45 2 BIOCHIMI(TA E'r BIOPHYSICA AC'I'A

AMINO ACID C O M P O S I T I O N OF A N E W R U I ~ B E R - L I K F .

P R O T E I N , R E S I L I N

K E N N E T H B A I I . E Y AND T ( ) R K E I . KVEIS-F()Gt l"

Department o/Biochemistry and Department o/Zoology, Cambridge (E~Tgland'

(Received October I l th , 190o)

S U M M A R Y

The chemical nature of an insoluble, rubber-like substance recently found in insect cuticle (e.g. prealar arm and wing-hinge ligament of locusts, elastic tendon of dragon- flies) has been investigated. It consists entirely of a cross-linked protein of unique composition and has been termed resilin (rez' i lin). Of the total residues, it contains 66 ~I~, of non-polar groups, 31 o glycyl, 16"0 from dicarboxvlic acids (mainly ami- dized), and 14 (~, from hydroxy amino acids but no sulphur-containing residues, no hydroxyprolyl and only traces of tryptophan.

In a few places, resilin is present as pure masses of protein but, typically, it occurs as continuous layers (2-5 l* thick) separating thin (o.2 t*) chitinous lamellae from which it can be extracted by hot dilute acid. The residue consists of chitin with only about 2 'Io protein. Fresh rubber-like cuticle contains less than o.3 % water- soluble protein.

The amino acid pattern of resilin is discussed by comparison with those of collagen, silk tibroin, and elastin from which it differs distinctly.

INTRODUCTION

It has recently been found that certain parts of insect cuticle are rubber-like and that this property is caused by an unusual protein. In lamellar cuticle, the protein forms continuous sheets (2-5~* thick) placed between thin lamellae of a chitinous, solid material (o.2 ix) but a few structures are known in which the epidermal cells have deposited thick elastic cushions of apparently pure rubber-like protein ],2. In both cases the protein parts are colourless, hyaline, swollen with water and completely deprived of structure in the ordinary, as well as in the electron microscope. Histo- chemical tests provided no evidence for the presence of lipids, polysaccharides, sulphates or metaphosphates ~.

In all physical respects (thermoelasticity, mechanical and optical properties) the swollen protein behaves as a typical cross-linked rubber and shows no tendency to crystallize at all 2 (X-ray analysis). I ts main characteristics are as follows ~. The

• Presen t address : Zoophysiological l . abora to rv B, 36, Ju l i ane Maries Vej, Univers i ty of Copenhagen (Denmark) .

Biochim. t]iophys...Iota, 48 11961) 45z--459

A NEW RUBBER-LIKE PROTEIN, RESILIN 453

strongly hydrophilic protein is insoluble in all media which do not attack the peptide backbone, including agents which reduce or oxidize disulphide groups. It remains rubbery and mechanically intact even after treatment with strong protein coagulants, histological fixatives or with heat up to I4 0°. Since the samples do not flow when strained for prolonged periods of time and since the material is so insoluble, nearly all protein chains must be firmly held in a network by means of stable cross-linkages, involving co-valent bonds the nature of which is unknown so far. On dehydration the protein becomes glass-like but resumes its former rubberiness when again wetted with water or such polar solvents as glycol, glycerol, glacial acetic acid and formamide. In dilute buffers the degree of swelling varies strongly with pH and is minimum at pH 4. This and the staining properties with basic dyes (methylene blue and toluidine blue) indicate an isoelectric point at about this pH. In contrast to elastin, the native protein does not form fibres and is easily digested by all proteolytic enzymes tested (pepsin, subtilisin, trypsin, chymotrypsin, elastase, papain).

The so-called prealar arm of locusts consists entirely of the lamellar type of rubber-like cuticle whereas the main wing-hinge ligament is composed of lamellar cuticle plus a pad of pure rubber-like protein (up to ioo/~ thick). In addition, there is a small amount of tough, tendinous protein which could be removed separately. In dragonflies there also occurs a small elastic tendon consisting almost wholly of rubber- like protein, and with this preparation it was possible to prove beyond doubt that the protein really exists in the rubber-like state of matterL In large dragonflies (Aeshna spp.) the tendon contains 5 to 7 t~g of dry protein.

Since the appearance, general properties, swelling behaviour and isoelectric point are nearly the same in the three kinds of preparation, the protein was considered to be of the same general type 1. This is corroborated by the present study which shows that, in spite of important similarities, it differs distinctly both from elastin and from other structural proteins. We therefore propose to name it resilin, derived from the Latin resilire, i.e. to spring back (pronounced rez' i lin). (We are indebted to Professor D. S. ROBERTSON, Cambridge, who suggested the name.)

Resilin is not only rubber-like but it seems to approach the ideal cross-linked rubber more closely than other natural or synthetic products*, 3. It is probably present in the cuticle of all winged insects where it is responsible for spring actions of a variety of sorts, characterized by precision and quickness. Examples are the elastic recoil of the insect wing a, the inspiratory movements in beetles and the long-range spitting of certain predacious bugs (Dr. J. S. EDWARDS personal communication).

Apart from the mechanical perfectness of the material, the general interest in resilin arises from the fact that it represents a three-dimensional protein network of high stability, resembling a giant molecule whose chains are thermally agitated and linked to each other at regular intervals but with little or no secondary protein structure of mechanical significance.

MATERIALS AND METHODS

The insolubility of resilin made it necessary to isolate the fresh rubber-like patches by dissection in dilute buffer solution or in 7 ° °/o ethanol, rinse and mildly hydrolyse the cut samples from a large number of animals and to analyse the residue and the extract separately. The prealar arm and the main wing-hinge ligaments from the

Biochim. Biophys. ,4cta, 48 (i961) 452-459

454 K. BAILEY, T. WEIS-FOGH

forewings of full-grown desert locusts (Schistocerca gregaria ForskM, phasis transiens) were used for quanti tat ive estimates and some elastic tendons from the dragonfly Aeshnajuncea L. for qualitative comparisons. For quanti tat ive analysis (Runs I to 3), the fresh cuticle was stored at - - 1 8 ° before dissection in o.oi M phosphate buffer pH 6. 7 in order to facilitate the removal of epidermal cells and other non-rubbery components.

Qualitative tests on lamdlar materials

A number of wing-hinge ligaments were dissected out and stored for some days in a large amount of 7 ° % ethanol, the adhering epidermal cells were carefully re- moved and the sample was dried (wt. 2.6 mg). The material was extracted with I N HCI for 2 h at 7 o°, and both extract and residue (wt. 0.8 rag) were freeze-dried. The extract was rehydrolysed at IOO ° in I N HC1 for 4 h and taken to dryness. No hexos- amine could be detected by the Elson-Morgan reaction and no sugars after chroma- tography on paper (ethyl acetate-pyridine-water mixture of JERMYN AND ISHER- WOODS). After further hydrolysis in 5.7 N HC1 (24 h at IOO °) a two-dimensional chromatogram (butanol-acetic acid, phenol-borate) revealed many amino acids: Asp, Glu, Gly, Ser, Ala, Tyr, Pro, Phe, Val, Leu, Thr. I t thus seemed that the material was entirely protein, and further work was directed towards its extraction by mild methods of hydrx)lysis from the wing-hinge ligament and the prealar arm.

Material for quantitative amino acid analysis

Three separate Moore-Stein analyses, designated Runs I, 2 and 3 have been carried out. The initial t reatment of the cuticle has varied and must be described in- dividually.

Run z: The mesothoracic wing-hinge ligaments from 34 locusts were isolated, yielding 8.4 mg dried material. This was extracted for 6 h with I N HC1 at 60 °. After drying, the extract weighed 6.8 mg and the chitinous residue 1.6 mg. Each was hydro- lysed in boiling 5.7 N HC1 for 24 h. The original extraction conditions here may have been too drastic as the chitinous residue under the microscope seemed "gluey". In addition, some contamination of the residue may have been introduced because of the small amount of non-rubbery material from the lateral, tough part of the ligament.

Run 2 : The prealar arms from 80 adult locusts were treated to remove first of all any water soluble nitrogenous substances. The rinsed rubber-like pegs were cut int~ small slices (about o.I mm thick) and heated in o.oi M phosphate buffer pH 6.7 at 95 ° for 1.5 h and "dialysed" by placing the pieces in distilled water at 4 ° overnight. This t reatment does not affect the rubbery properties of the inter-lamellar protein t. The total extract (A2) was freeze-dried. The residue was now treated with o.I N HC1 at 97 ° for 6.5 h to extract the resilin, the extract being then separated from the residue and each dried. The resilin fraction (Bz, wt. 3.4 mg) was then hydrolysed completely in boiling 5.7 N HCI for 24 h. The residual lamellae (C9, 0.9 mg) appeared clean, glossy and continuous, giving negative Millon and xanthoproteic reactions, but a positive chitosan reaction. This too was hydrolysed as above.

Run 3: The wing-hinge ligaments from the mesothorax of 80 adult locusts were excised and freed from tissue as above. Each ligament was then split longitudinally and the lateral non-rubbery tough part discarded. The remaining median part con- sisted of the pad of pure resilin plus the lamellar rubber-like cuticle which inserts on

13iochim. Biophys. Acta, 48 (x961) 452--459

A NEW RUBBER-LIKE PROTEIN, RESILIN 455

the median aspect of the second axillary sclerite, in about equal amounts. Apart from the lamellae, this material should contain no non-rubbery substance. It was then soaked in distilled water for 2-3 days at 4 ° and extracted at 960 for 5.5 h with o.I N HC1 (fraction E s, 5.4 mg). This was fully hydrolysed with 5.7 N HC1 as above. The residue (F 3, 0. 4 mg) was hydrolysed under milder conditions than C, above to minimize destruction of glucosamine derived from chitin, i.e. in 3 N HC1 at IOO ° for 18 h.

Amino acid analysis

The protein hydrolysates were analysed by the improved procedure of MOORE, SPACKMAN AND STEIN e on a column of Amberlite CG 12o. Two columns are used, one of 15o cm for estimation of amino acids other than arginine, histidine and lysine, and a short 15 cm column for estimation of these latter and ammonia. The yields of serine and threonine were corrected for destructive losses. The presence of tryptophan was sought by the method of SPIES AND CHAMBERS 7, using crystalline edestin as standard.

Since resilin can only be extracted and separated from the chitinous lamellae by mild hydrolysis, its absolute N content is unknown. The results of the amino acid analysis can nevertheless be expressed on a protein weight basis by calculating the anhydro weight of amino acid corresponding to the a-amino N of each amino acid as determined by the ninhydrin reaction. The anhydro weights are then summated to give the reference weight of protein. The results have been finally expressed as weight of anhydro amino acid/xoo g of protein. It seems to us that this latter method of tabulation is more meaningful than the conventional expression grams amino acid' IOO g of protein, since it gives at once what proportion of the absolute weight of protein is occupied by a given amino acid. The theoretical N percentage of the protein can be calculated from the expression (ZN of anhydro amino acid + ammonia N/2," weight of anhydro amino acid) × ioo.

RESULTS

Amino acids

Resilin contains no methionine, cystine, hydroxyproline, and less than 0. 3'!o tryptophan. Considering that the material needs to be derived by mild hydrolysis from the various anatomical structures, the uniformity of the results in Runs I, 2, and 3 (Table I) is satisfactory. For the computation of the proportion of certain types of residue (e.g. total base, free acid groups and non-polar groups etc.) as percentage of the total residues, only Runs 2 and 3 have been used, since the initial extraction process gave a cleaner separation of resilin from the chitinous structures.

It is clear from the analyses that resilin has a unique composition, differing from other structural proteins such as collagen, silk fibroin and elastin (Tables I and II). The proportion of hydrophobic side chains in Pro, Gly, Ala, Val, iLeu, Leu, and Phe is high (66 % of the total residues), but the amount of dicarboxylic acids is greater than in collagen, these being negligible in elastin, and hydroxyproline is quite absent. The very large amount of glycine is worthy of note.

No great reliance can be placed upon the yield of ammonia as representing the amidized carboxyl groups, even when corrected for deamination of serine and threo- nine. In our experience it tends to be greater than the value derived by direct mild

Biochim. Biophys. Acta, 48 (i96t) 452-459

456 K. BAILEY, T. WEIS-FOGH

hydrolysis. Even so, the total acid groups are greater than the base ~- "amide" groups, suggesting that at least 4 % of the total residues exist as free acid groups. Resilin would appear therefore to have an acid isoelectric point, a finding in accord with its staining and swelling properties 1.

The smallest "molecular weight" unit calculated from the lysyl content is approx. 20,000, and i3,ooo from the histidvl content.

]'he chitinous residue after extraction of resilin

The residue of Run 2 (C2) and of Run 3 (F3) were each hydrolysed to liberate glucosamine and amino acid (if present). C, was treated with boiling 5-7 N and F, with boiling 3 N HC1 each for 18 h. The hexosamine content was determined directly by the Elson-Morgan method as modified by R I M I N G T O N s, and the ninhydrin colour before and after removal of ammonia at pH Io. The rest of the hydrolysates were used for paper chromatography, using butanol-acetic acid, and running with known quanti- ties of amino acid markers and also with glucosamine. In both samples, the glucos- amine N is approximately equal to the "a-amino" N as estimated by ninhydrin, and

m

_ m

T A B I , E i

AMINO ACID COMPOSITION OF RESILIN FROM "~'ING-HINGE (RUNS I AND 3) AND PREALAR ARM (RUN 2) OF THE LOCUST Schistocerca gregaria

Resiltn: Comparisons ."

W t . anhydro amino acld/ R c s i d t w s ] l o ~ g z ¢.,,,. ~ g protein protein residues I¢? g o[:

E l a s t in o, .'i ilk f ibro l n " '

( 7e l l a g e n I . (o:~ ( B o m b v Run r Run 2 Ru~l I R Io$ 1 l~t4t$ : R u n ? f o x . h i d e ) ligamentum

nudhae~ m o r n ,

m .

A s p 12. 5 14.1 I3 . 3 xo9 i 22 l I6 52 4 .5 tO T h r 3-7 3 .4 3.4 37 34 33 .5 I9 8 12 Ser 8.o 8 .0 7.95 92 91 89 41 ~ i t[)o G l u 6.2 7.0 7-3 48 54 ,57 76 t 4 14 Pro 8 . - 8. 7 8 .8 84 80 9o 125 ~ 5 ~> 5 G l y 26 .9 23.7 24 .3 47 x 414 425 354 398 590 A l a 9-45 8.5 8 .9 x33 I 2o 125 116 212 389 Val 3-I 2 .8 2 .9 31 3(7 29 2 x t 48 30 M e t N i l Ni l N i l N i l Ni l Ni l 6.5 ¢ ~ ~} Nil i L c u 1.9 2. I 2 "55 16-5 19 23 14 31 9 l . eu 2 .0 3. I 3 .25 23 27 20 - 8 6 6 7 T y r 4 .0 .5- 8 5.3.5 24..5 3.5 33 ,5 0 ~)~) P h e 4.4 3 .95 4 .6 3 ° 27 3 t 14 3 o S . \ m i d e N (126) - - (78 ) (46) (3)

T o t a l T o t a l 90 1o7 H i s 1.7 c a l c u l a t e d l .o 12.2 c a l c u l a t e d 7.4 4.5 0 .5 2 Arg 6. 5 f r o m 6.0 4 t . 5 [ f r o m 38 47 5 ~>

R u n I ~ R u n x

T o t a l IOO lOO IOO I I 5 9 1128 I t 3 I 1063 rO93 132~ ( incl . 106 h y d r o x y -

C a l c u l a t e d N c o n t e n t (°/o) x9.5 19.5 pro l ine 4 7 h y d r o x . v -

lys ine) A v e r a g e re s i due w t . 88 .5 ( R u n s I and 3) 94 .0 0 t . 5 75.7

I~iochim. Biophys. Acta, 48 (lq(>x) 4 5 " - 4 5 0

A NEW RUBBER-LIKE PROTEIN, RESILIN 457

T A B L E I I

N U M B E R O F R E S I D U E S O R G R O U P S P E R I O 0 R E S I D U E S IN S O M E S T R U C T U R A L P R O T E I N S

R E S E M B L I N G R E S I L I N IN C H E M I C A L C O M P O S I T I O N

Resilin, Collagen Si lk fibroin Elastin Residue or group: Runa 2 -

( insrct ) (mammal) ( insecO (mammal)

Non-polar ° 66 63 78.5 95 Gly 31 33 45 36 Basic 4.5 8.o*" o.9 o.9 Tota l a c id " " " 15.5 I 2.o 2.3 i .7 Free acid§ 4.o o. 3 - - o.6 To ta l h y d r o x y l 14.o 16.7"* 18.6 2.3 H y d r o x y p r o l i n e w h y d r o x y l y s i n e Nil IO.6 Nil Trace t7 Tvr 3.o o.5 5.2 o.8 Met Nil o.6 Nil Trace 17 Cys Nil Nil Nil Trace t~

" Pro, Gly, Ala, Val, Met, iLeu, Leu, Phe. *" Inc lud ing hydroxy lys ine .

"** Dica rboxyl ic acids + amides. § Tota l acid - - (bases + amide).

if for sample C, the ammonia N be assumed to arise by decomposition of glucos- amine, the total chitin content of the residue is 8 5 °/o. This procedure is of course not very accurate (Table III).

In both C2 and Fa, the amount of amino acid revealed by chromatography was negligible, though the glucosamine spot was very intense. Only traces of Glu, Gly, Ala, Val and Leu-iLeu were evident, and the probable protein content of the residues appears to be no more than ~ z %. Lamellar rubber-like cuticle is therefore unusual by the ease and effectiveness with which the protein component can be separated from the morphologically distinct lamellae, consisting mainly or exclusively of chitin.

Material extracted from the intact arm by hot buffer and water (Run 2)

A direct ninhydrin reaction on the extract gave negligible colour which increased after complete hydrolysis to the equivalent of 9 tzg of protein, i.e. 0.3 % of the dry weight of the dissected material. Paper chromatography (butanol-acetic acid) gave traces of slow-moving material (Gly.Ser.Ala) and very faint spots of Leu-iLeu, Val and Pro. There occurs therefore only traces of water-soluble material in rubber- like cuticle.

T A B L E I I I

A P P R O X I M A T E C O M P O S I T I O N O F C H I T I N O U S R E S I D U E S A F T E R E X T R A C T I O N O F R E S I L I N

A nhydro acetvl- Estimated Glucosamine Ammonia N Amino N gluzosamine,

N (rag) [rng) (me] a,~ °o protein total residue content. %

Residue C 2 (0.9 mg) o .o i9 0.034 o.oi 8 85 i .4 Res idue F 3 (o.3-o.4 rag) o.o 15 Nil o.o21 55-73 0.3

B i o c h i m . B i o p h y s . A c t a , 48 (I961 452-459

458 K. BAILEY, T. WEIS-FOGH

Comparison of resilin Jrom prealar arm of locusts with the elastic tendon of dragonfly

The elastic tendon of dragonflies consists of a thick-walled tube of rubber-like protein surrounded by two extremely thin tubes of different materials, an inner and an outer tube 1. About 2o t*g dried tendon from Aeshna juncea was hydrolysed with 5.7 N HC1 for 24 h at IOO ° in a sealed tube and also a similar amount as extracted in Run 3 from the prealar arm ot the locust. Simultaneous two-dimensional chromato- grams were then carried out in butanol-acetic acid, followed by phenol-borate. After spraying with ninhydrin containing 3 % collidine to diversify the colour of the spots, the resulting patterns were indistinguishable: Ser, G l y - T h r + + + , A l a + + , Leu- iLeu +, Val + , Glu + , Asp +. This evidence, considered in conjunction with a general comparison of the properties of each type of materiaP makes it almost certain that the elastic tendon of Aeshna is also composed of resilin.

DISCUSSION

Tile four structural proteins in Table II are clearly different but may be grouped together because of a number of common features. One third or more of all residues is glycyl while tryptophan and the sulphur containing residues are absent or nearly absent (impurities?). Collagen, elastoidin, and silk fibroin are truly fibrous with distinct X-ray diagrams in the normal state but heat and/or formamide and other solvents make them shrink and become rubbery, then resembling elastin and resilin which are both unable to crystallize.

As to bulk properties, there is a superficial similarity between resilin and collagen (Table II) but the large amount of hydroxyl groups in collagen (17 % of the side chains against 14 % in resilin) is made up predominantly of hydroxyproline which is quite absent in resilin. Compared with the other insect protein of epidermal origin, silk fibroin, the extreme tendency of fibroin to crystallize is in sharp contrast to the complete absence of this property in resilinL The most pronounced differences between the two are the abundance of basic and acid groups in resilin, 20 % against 3 % in fibroin, and the small amount of proline in fibroin.

It has been suggested 9, lo that the rubberiness of elastin is caused by the scarcity of polar groups (5 %) and the presence of a large number of non-polar, "self-lubri- cating" side chains. In resilin, the only other truly rubber-like.protein known so far, one third of the side chains contain polar groups. In the body, the two proteins are swollen to nearly the same extent and for these reasons the above suggestion cannot be upheld. In fact, Tables I and II show that the chemical resemblance between the two rubber-like proteins is smaller than between resilin and each of the two fibrous proteins.

Silk fibroin contains very little proline (0. 4 °/o) while 8 % of the residues in resilin and 14 % in elastin are prolyl. Proline residues may introduce considerable constraints as to the possible configurations of the peptide backbone, depending upon the neigh- bouring residues n. It is therefore conceivable that the lack of crystallinity of resilin and elastin and the ensuing rubberiness when wetted by solvents is caused by an arrangement of prolyl so as to prevent larger segments of neighbouring chains from forming carbimino hydrogen bonds, such as prevail in crystalline fibroin.

For the time being, it is difficult to compare resilin with other proteins from insect cuticle since most preparations seem to be polydisperse. It is generally agreed

Biochim. Biophys. A cta, 48 (Iq6t) 452-459

A NEW RUBBER-LIKE PROTEIN, RESILIN 459

that sulphur containing residues are scarce or absent12-14; in the soluble fractions, the content of tyrosyl may vary from 4 % by weight to 13 % (5 % in resilin), according to HACKMAN AND GOLDBERG 15. These authors found tryptophan and hydroxyproline in all soluble fractions from larval cuticle of a beetle, but no histidine, in contrast to the present findings on resilin. By means of microbiological methods, DUCHATEAU AND FLORKIN le estimated the relative amounts of 15 amino acids in the water-soluble ("arthropodine") and the water-insoluble fractions of the cuticle of two decapod ('rustacea (crayfish). The soluble proteins contain much less glycyl than found in resilin and, apparently, than in insoluble scleroprotein.

The analysis did not offer any clue as to the chemical nature of the cross-linkages. I n conformity with elastin, disulphide bridges are out of the question I~. The mechanical analysis indicates that there are of the order of one hundred residues in the backbone of a chain between two cross-linkages 2. Provided all lysyl is involved in cross-bonds, it is therefore just conceivable that they are similar to e-N lysine peptide bonds now known to occur in collagen 18. However, preliminary experiments by S. O. ANDERSEN,

Copenhagen, do not support this hypothesis. He was also unable to demonstrate any phosphate in resilin so that phosphate bridges are ruled out. I t should be stressed that small amounts of groups not normally present in proteins may have escaped notice, the number of cross-linkages being so small.

ACKNOWLEDGEMENTS

We are indebted to the Anti-Locust Research Centre, London, for the supply of locusts, to Mr. F. J. BLOY, Cambridge, for dragonflies, and to Mr. S. O. ANDERSEN,

Copenhagen, for permission to quote unpublished results.

R E F E R E N C E S

l T. WEIs-FoGH, J. Exptl. Biol., 37 (I960) 889. 3 T. WEis-FoGH, J. Mol. Biol., in the p re~ . 3 M. JENSEN AND T. WEIS-FOGH, in preparat ion.

T. WEIs-FoGH, Proc. XVth Intern. Gongr. Zool. i958, 1959, p. 393. 5 M. A. JERMYN AND F. A. ISHERWOOD, Biochem. J., 44 (1949) 4o2. 8 S. MOORE, D. H. SPACKMAN AND W. H. STEIN, Anal. Chem., 3 ° (I958) ix85. 7 j . R. SPIES AND D. C. CHAMBERS, Anal. Chem., 21 (1949) 1249. s C. RIMINGTON, Biochem. J., 34 [1940) 931. 9 D. J. LLOYD AND M. GARROD, Fibrous proteins, The Soc. Dyers and Colourists Syrup., 1946, p. 24.

i0 j . C. KENDREW, in H. NEURATH AND K. BAILEY', The Proteins, Academic Press Inc., New York Vol. 2B, 1954, p. 845.

11 B. W. Low AND J. T. EDSALL, Currents in Biochemical Research, (1956) 378. 13 V. a . WIGGLESWORTH, Ann.-Rev. Entomol., 2 (1957) 37- 13 A. G. RICHARDS, Ergeb. Biol., 2o (I958) I. 14 R. H. HACKMAN, Proc. 4th Intern. Congr. Biochem. Symposium I2, 1959, p. 48. 15 R. H. HACKMAN AND M. GOLDBERG, J. Insect Physiol., 2 (1958) 221. 16 G. DUCHATEAU AND M. FLORKIN, Physiol. Comparata et Oecol., 3 (I954) 365 . 17 S. M. PARTRIDGE AND H. F. DAVIES, Biochem. J., 61 (1955) 21. is G. L. MECHANIC AND M. LEVY, J. Am. Chem. Soc., 8i (i959) 1889. i9 j . H. BowEs, R. G. ELLIOTT AND J. A. Moss, Biochem. J., 6i (1955) 163. z0 R. E. NEUMAN, Arch. Biochem., 24 (1949) 289. 31 F. LUCAS, J. T. B. SHAW AND S. G. SMITH, Advances in Protein Chem., Vol. I3, Academic Press

Inc., New York, I958, p. lO 7.

Biochim. Biophys. Acta, 48 (I961) 452-459