

46

39

9.8 10.8 7.6

2700 2400 120

370 530 57

370 490 30

323

247

pI

Non (fjmoI/g)

Ca2 + (fjmol/g)

Nonpolar: D,L-Ala D,L-Val D,L-Leu D,L-Phe

6.0 6.0 6.0 5.5

80 56 27 100

Neutral polar: D,L-Trp

5.9

Acidic: D,L-Glu D,L-Asp Basic: D,L-Lys-HCI D,L-Arg-HCI D,L-His-HCI

I

Mn 2 + (fjmol/g)

Amino acids

Amino aCid-water soluble CM-chitin mixture was dialyzed against deionized water to remove free amino acids, and then adsorbed amino acids were released by pH shift(pH 1.0), and it was redialyzed against deionized water to quantify adsorption capacities.

82

Table XII-b Adsorption of amino acids on water-soluble CM-chitins in the presence of Ca2 +

6-0-CM-chitin from a-chi tin Amino acids

6-0-CM-chitin from {3-chi tin

Ca 2 +

D,L-Ala D,L-Val D,L-Leu D,L-Phe

Ca2 +

Non (/..tmol/g)

(/..tmol/g)

Non (/..tmol/g)

80 56

17 89

33 22

27 100

110 390

36 14

60

32

17

20

247

282

Nonpolar:

-

I I I

18 27

34 21 17

13 21

I

Ca 2 + (/..tmol/g)

100

8

13 19 18

30

17

262

626

I

Basic:

D,L-Lys-HCI

Non (/..tmo II g)

(/..tmo II g)

Acidic:

D,L-Glu

3,6-0-CM-chitin from a-chitin

2700

370

83

I

I

D,L-Phe

and D,L-Leu

are

increased

about

fourfold,

and

a

slight influence on the adsorption of D,L-Val was observed among ion.

the various

amino

acids

adsorption

D,L-Ala

negative effect

in

was

the presence of

decreased

by

calcium

calcium.

The

was observed for the adsorption of acidic

and basic amino acids

in

the presence of calcium ion. The

adsorption capacity of CM-chitin from ex-chitin for D,L-Phe increased

about

threefold

and

hardly

any

effect

was

observed on the adsorption of amino acids in the presence of manganese ion. But the adsorptions of neutral amino acids to CM-chitin from

ex-chitin

in the presence of barium ion or

magnesium ion did not give the similar phenomena to those of calcium ion and manganese ion. The capacities of Phe in the presence of both ions were lower than D,L-Leu. amino

However,

acids

adsorption manganese

adsorption

were site

ion

higher

capacities

than D,L-Ala.

formed

is

that of D,L-Val and

by

the

d if feren t

from

for It

presence tha t

by

these

seems

three

that

the

of

calcium

and

the

presence

other alkali-earth metal ions. It will be discuss

0

f

in detail

in next section. The

adsorption

fro m t1- chi tin

profile is

a I m0 s t

chitin from ex-chitin. 6-0-CM-chitin-Ca complex)

is

of

acids

d i f f'e r e n t

to

6-0-CM-chitin

fro m tha t

to

6-0-CM-

It seems that the surface property of

complex· from

quite

amino

ex-chitin(Ca-6-0-CM-chitin(ex)

distinguishable

from

that

of

6-0-CM-

chitin-Ca complex from t1-chitin due to the distribution of carboxyl group and d i spers i ng chai ns,

though

the mechan ism

of calcium adsorption is very similar in both 6-0-CM-chitin

84

described above. Since

the

chi tin side

capacity

of

decreases wi th

chain

in

the

neutral the

to

acids

to

3,6-0-CM-

increase of hydrophobici ty

absence

charge density seems

amino

of

metal

inhibit

ions,

the

high

neutral

of

negative

amino

acids

adsorption to 3,6-0-CM-chitin.

SPECIFIC ADSORPTION OF NEUTRAL AMINO ACIDS TO 6-0-CM-CHITINCA COMPLEX As the affinity of neutral amino acids toward the 6-0-CMchitin(a)-Ca complex was suggested to depend on the hydrophobicity plotted

of

its

against

side

chain,

the

hydrophobicity

adsorption of

capacity

neutral

amino

was

acids

according to Nozaki and Tanford(45) as in Fig.IS. This data shows ac i ds

tha t 0

the

ther

relation

additive

capac it i es

than Phe were found

to

abnormally

adsorp ti on

the

hydrophobicity

high

adsorption

factor,

probably

0

f

neu t ral

ami no

to be simp I y propor t i onal of

the

side be

chain.

caused

by

The

of

Phe

would

an

by

the

aromatic

hydrophobicity

amino acids,

the adsorption

effect. On

the adsorption of neutral

constant

depending on

temperature by

temperature was

measured

at various

equilibrium dialysis to achieve the thermo-

dynamic study of complex formation.

The results are listed

in

of

Table

XIII.

The

specificity

6-0-CM-chitin(a)-Ca

complex for Phe was confirmed by higher value of Ka for Phe comparing

that

amino acids,

Val

for

Val.

Adsorptions

and Phe,

of

are suggested

85

non-polar

neutral

the conformational

---

400 P

CJ)

I

'-....

I

r--t

I I

0

E

-~

I I

300

·rl

I

I I

/

I

I

,

>-

+J

A

I

/

I

I I

I

200

I

I

,,

I

U

co 0.. co u

I

I

I

I

I

I

I

I

100

-

p ....

~

-

....

."*,,,,"

....

~

,

I

~d

I

0

I

~

~._._._.

__ ._.-. -.-·a

.-.--

0 0

500

1000

1500

Hyd rop hob icit y Fig.1 8

2000

2500

3000

( ~ft)

Rela tions hip betw een adso rptio n capa citie s of amino acid s for wate r-sol uble 6-0-C M-ch itin from -chi tin and hydr opho bicit y of the side chain In the prese nce of Ca 2+ , ( 0 ) : in the prese nce of Mn , ( 0 ) : in the absen ce of meta l ion ( c. ):

2+

86

Table XIII

Dependence of association constant on temperature

Ka (W

Phe

Val

l

1 )

6G (kcal/mol) aH (kcal/mol) 68 (calK/mol)

30

-4.0

40

-3.6

50

-3.0

30

2.8xl0

-2.0

40

1.0xl0

-1.5

50

0.7xl0

-1.3

~ ~+---+-------1

I '

40

I

I

50

I

I Ka:

7.5xl0 2

-4.1

-14

-38

5.7

31

-4.2

i

I

association constant was

dialysis

-48

-3.7

30

Trp\

!

-18

equilibrium

buffer(pH7.4,

calculated by Scatchard plot after

was

10mM CaCl2)

carried

out

in

ImM

Tris-HCI

for 24hours with shaking and concent-

rations of amino acids were measured by fluorescence spectrometry using o-phthalaldehyde method. plot.

6H

was

calculated

by Van't

Hoff

change

of

CM-chitin-Ca

complex

to

reach

ground

state,

because aH for both amino acids show negative value. As it for Phe gi ves chi tin-Ca-Phe

1 arger aH val ue complex would

phobic

interaction

polymer

molecule.

with Since

than

be

formed

the aH

that

for Val,

through

the

conformational

for

Trp

gives

the CMhydro-

change

positive

of

value

because of polar indole ring as side chain and insensitively to the calcium ion, the conformation of CM-chitin-Ca complex might be in unstable state. It suggests that specific domain formed

in CM-chitin-Ca complex might

just fit

for benzene

ring but not for more bulky group. than benzene ring. A preliminary lH-NMR measurement was

carr i ed ou t

by

for CM-chitin(ex)-Phe-Ca

500MHz_l H-NMR as

shown

in Fi g. 19.

As

chemical shift of meta-proton of phenyl ring seems to reduce and all

line width in aromatic ring are broaden,

also suggests

its study

that phenylalanine side chain surely contri-

butes to the adsorption to 6-0-CM-chitin-Ca complex. The adsorption

capacity of benzene to the low substituted

6-0-CM-chitin(from

ex-chitin)

was

investigated

in

several

concentration of calcium ion to clarify the contribution of benzene ring on

the Phe adsorption.

was

be

applied

to

suspended

in

O.5g of 6-0-CM-chitin

45mL

of

several

aqueous

calcium chloride solution. 5mL of saturated

aqueous benzene

solution was added to the mixture and was

shaken for 24h.

The

concentration

of

benzene

calculated by the adsorption

in

the

at 253.7nm

supernatant

was

using the equation

mentioned by Franks et.al.(46), because the concentration of ben zene

does

no t

obey

the

88

equa t i on

0

f

Beer-Lambe 1 t.

The

D. L-Phe

Ca-CM-chitin-D.L-Phe ( 23°C)

Ca-CM-chitin-D.L-Phe

.,

f

•

7.7

Fig.19

1

I

•

'

7.6

7.5

I

.,

7.4

,

I

7.3

ii"

7.2

I

7.1

i

, I

7.0

(ppn)

H-NMR-spectra of Ca-CM-chitin-D.L-Phe and D.L-Phe

in the aromatic region using 500MHz NMR spectrometer

capacity

is

estimated

concentration in

from

the

difference

deionized water and that

between

the

in the presence

of calcium ion. The

adsorption

capacity of

benzene

to

CM-chitin

presence of calcium ion is higher than that in increased

in

the

water. The

capacity is 28t,tmol/g in ImM of calcium ion, and

39 t,tmol/g in

lOmM of calcium ion.

capacity of benzene As benzene,

This suggests

that

the

depends on the calcium concentration.

lacking ionic functional groups,

is adsorbed to

the CM-chitin-Ca complex, benzyl group in the side chain of phenylalanine would contribute the Phe adsorption to 6-0-CMchitin(a)-Ca complex. When aromatic ring forms the metal ion complex, Ca

1r

electron induces the formation of

complex.

The

adsorption

of

phenylalanine

1r

complex for seems

to

be

contributed by the similar mechanism. This assumption would explain fairly well chitin(a)-Mn

to

the higher capacity of Phe for CM-

complex than

that of Phe in the presence of

other non-specific metal ions, since manganese ion seems to form d

1r

complex easily because of insufficient of electron in

orbital.

SPECIFICITY OF CM-CHITIN FOR THE OPTICAL

ISOMER OF

PHENYLALANINE As the specificity of 6-0-CM-chitin(a)-Ca complex for D,LPhe was found,

the affinity of its complex for the optical

isomer of phenylalanine was

investigated to obtain further

information about the geometrical arrangement of the contributed functional group on CM-chitin molecule.

90

Since times

the

as

amount

much

of

as

that

adsorbed

to

the

analysis

of

adsorbed

shown

in

Table

selectively to

adsorbed D,L-Phe of

CM-chitin-Ca

XIV,

and

L-Phe

was

a

after

L-Phe

was

three

little D-Phe

complex(as

Phe

almost

estimated

was

by

UV

equilibrium dialysis,

as

suggested

to

be

adsorbed

the CM-chitin(a)-Ca complex without optical

resolution. It was assumed that the adsorption site for D,LPhe

is

both

predominant

L-site

and

weak D-site

which

op t i ca I i somers were conj uga ted wi th respec t i ve enan t i orner, because the amount of adsorption for D,L-Phe is nearly equal to twice as much as the summation of them. At

the

same

time,

optical

rotation

mixture solutions were measured.

of

the

adsorbed

It was observed that the

optical rotation for L- and D,L-Phe adsorbed CM-chitin was higher

than

that

of

calculated

from

the

UV

absorption

analysis. Especially L-Phe adsorbed one gives enhanced value sixfold higher than that from UV. This result induction

of

the

optical

rotation

by

the

suggests

the

adsorptions

of

optical isomers. The effect of D,L-Phe to weight

on

the

CM-chitins of various molecular

induction

of

the

optical

rotation

was

investigated by UV and optical rotation to elucidate whether polymeric effect or the chemical structure of CM-N-acetylglucosamine

residue

was

ascribed.

The

experiments

were

carried out refering with that for carboxymethyl-cellulose (CMC) as shown in Fig.20,21. The

induction

of

negative

optical

rotation

with the decrease of molecular weight. This

91

increases

effect for CM-

Table XIV

Induced optical rotation by the adsorption of Phe to Ca2 +-CM-chitin in solution

D,L-Phe Adsorbed D,L-Phe .:1X

1.2mM es t imated from OD260 (390 j.lmol/g)

= -0.007

1.2mM es t ima ted from optical rotation as L-Phe

L-Phe Adsorbed L-Phe .:1X

0.4mM es t ima ted from OD260 030 j.lmo 11 g)

= -0.013

2.3mM es t imated from optical rotation

D-Phe

I

O.lmM estimated from OD260 (. 40 j.lmo 11 g)

Adsorbed D-Phe .:1X

.:1X

=0

= Xobs

-

XRef

,Xobs

;Optical rotation was obtained from Ca 2+-CM-chitin and Phe solution

XRef

;Optical rotation was obtained from only Ca 2 +-CMchitin solution

The concentration of its solution obtained by optical rotatjon was calulated as the concentration of [-Phe. Water soluble CM-chitin was mixed with amino acids in the presence of Ca 2 + and then dialyzed against deionized water thoroughly at room temperature.

[D,L-Phel/[COOHl

= 0.14,

[D,L-Phel/[COOHl

= 0.05

6X

+0.OCJ5

('\

,

~ .,/'

o ~

...---

(0

,

0

-

------- - - -6-"-_

100

-

-- - -

-c--'--

0

~

[DL-Phe]xlO-SM

-

--

.

C\)

Fig.20

2 Induced optical rotation on the DL-Phe adsorption to Ca +-CM-saccharide complexes. -o-:CM-GlcNAc -~-:CM-chitin

oljgomer

_.1'::.-.: CM-chi tin

-A-:CMC-Na 2 The concenration of CM-saccharides is 0.2% and the concentration of Ca + is 25mM.

O. 1

-..

.-~------

~

..

0.0 Q)

......

u

c::

~.\

I-f:. ,

C1l ..0 h

--

~

......

----- - --

-

... -

--4

0 V'l

..0



>

·H-G ly-Ly s-Phe -Gly- OH Z-G ly-O H-- ---- ---- ---- ---- ---- ---- ---- --> Boo-L ys-Ph e-Gly -OH HCI/dioxan9~ H-Ly s-Phe -Gly- OH >---I

I

Z

------~~~

Z

MA Z-GI P d -Phe-C --;.--. ,] y-Lys -Phe- G I y-OH H 2 • (H-G I y-Lys G I y-O~ I Z

·H-Gl y-Lys -OH Z-GIY-OH~

H-Lys-OH I

Z

Fig.2 4 Z:

. /Pd C Z-GI y-Lys-OH H.t!/ - Jo [H-GI y-Lys-OH] I

Z

Synt hetic route to pept ides.

Benz yloxy carbo nylgr oup.

Z-CI :

Boc:

Benz yloxy carbo nyl chlo ride. Boc-S DP: S-t-B utylo xyca rbon yl-

t-Bu tylox ycarb onyl grou p. 4,6-d imet hyl-2 -mer capto pyrim idinc . DCC: Dicy clohe xylca rbod iimid e. H-Ty r-Gly -OH, H-Gl y-Ty r-Gly -OH, afld H-Gl y-Tyr -OH were synth esize d by meth ods simi lar to those for H-Ph e-Gly -OH, H-Gl y-Phe -Gly- OH, and H-Gl Y-Ly s-OH ,resp ectiv ely.

106

affinity chromatography(47-49) detail

described

in

chapter

according The

1.

to

the

adsorption

method

in

capacities

were estimated from UV absorption at 257nm using flow cell and fluorescence intensities of o-phthalaldehyde derivatives of

amino

acids

using

340nm

as

excitation

and

455nm

as

emission wavelengths(see chapter 2).

THE HYDROLYZED PROFILE OF GLY-PHE-p-NITROBENZYL ESTER O.8mL of 2.5mg/mL 6-0-CM-chitin from a-chitin in 30mM of aqueous calcium chloride solution was added to 2mL of 300mM glycylphenylalanine p-nitrobenzyl ester hydrochloride (HCI· H-Gly-Phe-ONBzl). 50min

and

then

shaking

to .be

peptide.

Then

The solution was

stood

at

formed O.2mL

room

complex of

kept

in a refrigerator

temperature and

O.2mg/mL

to

in

avoid

occasionally autolysis

chymotrypsin

or

of

binary

enzyme (consisted of O.2mg/mL chymotrypsin and Img/mL lysozyme in O.03M of calcium chloride solution) was added to the substrate solution. The release of p-nitrobenzyl alcohol as a

enzymatic

hydrolysis

product

was

monitorred

using Hitachi U-3200 type spectrophotometer.

107

at

300nm

RESULTS AND DISCUSSION

ADSORPTION OF PEPTIDES TO 6-0-CM-CHITIN Various peptides were examined to confirm the adsorption sites

of

amino

acids

for

the

CM-chitin-Ca

complex(water

insoluble), as listed in Table XVI. Since

the

adsorption

capacity

for

H-Gly-Phe-Gly-OH

is

maintained at almost the same level that of H-Phe-Gly-OH and H-Phe-OH,

the a-amino and a-carboxyl groups of Phe do not

likely to affect

the adsorption of Phe significantly.

The

increase of the adsorption capacity of the peptides containing tyrosin suggest a contribution of aromatic ring to the adsorption. Though there is a positive contribution of the calcium

ion

on

the

adsorption

of

a-N-blocked

lysine,

a

negative effect of calcium is still shown on H-Lys-Phe-GlyOH and H-Gly-Lys-Phe-Gly-:OH. enhance

the

calcium

The change of basicity might

effect

in

spite

of

a

significant

decrease in ionic linkages. These

results

would

suggest

the

contribution

of

only

benzyl group for the adsorption of Phe even in the peptide form.

These

results

proposed model rated

in

the

that

might

reinforce

to

reasonableness

of

the side chain of Phe justly incorpo-

adsorption

domain

formed

by

the

calcium

adsorption, and main chain of the peptide is exposed on the surface of polymer chain. The functions given by the carboxymethylation are seemed to have right to develop for biomimetic material utilizing its high biocompatibility described in introduction section

108

Table XVI

Adsorption of peptides to low-substituted 6-0-CM-chitin from a-chitin.

Peptide name

Ca2 +

CapacitY,llmol/g

D,L-Phe

+

33

H-Phe-G 1y-OHB

-

7

+

15

-

8

+

12

-

27

+

32

-

23

+

25

-

22

+

26

-

37

+

46

-

230

+

100

-

90

+

34

H-Gly-Phe-Gly-OHB

HCI-H-Tyr-Gly-OHB

H-G 1y-Tyr-OHB

H-Gly-Tyr-Gly-OHB

H-GI y-Lys-OHB

H-Lys-Phe-Gly-OHa

H-Gly-Lys-Phe-Gly-OHb

BIg CM-chitin was packed into a column(1.0x10.4cm), and 4.0mM peptide solutions were passed through the column at a rate of about 0.2 mL/min. bColumn contained 0.5g CM-chitin;flow rate as above.

lOti

and

biodegradability,

described account,

in

wh i ch

chapter

model

higher

Taki ng

1.

synthesis

is

for

these

than

chitin

properties

into

sustained release system

of

drug without side effects was planned. The adsorbed molecule would be released with the digestion of CM-chitin in animal body,

and

CM-chitin

would

disappear

in

the

system without any toxicity in animal body.

metabolism

It was examined

to obtain fundamental informations about sustained release a synthetic chymotrypsin substrate in two enzyme systems. result will be

Its

discussed in next section.

HYDROLYSIS OF SYNTHETIC SUBSTRATE CONTAINING PHE The hydrolysis rate of HCloH-Gly-Phe-ONBzl, substrate for chymotrypsin, was

was measured

chymo tryps i n

sys tern

in

and

two the

enzyme systems, 0

ther

was

the one

binary

enzyme

system consisted of chymotrypsin and lysozyme being closer system for physiological condition. The

initial

velocities

HCloH-Gly-Phe-ONBzl sys terns as

shown

chymotrypsin

hydrolyses

the presence of CM-chi tin

were higher than tha tin the absence in

Fig.25.

peptidyl substrate to

in

of

extremely

slow

But

the

initial

0

of

on both

f CM-ch it i n

hydrolysis

of

the

in the presence of CM-chitin was settle rate

hand, af ter 72 hours,

after

five

minutes.

On

the

the hydro I ys i sin the absence of

other CM-

chitin was continued slowly. These results seem to indicate that

CM-chitin

accelerate

the

hydrolyses

of

chymotrypsin

apparently. Since autolysis of a-chymotrypsin is reduced by the presence of calcium ion(57) and CM-chitin-Ca complex had

110

( a)

0.3 (b )

(c )

.

U I:

•..

.a ~ ~4

~

o

t"