Conformation of silk sericine in solution, Bombyx mori L.

SHORT COMMUNICATIONS

477

BBA 3 3 1 5 5

Conformation of silk sericine in solution, Bombyx mori L. Although extensive investigations have been carried out on the conformation of silk fibroin, there has been almost no work published on the conformation of silk sericine, especially in solution. In this paper, the conformation of silk sericine, Bombyx mori L., studied by optical rotatory dispersion (ORD), by circular dichroism (CD) and by infrared absorption measurements is reported. Native and regenerated silk sericines were tested and compared. Native silk sericine. Middle silk glands were removed from mature sericine silkworms whose posterior silk glands degenerate and, supposedly, are unable to produce silk fibroin. With forceps they were stripped from their cellular membranes, dispersed and dialyzed with pure water in cellophane tubing overnight. Silkworms whose posterior silk glands were removed on the first day of the fifth instar were also used. Regenerated silk sericine. Silk sericine was extracted by OKAMOTO'S1 method from shells of unheated cocoons with 0.04 M NaOH at 30-35 °. It was separated into two components by adjusting the solution to pH 4.0 with acetic acid. The soluble part, sericine A, was adjusted to a neutral pH and dialyzed against pure water overnight, and the insoluble part, sericine B, was redissolved with o.04 M NaOH and dialyzed. Sericines A and B were in the ratio 5:2. These crude silk sericine solutions then were clarified by centrifuging them 20 rain

6

I

I

I 200

I 220

I

I

1

I 260

I 280

1

2

? o

2

x ,rTn

&

///

2:

zl

__

i -J

-1.5--

L

-30, __ I. 180 200

.

I . _ ~. _ 220 240 ?~(rn!J)

I 260

I~ 280

I

o

-2--

-1.5 -3.0 180

I 240 Mm!J)

Fig. i. R e d u c e d m e a n residue r o t a t i o n and m e a n m o l a r e l l i p t i c i t y of n a t i v e silk sericine in a q u e o u s solutions. Line I, p H 7.3; 2, 8 M u r e a ( p H 7.3); 3, 3.6 M NaC1 ( p H 7.3) ; 4, p H 5.o; 5, p H i i . o . Fig. 2. R e d u c e d m e a n residue r o t a t i o n a n d m e a n m o l a r e l l i p t i c i t y o f t h e t w o c o m p o n e n t s of r e g e n e r a t e d silk sericine in a q u e o u s solutions. L i n e I, sericine A; 2, sericine B. - - , p H 7.3; ------, 8 M u r e a ( p H 7-3). A b b r e v i a t i o n s : O R D , o p t i c a l r o t a t o r y dispersion; CD, circular dichroism.

Biochim. Biophys. dcta, 181 (1969) 477-479

478

SHORT COMMUNICATIONS

at 2000 × g before p r e p a r i n g stock solutions. O R D a n d CD were m e a s u r e d at 27 ° with a J a s c o O R D / U V - 5 recording s p e c t r o p o l a r i m e t e r , and infrared a b s o r p t i o n was m e a s u r e d with a J a s c o D S - 3 o I recording s p e c t r o p h o t o m e t e r . The m e t h o d s for measuring the O R D , CD a n d infrared a b s o r p t i o n of t h e solutions h a v e been described elsewhere 2. I n o r d e r to characterize the c o n f o r m a t i o n of silk sericine, the O R D a n d CD of silk sericines were m e a s u r e d at various p H ' s a n d salt concentrations. The O R D of n a t i v e silk sericine in aqueous solutions d i s p l a y s shallow t r o u g h s n e a r 23o a n d 2o5 m # a n d a p e a k at 19 ° m#, as shown in Fig. I. The f e a t u r e of t h e O R D curves resembles t h e c a l c u l a t e d dispersion a of a h y p o t h e t i c a l m i x e d c o n f o r m a t i o n of the/5-form a n d of

TABLE I AMINO

ACID COMPOSITION

OF SILK

SERICINE

The v a l u e s in th is t a b l e are g i v e n in mole %, refering to t he d a t a gi ve n b y SHI~UZU et al. 4.

Gly

Ala

Ser

Tyr

Asp, Glu

Lys, A rg

Cyg

Pro

Others

11.6

5.0

28.1

2. 7

19. 3

6.2

0.2

0. 4

11. 9

t h e coil of silk fibroin. The CD curve of n a t i v e silk sericine in w a t e r d i s p l a y s two shallow, n e g a t i v e b a n d s centered at 217 a n d 2oo m#. These results i n d i c a t e t h a t the conform a t i o n of silk sericine is a coil c o n t a i n i n g a small a m o u n t of the/5-form. I n T a b l e I, the amino acid composition of silk sericine is shown. The presence of the/5-form in silk sericine could be due to a high serine content. A d e t a i l e d discussion, however, will be impossible because t h e a m i n o acid sequence in the p o l y p e p t i d e chain is still unknown. Moffitt p a r a m e t e r b0 of n a t i v e silk sericine at n e u t r a l p H ' s is o, consistent with the result t h a t the a-helix is n o t present in silk sericine, t h e p a r a m e t e r a 0 being - - 2 7 o. Tile a0 a n d b0 r e m a i n u n c h a n g e d in a p H range between 5 a n d I I . No difference in the Cotton effects is seen (see Fig. I) besides the c o n t e n t s of acidic and b a s i c residues would balance the dissociation of the p o l y p e p t i d e chain. W h e n NaC1 is a d d e d to the solution up to 3.6 M, the a 0 value s t e a d i l y becomes less negative. As for poly-L-glutamic acid 5 a n d silk fibroin 6, this would i n d i c a t e a c o n t r a c t i o n of t h e p o l y p e p t i d e chain. In 8 M urea, on the c o n t r a r y , the a 0 value becomes more negative, p r o b a b l y i n d i c a t i n g unfolding of the p o l y p e p t i d e chain. The O R D a n d CD curves indicate a d i s a p p e a r a n c e of the fl-form. I n zH~O, silk sericine has a single a b s o r p t i o n m a x i m u m a r o u n d 166o cm -1 with a slight rise n e a r 162o cm -1. This rise m a y or m a y not be i n d i c a t i v e of the presence of the/5-form. In a n y case the c o n t e n t of the/5-form is n o t enough to be d e t e c t e d u n a m biguously from the f e a t u r e of the a m i d e I b a n d . W h e n 5o % (v/v) dioxane or m e t h a n o l is a d d e d to aqueous solution, a new a b s o r p t i o n m a x i m u m a p p e a r s a t 162o cm -1, i n d i c a t i n g an increase in the/5-form content, which is now a b o u t 5 o % as e s t i m a t e d using the m e t h o d described elsewhere 6. I n f r a r e d dichroism of s t r e t c h e d films cast from the m i x e d solvents shows t h a t the/5-form which a p p e a r e d in the solutions is t h e cross/5-type, as is t h e case in silk fibroins 2. I t was n o t possible to d e t e r m i n e if the /5-form originally present in t h e n a t i v e silk sericine is the cross/5-type. Biochim. Biophys. Acta, i 8 I (1969) 477-479

479

SHORT COMMUNICATIONS

ORD and CD curves of sericines A and B are shown in Fig. 2. Based on arguments similar to those for the conformation of native silk sericine, both components are known to exist in a disordered conformation with a small amount of the/5-form. Mean molar ellipticity, [0], of IOO~o/5-form is estimated from our previous work 3 to be about -- 16 ooo at 218 m/~. The content of the Or-formis now calculated as between 5 and IO %, although any quantitative estimation of the/~-form by CD and ORD is hazardous at the moment. It is thought that 1,~-9 there are some small differences between the two components in the amino acid composition. As their conclusions do not always coincide and are inconsistent in m a n y cases, the cause of the difference in the/5-form content can not be discussed. The Or-form of sericine B is known to be the cross/~-type. In the visible ORD, sericine A is less negative than sericine B, which is less negative than native silk sericine. Nevertheless, the Cotton effects of native silk sericine are expressed as the mean weight of those of the two components of regenerated silk sericine in proportion to their content. This, however, is not the case in the visible ORD. This discrepancy could be due to the expansion and contraction of the polypeptide chain without any change in the ordered structure. The author thanks Dr. H. Takeda and his co-workers of this Faculty who donated silkworms. He is also indebted to Miss Y. Takeuchi for handling the crude data. This work was supported by a grant from the Ministry of Education.

Faculty of Textile Science and Technology, Shinshu University, Ueda, Nagano (Japan) 1 2 3 4 5 6 7 8 9

EISAKU IIZUKA

S. OKAMOTO,in T. ITO, Structure of the Silk, Chikuma-kai, Ueda, 1957, p. 88. E. IIZUKA, Biochim. Biophys. Acta, 16o (1968) 454. E. IIZUKA AND J. T. YANG, Proc. Natl. Acad. Sci. U.S., 55 (1966) 1175. M. SHIMIZU, T. FUKUDA AND J. KIRIMURA, in S. AKABORI AND S. MIZUSHIMA, Protein Chemistry, Vol. 5, Kyoritsu, Tokyo, 1957, p. 317 • E. IIZUKA AND J. T. YANG, Biochemistry, 4 (1965) 1249. E. IIZUKA AND J. T. YANG, Biochemistry, 7 (1968) 2218. J. T. B. SHAW AND S. G. SMITH, Nature, 168 (1951) 745. F. BRYANT, Textile Res..[., 22 (1952) 523 . K. KOMATSU, J. Sericult. Sci. Japan, 35 (1966) 125.

Received February 24th, 1969 Biochim. Biophys. Acta, 181 (1969) 477-479

BBA 33153

Stoke's radius and frictional ratio of human prostatic acid phosphatase In previous papers1, ~ the determination of the sedimentation coefficient (s20' w 5.73) and the molecular weight (M = 95 800) of human prostatic acid phosphatase (EC 3.1.3.2) obtained by ultracentrifugation in a sucrose gradient were reported. The molecular parameters of the enzyme such as the Stoke's radius and the frictional ratio have been determined using gel filtration techniques. Human prostatic acid phosphatase was obtained by the method previously Biochim. Biophys. Acta, 181 (1969) 479-482