Collagen, are fibrils arranged in overlapping fashion too just like tropocollagen?

Collagen, are fibrils arranged in overlapping fashion too just like tropocollagen?

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Collagen molecules (tropocollagen) are interlinked into fibrils, with a banded structure showing the spaces ("lacunae") between the molecules. Do fibrils in turn also interlink in a similar way, or, do fibrils run from end to end in the fibers?

Collagen D-spacings in fibril bundles

Methods and systems for diagnosing a bone disease or other condition related to collagen in a subject are provided. These include providing a bone sample from the subject and determining a quantitative collagen morphology value of the bone sample. A reference value is provided from a non-affected control subject where the reference value is a quantitative collagen morphology value from the same type of bone sample obtained from a population of non-affected control subjects. The quantitative collagen morphology value of the subject's bone sample is compared to the reference value. If the collagen morphology value is altered versus the reference value, the subject is diagnosed as having a collagen related bone disease. The collagen morphology value can include mean fibril spacings and distributions of the fibril spacings taken from a subject's bone sample.

Enzymatic investigations into extra helical and terminal structures of collagen

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I hereby declare t at the following thesis is based on work carried out *y ne, that the

thesis is ray own composition, end that no part of it has b en presented previously for a higher degree*

The research was carried out in the

T hereby certify that David Steer hrs spent nine terras engaged in research work under ray direction, and that he has fulfilled

the conditions of ordinance No.16 (St.Andrews), and that he is qualified to submit the

z accompanying thesis for the degree of Doctor

I matriculated at the University of 3t. Andrews in October 1959, and graduated with the degree of achelor of Science, Second Class Honours in Biochemistry, in June 1963. My subsidiary


Aggregation States, the fibrous form b. and electron microscopy

The Primary Structure of Collagen 12. The ef ect of proteolytic enzymes 30.

N-termlnal residues of collagen 33• The availability of -lysyl amino 37.

^-Glutamyl end ^-aspartyl linkages b3. Jubunit composition of collagen **5.

The Cross-linking of collagen 56.

1. Acid Soluble Collagen 6b.

(1) Investigations into the N-terrainal 66. residues or soluble collagen v

Isolation of _ther-soluble D.3.P. amino 67. acids

(2) Investigations into the activity o^ 75. <x -amylase on collagen

Solubilisation of mature collagen by ^reatmont with o< amylase

Preparation of soluble collagen by 76. amylase treatment of Insoluble

Carbohydrate content of amylase 76. solubilised and normal collagen

Assay ror proteolytic activity of the 77. ensyme

Action of pepsin on amylase solubilised 33. collagen

Column fractionation of amylase 3l. solubilised collagen

H-terminal residues of amylase 32. solubilised collagen

Investigation of the low molecular 85. weight products of amylase

(3) The availability of .-lysyl residues 33. Pinitrophenylation of heat-denatured 33.

Hydrolytic release, destruction and 90. total amount or R-D.H.P. lysine

Dinltrophenylation of Hippuryl-lysine 93• Titration of D.H.P. collagen 91*.

(M Experiments with the peotides liberated from soluble and

insoluble collagen by the enzyme collagenaso

Dinitrophenylation of peptides Amino acid analyses

Separation of free collegenase- liberated peptides

Dinitronhenylrtion and fractionation of peptide fraction P

3. Availability of ..-lysyl residues . xperiments with collagenase

liberated peptides Summary


classification is one based on secondary structure,

collagen having a unique triple heller 1 polypeptide

"backbone0 quite different from the ex- I

e feature of most pi'oteins (Fiich r-nd Crick 1961).

Th amino acid composition of different collagens

hydroryproline which is compatible with the existence

of the collagen fold in which these amino acids play

In most collagenous tissues the majority of the

collagen is highly insoluble. Usually

protein can b extracted by procedures involving

relatively mild solvents the amount depends on the

tissue concerned anti the solvent used, fhus with

calf-skin and rat-skin collagen, extraction .ith

dilute salt solution produces a fraction which


methods which probably Involve the rupture of covalent

bonds, is regarded <s t e mature form, and in most

tissues represents the hulk of the


fhe tern trnpocollageo hrs veen used to designate

the single collagen molecule* It is believod that

nature collagen, which is always laid own in a

fibrous form, consists of tropocollagen units

polymerised by intemolecular crosslinking. fhus the

limit for solubility will be one of size of aggregate,

some of the poly .©rs still being sufficiently small to

pass into solution. Howev r, it is thought thrt neutral

salt soluble and acid soluble collagen solutions

contain mostly tronocollngen monomers, the former

approximating most closely to the homogeneous condition,

of rabbit skin collagen in solution lehnve as rigid ro s

of length U ) - 6 30°A and width 1 v3 - ? '^Af this

An average aggregate


■Jcutrrl salt soluble collagen and acid soluble

collagen have b en proposed as precursors o mature

collagen, however the r >le of acid soluble collagen in

this connection has b<en questioned by Harkness

of (A1* glycine into neutral salt soluble collagen than in*

against a mechanism of ^om? tion of tissue collagen o<’

the forw,i* neutral salt lubla collagenacid soluble

between the different forms of soluble collagen is

probably not a hard and fast on^ (Jac’/son and ' ontley

-19>0)» Thus there probably exists a continuous

"orated and most easily solubilised molecules, to the

older, p rtirlly polymerised ones, which are dissolved

with ar»st difficulty. Viewed in this light, the

observations of Harkness can be explained by the

probability that acid soluble collagen will contain a

proportion of "older” molecules which have escaped

Lagan fibres

exists in cornea where sheets

ibres are laid om* on the other with their grains at

right angles in I laminated st: c' re (Gross 1961). *hen suitably stained and era-alned with the electron


bandinr pattern. ?he technique o positive staining

attaches heavy metal ions to polar regions of the


the distribution of basic and acidic aide chains

respectively. Tn both cases an rxial nerioJ of about

6bO°A is observed in which 12 or 13 bands are normally

present. The most prominent of these h^s been called


by precipitatin£ collagen from solution under specific

•) and ’*-‘i bfous

The f'ormer

of length 2, the latter as itros similar to

The .lost widely accepted interpretation of these data

is that whilst in m tive collagen, the tropocollagen

molecules ov rlrp by 6MnA$ in S*L« • aggregates the

molecules are joined only side to side, and are in

register. In the .L.'. forms the molecules are

attached both side to side and end to end, • ut there

is no overlan the individual molecules being laterally

in register. ?hus the length of the «L. • crystallite

Is equal to the length o' the trooocollagen molecule,

and the peri'dicity o the .L. . is “oir tin-s that

of the native fibre. The discrepancy between the

periodicity of .L.-. and the total length o the

molecule, can le accounted or by the act that co-ryiel

molecules, overlap or "interlock” at their ends by

pprox. 3 ' . . ( ch'.itt st. al. 19 3, Tall otg 1953,

Hodge & . ch tilt 196 '). It has been suggested ( ?c tker

<! Doty 195> and Hodge and ehmi^t 1953) that terminal

pent! e apo -n r es are concern >d in this overlap region.

by examination 0 ’ dimorphic forms in which native*

type fi rils were use as nucleation sites for

subsequent growth of .L. . crystallites, H dge and

fchmitt 196 j demonstrated the exact correspon ence 0*

bands with those of m tive ' ifcrlls with rested

to axial location, but not with respect to intensity.

type pntt- rn arise by summation o sets o equivalent

bands which contribute to the stainlnr intensity by

virtue of their lateral apposition In the stsrgered

array*" I" it is accented that the bauds are cine

solely to distinct polar regions in the molecule

(Ftihn 19 o)) then fhls hypothesis requires that the

tropocollagen mrcronnleculo cen he divided into four

regions o' equal length, each containing a tot'1 of

approx. 12 polar sones whose axial positions are

identic.'1 in each of the? four regions. In each molecule

there ill be a total of nppror. polar locations,

possibly a fow aore if the terminal peptide overlaps

contribute to the handing at all. rom amino acid

analyses of collagen there are about 009 Acidic and

basic side chains i.e. if all of the polar residues

are involved in the "ormetion of the polar sones these

would contain an overage of 12 polar retinues each (as

veil i s any other amino actus present). Only one half

of this average o** 12 residues will be responsible for

the bond Intensity at any one time depending upon

whether staining is for acidic or basic residues

(collagen contains approx, equal nun) ers of eci ic anc

contribution of each of the three constituent polypeptide

Eh'ins of tropocollagen> to

There Is good evidence that these three chains pre

chemically distinct (lies 196?)> so their nrlmrry

structure must be such that when assembled as a

triple-hellr, the 'our equivalent s* ts of br ids result.

Tf all three chains are involved in the formation of

each ^endt the number o polar residues per hand from

each chain will average 'our. If only one chain is

involved then there is the question o' the distribution

of the polar regions between the three of them. The

‘orner possibility is probably closest to the true state

of a fairs, for all mmont o* the constituent chains

of tropocoJLlrgon could occur initially

between acidic nnd basic areas of the polypeptides.

This wouH account for the identity of the bands

resulting "rora the two methods of positive staining viz.

or acidic and basic groups.

Tn the final construction of fibres from the

tropocollagen uolecul sf it is necessary to postulate

a two stage process. The molecules ere irrt polymerise!

by ond-to-en linkage through interaction o the

terminal peptides to form proto fibrils and these ere

subsequently displaced by .25 of their lengt relative

Obviously to explain the el


collagen ibres in term o'* a quarter-staggered

arrange wnt an! an orderly distribution of polar

locations, nece sltntes a knowledge of the organisation and str cture o the nmlec le at several levels.

Current theories o4* protein bi sy thesis con account

for the m^st specific requirements of primary stricture

species differences 4c occur, btt1

Always those residues

in vital positions !*or the correct



Tt is unlikely though that proteins ore synthesised as

units of Molecular weight greater than about 6

the raoleculnr weight or each o’ the three constituent

chains of tropocollrgen is approx. DO,*) Thus it

seeras likely thet collagen is synthesised from a num or

of su-units these cannot ie identical because o the

dissimilarity o the three chains of tropocollagon, and

also because the f^ur equivalent sets

in position but not in intensity. Nevertheless, the

in respect to the spacing e oolar residue , • s

equivalent bauds are to exist, loss this stretch concepts op 1lochamical evolution too ar?

f'r >n subunits which although di fer att hear a

mathematical relationship to one


explain 1 ut biology has pro’ably had greater surprises

or the biochemist in the past.

An attempt to explain electron micrographs of


Grant et. rl. (196*0. Pointing out that a completely

quarter staggered arrangement has been demonstrated to

ve theoretically impossible in a three dimensional

system where more than two molecules can be mutually in

contact (Gmith 1965) > they suggest an essentially

random aggregation based on the alignment of so called

’•bonding regions”> 1 Ive of which are present in the

length o each tronocollsgen molecule.

This theory can not only account for the SUo°A

peri dicity o positively stained colleger^ it also

explains the pattern obtained with negative staining

which has been something o en embarrassment to

protagonists of the "quarter stagger” system. The idea,

of Grant ot. al. is that negative staining Invol/es

retention of heavy netrl in a neutral 'ora in the

regions of loose packing o the material. Gegetively

(stands) alternating with crk bands (b-ban s). In a !stance correspon lag to the length

there orc five ©•bands and four b-bnnds. The llrht

zones correspond to bonding regions with © high content

of polar anino acids ieh by polar and hy *rogcn

bonding, hold these parts of the molecule tightly

together. In these brads there will be no room ’or

the entrance o‘ molec lies of stain. Conversely the

dark brads are more loosely packed non-bonding sones

where there has been ready access for the negative stain

upport ‘or this concept o" negative staining c >31 s

from examinations performed by Grant on glmtar* 1 y

treated collagen* In this material the sis© an density

of the a-bandc is Increased whilst the b-bands becomes

less distinct. This is thought to be due to additional

lnt ©molecular crosslinks beln introduced by the

glutavaldchyde. Thus negative staining reflects the

molecular packing of collagen fibres and beers little or

no relationship to the variations in crystallinity

vithin individual molecules.

The length o*‘ the ©•bands is approx. 26 5° A and of

the b-ban?s 375°A. hen the collagen molec les are

aggregated into ibrcs there is s random choice as to

which bon inr region of one molecule crosslinks ’ th one

ban s Is in the region o' 6Uo°A,

A periodicity o this

An interesting point raised was that the collagen

molecules in negatively stained preparations appear to

B bands.

A hypothesis

giving an essentially random mode of aggregation, is

that there is no n ed to postulate s two stage process

of end to end linkage into protofibrils followed by


does no store to holn explain the 12 - 13 bands in the

6Vo^a period of positively stained collagen. The only

mention is that these characteristic spacings car. be

considered to be determined ty a basically random process

o' aggregation of tropocollagen units. It woul appear

to be again necessary to assume the presence of four

equivalent sets of bands and in this case they would n ed

to be compatible ’wi th the concept of the si'lultrne >us

Amino acid analyses o soluble collagens rive quite reproducible results, whilst nature collagen and Jerlved

gelatins tend to give inconsistent values, due no doubt

to the difficulties in purification of insoluble

proteins. Thus reliable comparisons have been possible

only "'or the amino acid analyses of solu'le "crms of

differences h^ve cone to light.

the earliest amino acid analyses were achieved by

iscro-cheaical methods anr although those techniques have

^een outdated ’or some tine, the analyses oz C’ itnall

and co orfcers (Chifcnell 19- S) and "’owes and lenten (lf^+3)

have certainly stood the test of tine. Ton exchange

chromatography as developed by Moore and ‘'teln (19rl)

hrs revolutionised methods of amino acid analysis an

the modern automatic analysers tas^d on this prindole

have improved the speed an I accuracy of analyses

enormously* ast e (1955) usln^ the original Toore and

teln technique, and published erte sive data on the

a 'ino acid exposition of collagen an g latins. A large

Result of

s amide nitrogen mrsks in t v region of U5 of the

anionic side chains cf rspastlc acid and glutamic acid,

there is a slight excess o cationic groupings in

collagen 'hich is therefore a slightly basic protein.

*'ost values for the iso electric point o' collagen lie

between pH7.5 - 3 which woul be in agreement with this

concept. Other factors oust al3o v© considered in

evaluating iao electric points, inclu ing non-covalent

interactions involving ionisablegroups. fhese

interactions may affect the dissociation of the groups

concerns:• Another factor which lust be considered is

Grettle (1965) produces evidence that the true

iso electric point of collagen is only just above pH7»

This could bo accounted for by anion binding, or by

interactions involving I asic groups in excess of acidic

.7roups. he anomalously steep titration curve of

collagen in th » region pHL - pH> r-tses the possibility

of masked groups. nly part of the lysine appears to ve

titrated, so the possibility that some of th?se are

masked must be considered (teinhardt and falser 19 5).

The most lntcrestin erture o’ the amino acid

composition of collagen is the high proportion of glycin

an I o*‘ the imino acids. Hydroxy roline and hydroxylysine

hydroxy roline, bu otherwise they are ■ound In no other commonly securin’ proteins. It is generally accepted

*hst collagen contains no tryptophan or cysteine. The

tyrosine content is very low rnd it has teen suggested

that this amino acid is confined to terminal regions o

the molecule or to c 'val^ntly attached peptide

apanages (Ho ge et. el. i960). Arginine and Gluts tic

acii give probably the most consistent values for


quite different sources their amount seems to

Although the content of glycine, proline and

hydroxyproline varies to a certain extent, their amounts

composition o other proteins. ( or collagen glycine

usually represents approx.

An proline *


feature is a consequence of the unu ubI

Configuration of

the polypeptide chains in collagen. Glycine

imino acids tend to distort the normal ©rraigeaent of

os high as it is in collagen then the unique

con igurrtion known as the


•pi « ’ he belly is much wid r ? ’ <x

•CrlHH o a o o o -4 CO o to

•o q -h © c •H Ord © 44 < ,© bO to q ©>

HHHH <M ctf OH H 43 00 o

o q © « •h tn

=J 34 as o 0 rH A

44 rd C O i-l O a o o 9 o M Hl § q u> ♦H ixJ 44r-»

tO fH Q O 9 a o E o a Hi O 2& • • * *

q G UA CO -t Ch UA

O in cm o ia o <* r- cm r- v- cm cm At>-O Ch


Q Ch‘00 ACM CO trwOvO v- C**-fA-4-Ch CM A v- 40

ChiAr- cm v- CM *- CM A-4-4

-^CAfA AvO A*- O -4 3 <f r-CA C**. CA CM <M r-CM w— CM A O-4


CMCv-C0ChAv—Cs».tx*CM-4’CM'^? ca^4 AtO'O -4’1

CO *-CM <A CM O CM w- CM t- CM A r-

CM A COO v- O CA A v- A-r- A A AO* A-4 O Ch-4r-A!>CMAv


Ch -4 *— A >* CM At- CM r-CM

do© a 5 fi G © O 4 »rl *H C «H 43 rH O G -H Jd 3 O X <3 rH p

© 5 ©B © cd w G «H X

•H © C rH C © O © G X -H G «

H H X © 53 «H «>>oq«riqu© rH o o q q th 43 «h <4 *o o 3 Jh o *o e a boe-* «h

<a © >>xjk x*h q o q

The hydr x^proline content

approx • lb whilst that o reptiles lies be tv en

1 .? - 9.3 ant fish 5.3 - 7.9 . Cakehashi and Tasiakr

(1953) haw shown that the threshold temnerature 4 or

hy rotherual stability o' collagens also varies, from

6 - 7oa< ^or mam alien collagens to 33 • Ia5°C

For cold

•ater "lsh. They propose that the two ‘'actors are

related. ustrvson (1951 ) supported these views,

suggesting that the hydroxyl


Involved in hyirogen bondin? t giving rise to the extra


this amino acid. More recant studios by Pies and Gross

(19^^), including accurate analyses of collagens 4roa

a wide rang of animals have ho/ever, lead the a to

conclude that it is the total imino acid conten rather

than the hyiroxyprollne content which determines the

degree of stability of the protein. The determining

factor would therefore rppiar to be *ne of configuration

tn ther than amount o hy ro en bond i ng •

Astbury (19^ ), basing is conclusions on chemical

data and X-ray diffraction st oics proposed that the

c “lagon ^olocule is composed o' repetitions of the

Sequence - P - 1 -

and one o' the remaining reel u s). chrohcnl her et.

to K© the most Important single trioeptlde sequence,

securing frequently throughout the length o" the molecule.

y attacking heat ertnt ur <1 collagen wi th trypsin and

analysin ’ the resulting 11^ peptides, Grass iann et. al.

(1Q6p) deduced that as /5 th only one exception the

peptides all contained approx. ' of their amino acids

as glycine, this amino acid is distributed evenly through

rheoe observations sum up the most important aspects

o' collagen primary structure, but *hat they hold true for

the entire molecule Is open to doubt, as has been sho^n

by other work. Schroeder et. al. (1953 *>nd 19? ) and


And 1955)

peptides obtained by controlled acid and 1asic hydrolysis

of collagen, the composition of some of these is shown

Table 2.

Totolygad, .sqUam

L xaUttn*.

.m ^anrcLr.

-2EJ1U "hr Glr "cl C-ly Glu Oly

Pro Chr Gly Pro Ala Gly Gly Gly Pro Glu Gly Alo

Gly Pro Gly Ala Hypro Gly I J? Glu Hypro Gly

It is evident thrt glycine is often bound via its

dipeptide - Gly - Pro - In feet appears to be a very 'requontly occuring sequence. "he carboxyl group of

nroline is bound to amino acida other than glycine in

several peptides, and one of the dipeptides isolated

had the structure * gly - glv -. These observations

taken together show that the sequence * G * P - ft - of

Aatbury cannot be the rule. This sequence does however

account for 33 * 35 l®r*t of the primary structure

according to the results of Grassmann et. el. (19>1),

and must therefore be an important structural feature

Peptide sequences of four or five residues

containing no glycine have been found (Hannig and 'lord wig

196?) so the assumptions that glycine repeats in every

thlri position, rnd is distributed evenly through the

molecule can be no more than partially true.

^uch indirect evidence for the existence o polar

and apolar regions In collagen h^s come from electron

microscope studiest and bJlhn (1969) showed conclusively

that the dark staining bands giving the characteristic

cross etriationa of positively jtalned collagen fibres

contain m aceunulrtion of acidic and basic asino acids,

fhe 1rst chemical evidence came when Grassmnnn et. al.

(1957' isolated six peptides of chain length ran ing from 13.

show that on the whole areas which wer*> rich In proline

and hydroxyproline were devoid of polar amino acids and

collagen triple-hell^ is composed of region- containing

a preponderance of n n-polar amino acids (crystalline

regions) alternating with possibly non-helical or only

slightly helical regions with a

side chains (n lorphous sones), "he crystalline regions

are thought to consist largely of glycine and the imino

-as sums its major importance.

Grassmann pointed out that areas of some thirty

amino acids which are completely devoid of polar groups

may represent macro repeating units which could be

associated with a single turn of the complete collagen

helir (as opposed to the individual helices o three

constituent polypepti e chains). urther chemical


composition:-Ale - Fro (Gly3 Al«2 GIU2 Asp2 i h«2 Thr) Fro • composition:-Ale

and Gly - Fro (Gly/ Ala^ Val? . eup Lys^ OIU2 Asp. ihe

The areas within the brackets agree with the idea of


home of the most detailed pepti © analyses yet

,Tsing highly purified, chymotrypsin - free trypsin, they

ig©3tGd 62gae« roeollagen which had been denatured at


37° rn 1 trftriaetrlcally. hnly

C-terUn-arginine end lysine was found, and from the titration eta It appeared that 6 of the bonds Involving these

This hetle appro .

16o peptidfs of average chain length should e obtained

I • oolec dnr weight of 3 -» collagen.

The peptides were separated by continuous oreperetive

electrophoresis, lon-eachange chro:mtograpby,

aolecular-selve chromatography and paper c r


JJ peptidtfd, Of these

peptf t 55 fulfills all criteria for homo it r, 51

of which ’©re submitted to quantitative -mine acid

analysis, 13 to :ruxp analysis,

studies. The results of the end group an< sequential

analyses of some o the n ptl-

Dee is show

In tholr Interpretation of these results, Grassnann

et. al* extend the hypothesis of alternating pol? r rnd

apolar regions, addin' that the polar sones can he


large amount o' their evidence must come ’"ron the

percentage 'igurec for imino fields and polar $ ilno acids

Of the 51 peptides analysed, for actual sequences

__ i /m Sraao.uq aU

Te!xU Al§r

1. Toler Peptides without proline

azi HGlyAspGluGlyiyLys 3 Gly,?hr,2 Glu,Asp

eptides containing pr >line ♦ hydx'oxyprolino.

H»Jly-(-ly,3er,2 Asp, ilu)«*2 Mo, Glu, 13 3p, I -Zrr-OH

1* h —Slu-|12 Gly 2 Al- , ier,Leu-Asp- ' yr, Jlu j/.sp-'tf.

T71’ iZ:>y’( Hy’ '®r»'?,lu»-' 1 ’ *7/1*2 er,2*Asp^2*Glul 2L^g -

10 Pro,2 yPro,13 Gl$-Lys OH ly

H- ily.CCly,Ala, ? xlu,Asp)«9 Ala,3 al, Leu,2

5 i o, ?ypro, l r.lf -Ly 0 er,2 7rl,. sp,2 ±u. J - ry

!>ProJ Hypio,l Gly,7 Ale r)-2 Val, eu,2 Phe,3 er,

3 Fr">,' Hypro.r Ay,o Ala

3 Val, 2 Leu,fhe, 7

longest peptides which war free o imino acids /©re o

chain length 21 and 22 residues peptide o’ 13 nmho

acids and a sequence* of 13 eminn acids in pepti e

category, i’he imino acid ree sones represented ly the

four long peptides account for only 2.7 o- the protein

rsolecule. No p ptides ere isolated ron this i est «hich

wore ‘reo of polar amino acids.

Of the p: ptides which were scrutinised f or aaino acid

sequence ( ee Table 3 most c ntain d regions to w^ich can

be ascribed either a polar or apolar character. No

information is available ns to the way in which these

peptides fit together or about the order o: the amino acids

in the remainder of the peoti ies (the major portion in

most cases). Most of the polar areas of peptides shorn

in Table 3 contain aci ic amino acids rath r than basic

ones and the question arises as to the number of these

which are present as asparagine or glutamine some of these

areas may not be truly polart rrthcr free o imino celts.

That polar regions lo exist cannot be disputed, but the

chemical evidence to date is no au ficient to give any

information as to their frequency or distribution, ro it

is difficult to draw any cinclusions concerning the theories

for the banding patterns obtained with electron micrographs.

?he enzyme collagenase has been used to nrobe the

shown t ' t the enzyme ( leaves the 1 me -r-.-G- - •*’ -r? P » proline, G * Glycine and X one other residue which

in collagen Is usually hydroxyproline or alanln

(?*ich*-els et. ale 1953, fa al rn ?to-r 19*9, Grassm*nn

et* el. 19*9, Cchrohenloher et. al. 19*9, Gallop end

del ter 1962). Thus collrgenase digestion results in the

'ormation of a large number of di and tri peptides

containing glycine an: proline, nrigin* ting rom the

apolar areas o the molecule, as -ell as larger peptides

derived r‘rom the polar regions. ranzblau et. al. (196M

isolated these larger peptides by dialysis and by

chromatography on Sephade* G*2

these non-di&lysable peptides obtained from different

collagens revealed basic similarities and a pattern of

amino acids quite different fr-'W that o intact collagen*

Thus the non-dialysnble fraction ( ee ta 1: ) has

increased glutamic acid, aspartic aci and lysine, contains

the majority o' carbohydrate, al ehyde and tyrosine| but

has reduced amounts of imino acids arglnin and leucine.

It would be expected that collagenase only attacks

non-polar regions of the molcculo wher^ the hi best

proportions o glycine and proline occur, and that the

non-dldyeable fraction as a result should be composed

largely o* peptides from the residual polar areas. his

cannot be entirely true however, because the reputedly

stain with phosnh tungstic acid, r reagent specific for prrinine and llstidine, being readily washed off the

lysine residues. Thus the polar regions of the electron

fBieroseopist are rich In arginine whilst the non-dlrlysshle

indicating that there can he no staple identity between

Fsaa atlfitraU

Fch A9&S.


(As raaldues/l real lues).

Xcthyocol ifihblt kin Cal' Skin

5 Vi ---— —. ■. ———3.2 The non dialysable fraction has a hi h conton* of reactive

side chains, especially rsnartlc acid and glut ante aci : • That hexose nay be bound to ^ne of these residues in some

way Is a possibility as the majority of the heyose of

of this p ptid© portion, is also compatible with the theory that inter and intro molecular

Crosslinking an

any other covalent modi ications of the molecule will • ?>

'utlor and Cunnin.hara (1965) have isolated a

glycopeptide from guinea pig skin collagen the amino

acid composition of which (Table ) indicates that It

is of a highly oolar nature. Their results suggest

that the herose Is hound via an -O-^lycosldie on I to

i ?\_.Qly.saE 16 ,er qiri C’-wnindian .126,

Amino Acid. . r. JrftQBtntion 1. . Eranf.caU'in Hyiroxylysine

desul :s expressed as ratio to hy rxylysine.

Peptides liberated ^rom letlyocol by collagunsse

have b en investigated by Green’erg et. al. (196 )

using the .. dman degradation technique on the entlro

enzymic digest. ’>y ollowing the c urs© o the enzymic

Hydrolysis with a pi stat

hbviouslv a large nua' er o the s 411 r peptides 111

pepVde nap of” the products of complete collagenaae

digestion of lcthyocol observed only 3 - e inite spots.

The results o the d * an degradation showed that glycine I

Is nost avun ant in positions 1 and , proline in

position 2 and hy ru yproline in position 3« ee to* le 6.

, XLi-dasaLj:—Iffi. ' (As residuea per VM in digest)

Amino cidj Position T|Position II Position III Position IV 1 *1

here is a certain amount o glycine in position Ill

evidence hr peptides o’ the orn -C- -G-. However, on

concept that the sequence -.-p- - <oris the nost cocoon

hydroryprolinc occurs in position ITT ’-sust be regarded as showing a eclnite characteristic of the


sequence o collagen. Although r.lanine is wore abundant

thf-n hyiroyyproline In collagen, and is a common residue

in the non-polrr regions, it is less frequent th n

hyfroyyproline in position TIT.

Thus two uncisputable features of the primary

structure or' collagen hr ve come to light. 1. The

requenev of the <11 peptide - lycine - proline *t if the

observation of Grassmann 1961 that accounts for

35 o the molecule is accepted, then 9 ' o the proline

of collagen is in this arrangement, and there Is no

question of a random distribution of this amino acid.

2. he high prop rtion of hydroyyproline in the third

p-Apart from these two observations, the picture of

the primary structure of collagen emerging from

information obtained to date, Is one of considerable

h<terogeneity. The polypeptide chain can he divided into

distinct polar and crystalline regions hut within the

ormer areas the amino acid sequence appears to be fairly

random. 'he results o' Crassmann et. al. (196 ) show

that within the polar regions the polar rmia^ acids

themselves are arranged In no particular ordered fashion.

sones to correspond with the Vandins pattern of electron

micrographs o' collagen. This is difficult to explain,

considering th« spread of polar residues in the polar


is in fact a representation at the level of one a lino

acid restlue. If for example, collagen stained with

uronyl acetate is considered: there are approximately 75

side chains which will bind this stain in each o' the

constituent chains of tropocollagen (1 OO amino acids


glutamine and aspara ine). The banding pattern reed res

the presence o s nothing like 5'1 positions at which stain

is bound in the length o the molecule. Thus 3 x 75 * 22$

re s must give rise to Jb bru-s. /1th a proportion

bends arising ron tw or more aci ic or basic groups rt

a tine, as a result o either: (1) cl-^se proximity of

polar residues in the polypeptide giving no resolution o ‘

bands corresponding t each o^ them and (ii) the presence

or polar residues in corresponding positions on the

three constituent chains of tropocollagea? a so

non-staining o s me o t>e polar side chains due to

involvement in inter and intra-molt cola r ionic,

proportion or the banding pattern -.111 be due to single polar groups# This concept silovs for an essentially

random distribution of acidic

And *sslc

Involves a lower degree organisation of the so-called

polar regions, this is perhaps 'nore In keeping with the

present state of knowledge of the primary structure of

Collagen Is re larkaMo for its resistance to

proteolytic attack, only the highly apact ic enzyme

collagen' se brings about any large scale destruction

of th- molecule. There h-8 b-en a great deal o'

conjecture as to whether or not proteinases in general

hove any activity with collagen. 1 uhn et. al. (19 >1)

maintained that trypsin degrades only tyrosine-containing

iopurities and that the collagen molecules remain

unchanged. Conversely Hodge et. al. (i960) reported

that when soluble tropocollagen is treated with

proteolytic enzymes, extra-helical peptide *openiages

rre released which they term telopeptides. Concurrent

ith the release o telopeptldea, the interactions of

the molecule are moci*’ier thus Ibrous-Ion/-spacing

a/greg?tes can no Ion er bo formed but the ability to

om segm nt-long-s oacin» crystallites is unimpeire .

ubin et. ale (1963) cemonstrrte^ that pepsin liberates terminal or near terminal covalently

bon ed-peptides the amino acid composition o which is

quite di?'rerent ron that oc the residual major portion

0“ the molecule. hey observed that pepsin c inverts

m - r1 th < r! j I o(c'rw ‘ s, .• .<< 1 T . r

that the inter chain link Is external to the body o the

Grant find Album (196 ) showed that rat tall tendon

collagen coni be solu’ilised at p-i 7*L in the presence

o calcium salts or sallcllates by a variety o enzymes

including trypsin an 5 ehymotrypsin. Other chemicals

which eoul be present in in-vivo conditions e.g.

arginine an creatinine, enhanced this sola ilisation.

he significance of such in ings is di fic lit to

asses?, or rat-tail tendon colleger? is an unusually

soluble orm. However collagen is normally quite

insoluble at neutral pHs, so the solubilisation observed

in this work could be due to telop ptlde liber tion and

subsequent separation into -chains at the temperature

of the exp-rim nt (33°C) I.e. a combination o proteolysis

A picture o *

"hairy-rod", i.e. carryin a number o protru in: p< ptlde

chains hrs been put orward by ?oamus et. al. (i960).

hey suggest that there are at least 1? places where

peptide chains 0 low molocular weight are sticking out

rrom the molecule, and that these chains are o ten almost

identical in amino acid sequence.

Thus the majority o inf motion is in avour o '

the concept that proteolytic enzymes find points of

attack in pepti e app-on ares o collagen> whilst the

main body of the triple-heliy is resistin’ to proteases,

suggest that the peptide appen ages mi ht be important

in connection with cr ssI Inking and end to en polymerisa

tion interactions of tropocollagen molecules.

Pretreatment with the enzyme <X -amylase at

has been used as a method for the solubilisation of

ox-hide collagen (Nishihara 19>3)* -teven (1961 ■) has

used the technique to extract collagen ron human

connective tissue and suggests that the enayoe destroys

covalent linkages which stabilise connective tissue

:owes and foss (19 3) and Grassoann anct Horaano



procollngen nowes and Moss foun. small anouns of

initrophenyl (D. ’•?•) aspartic acl end . ,!J. . alanine

which they thought must arise from extraneous matter, not

representing true terminal groups,

found significant amounts of K-teminrl residues in

Solub o


Aspartic Acid and D,#,P, Glycine of which D,?i.P. Glycine

represented 31 using the ,N,P, technique with procollng-n

Chan rarejan and Hose (1965)» using the phenyl

isothiocyanate nethod, found l.lo moles aspartic acid

• nd .^3 ool glycine per 1000 moles amino acl in

similar amounts o’ the same amino acids in insoluble

teven and Tristram (1962), by hydrolysing the

entire reaction mixture after dinitrophenylaMon of acid

soluble collagen, obtained D. P.

Derivatives of

Ino acids (

procedure would detect the N-terminal


which would otherwise le removed, by ./ashing o the . protein after dinitrophenylatlon. It was suggested that

these H-terminal derivatives originate from a collagen

non-protein nitrogen fraction which is not removed from

the material by normal purification procedures. It was

found that the non-protein nitrogen could be more or less

completely removed by acetone precipitation or dialysis

at low pH, and It was postulated that the fraction was

Important in connection with the fibre forming interactions

:&Ue ?♦ rtapUtfl .rasVaaa .t, .-alaUft ■..^W an*

( rom Steven and Trlstra- 19 -2)

Just how ma iy K-ter linel residues are present in

collagen is rs much of a question no r ns It was ten years

ago. The non-proteIn-nitrogen fraction of teven and

obeerved by the numerous workers who have investigated this problem. Phe fairly constant amounts of

rlyclne and b.r2.P. aspartic acid observed by h3f lann

could easily bolon to bound peptides which are impossible

to remo e from the parent protein, no covalent linkages

Hermann et. al. do not comment on the oi ni icance

' ' ■ .1 mol. ' • <x ' ' ^1. of amino acid residues, beyond saying that it is only

one tenth oc that expected if one amino end group occured

in each o** the three peptide chains of collagen. On the

basis of etermin tions of acetyl groups present in

collagen in act, they propose that the peptide chains

of collagen consist o' an average of siv subunits, whose

* mlno end groups are acetylated.

they could detect no end groups in insoluble college*,

but alkali treatm nt liberated amounts similar to those

they had estimated in soluble collagen* They took this

to indicate that the N-terminal residues of insoluble

collagen are masked by herose, and actually isolated end

characterised a glycopeptide from insoluble collagen

containin galactose and glucose. Again there was no

attempt to rrtlonalise the low yield of N-terminal amino

per molecular weight. Possibly th only logical

explanation^ i" it is assumed that glycine end aspartic

’•cid are present as terminal residues in the amounts

stated, and that the theories for the molecular weight

and subunit composition o'* collagen are accurate is

that the terminal residues if collagen are masked at

some stage subsequent to its 1ioaynthesis, possibly by

an enzymatic process, and that this larking is either

not always comolete or occurs over s relatively long

period of time co that in any preparation there are bound

t c y? mol cj1-s ' r e : ’1 : - r u s.

^isJ^ii&LUUz J2__ '.-Iral xiQuaa__ x-sjitaurtia*

A ’undauntal problem In 'he chemistry of collagen

are not ell equivalent that some of them rre not free t->

react with substituting reagents, while the majority can.

This question hrs come to light as a result of studies

usinr the dinitrophenyl technique on anger, mostly in

conjunction with investi ations of N-terminal residues as

described above, owes and Moss (1953) in th* ir

erneriments reported that V of the lysvl residues

appeared to not be avails'le to substitution by 1. laoro

2,b linitro ©nzeno ( ,D,M, • ) the rearent us d in tle

D.u,F, technique of Banker ( an er 19' 5). The reaction

medium used in this case was 70 ethanol plus Lh

saturated sodium bicarbonato, at room temperature*

Several forms of collagen and gelatin were investigated

• ut in no case did the degree o ' substitution exceed

70 . These workers pointed out that in the case of

gelatin and formic acid treated colleger., the D.f.P.

derivatives wore soluble, so the low < egr e or

substitution observe! cannot he due to a low rate of

reaction with an insoluble substrate. However they were

only able to detect a small amount o'* lysine in their

hy rol cates (V moles/10 0 amino acids) loavin approx,

1 moles/lT'h amino acid residues unaccounted or. It

was put forward V at -D.Tf.P. lysine Is iuc> less stal l

to acid hy rrlysis when combined In c llogen than then

present ns the free amino acid or In another ■ • .P.

hther workers to report incomplete substitution of

-lysyl groups were Holomons and Irving (1973) who

obtained 30 recovery of - . .r. lysine anI Hallsworth

(197k) v’ho quoted percentage aval labilities o k -7

for differ nt types o collagen aggregate, using

corrections based on hydrolytic recoveries of

lysine o'* B3.b - 94.9 ‘ as determined by control

experiments, hallsworth was also able to show that under

his conditions of reaction viz. aqueous medium pH 7.b

and 37°C, the degree o substitution was proportional

Mechanic and Lovy (1979) isolated the trln^ptl e

LL H - (glycyl- <*- ' - r ' ^vl • > ’ v

ten'on. They postulated that the of lysine reported

to b© unassailable to • .K. • by Bowes and Hose 1953

may be present as J lysyl p ptides o this nature.

’owever It is a known act that un or certain conditions

o by rolysis peptide synthesis an J rearran ement can

occur, so it must ■ e considered unlikely that this p ptide

occurs in such large amounts if at all.

of the lysine of collagen Is substituted by . . . d»en the reaction is carried out in & denaturing medium

2.5H with respect to sodium perchlorate. The degree of

substitution was determined by separation of the basic

amino acids from the hydrolysate of the D.M.s. protein

on a column of amherlite 1 C5 • he results are given

fa-le 3. rom Hormann et. al. 196J.

Analysis of basic amino acids 'rom normal and dlnitrophenylate soluble and Insoluble Collagen. Yields in mol./l > iol. amino acid.

di nitrophenyl a ted d ired 2.5

asolu’ la Collagen. ■ ).O3 0. 7

d 1 nl t rophe ny 1 a t ed undenatured.

denatured HaCl 0.1" 0.1) 0.12 5.35

Heyns and /ol "f (195‘S) also suggest that with excess

bicarbonate an, denaturantc -lysyl residues o collagen

become more or less completely substitute'. Many other

workers h -ve recorded a whole range o" availabilities

a variety of reagents, for . lysyl residues. Leach (1966)

rives vislues or substitution b potassium cyrnate

from bo * 99 and VJ - 93 resp ctlvely. Harding (1966)

in a very complete survey of the subject gives data on

reactivity of l-lysyl groups to many reagents including

1-acetylation, ' enzoylation, benzenesulphonylation,

succinylatlon, guani inrtion, so lum bromoacetate,

2, k dinitro benzene, nitrous acid, nitr^syl c dori e, ninbyrin rnd trypsin. The figures vary enormously hut

some are very hi -h including 100 ‘or acetylation.

In the race of this wealth of information, one must

conclude that the -lysyl groups of collagen

region of 100 free or substitution reactions. Possibly

In native collagen, some steric hindrance or ionic bonding

prevents complete reaction with some reagents, but in most

cases this can be overcome by denaturetion or increasing

the molarity of the solution. The act that ionic

strength hrs such an effect on reaction of ..-lysyl

resi ues with .T.H. could incicrt© th't this orm of

m( aking is in fact due to ionic bonding of these residues

rather than a purely steric ef. act and that th©

observations In connection with denaturing agents could

also arise ’rom a simple increase in ionic strength causing

the enhanced availability.

The orperiments used in estlm? tiny, the reactivity of

-lysyl groups, in general ere not sufficiently sensitive

two ner molecule) -lysyl groups In covrlent llnkeges. These, whilst few in number, could bo highly importantF r features o the molecule* Thus the tri pen

Tide o"

Mechanic and Levy cannot te completely ruled out.

’’ranxblau (1962) produced evidence in favour o' the idea,

when he found that 12 o the lysine of a colla&enrs©

igest o 1< 1, is not free to react with . j

masking in such small p ptides is unlikely to be anything but covalent, although here egain ionic linkages could


this theory came from the work of Hormann

they actually estimated the free lysine in hynrolysetes

o T.N.P. collagen. Their figure of only . 'nl./l

mol. amino acid (see Table 3) corresponds to 2.1 lysine


3001*' <), so the hypothesis is not completely ruled out,

although of course this tiny ©mount

Have originated i rom

Lockhart and Abraham (1956), reported that -lysyl

peptides are extremely stable to acid by rolyais, having

^ound that on ^3 hrs. hydrolysis with 11 • MCI

At 3'^C

There was

Aspartic acid from an

-lysyl peptide o' the two amino acids. This could be a

special case the proximity of two free carboxyl groups

to the peptide bonds would tend to repel protons and

.4 characteristic o all -lysyl peptide bonds. However, if this stability is a property o' all


estinrtions of free lysine in hydrolysates of I .f.P.

proteins would be subject to considerable errors, and the

results o experiments sue as those

Of Horman

would be misleading* This is only a remote possibility,

The possibilities or covalent linkages Involving


hypothesis for crosslinkin: between the polypeptide

chains o collagen, by N-glycosyl linked carbohy rrte

residues taking on the *orm of a Schl f-base, and thought

that tho . amino group of lysine would form the amino

group donors in this system.



structures in which the & amino groups o lysine could

participate se igure 1. He pointed ou that in the

letter case (straight chain li) the lysine residue

e-tlons Into the ^-terminal amino /cids or collagen. This

is not completely true: with Sa iger’s technique the

lysine, which is a er soluble. he derivative o' lysine

-lysyl bon I would be the unusual

Possible hhkAggs wok/mj £-li^i txtrnwo ^rpu^S ,

CO— CH—NH--- — CO- Jh«nh2brtn&h ArtrA IS tirrrurtAL X

CH — CO---NW- CH — Co---NH — C.W — Co&H

NH rco - r

HH2- OH-CO---NW - CH— Co---NH-Crt-CoOW

6) M —CH — co--- NH&K^)— ”*NH---CH’NH^

C'li^ hh^CH-Co ----UH-CtHj), Cii'W--- Nrt CH'CcM

lysine would almost certainly escape detection in the

aqueous nhase of an analysis of a I.K.P. protein

hydrolysate, due to the large excers v

which would also be present.

_______ 'UnKMia*

Bl arable number o X * glutamyl


al. (1957) ©s a result o experiments using the thiohydmt In

method. The Investigations were n:*t quantitative but were

taken o indicate the pr A


O( B d land

proprlonlc acids respectively. In the gelatin obtained rom lchtyocol, the formation o' these compounds wa3 noted

as well ns a decrease in glutamic acid and aspartic acid,

an the tor nation o' succinic seiial lehy e and ammonia. '

criticism o this work was thet the anhydrous med1 urn used

coul-i promote ring closure by the o carboxyl groupst with

Su (1962) and ransbli

[ Prom GaMojo 0^ • 14&O-)

e0__ Mt aw/flc2o

i ---CCrl^') n gsl'e.riPiCtu't’ion

CcHx) n. ---> CC1r/) n I kO*C 30m»n. NR - CH- co - NH


C’H 2. 1 CH 2

hy irorauic acids from unoo i led proteins In aq eous conditions. Using water soluble 1 - cycloheyyl - 3

C - >rph n/1 * (' ) et’ •>> * c” ' Mil il c

o-toluone sulphonate at pH** and 25°C in an aqueous

medium, they suggested that there



O peptide


0< ?00 ’ rsi^? s V'' : >u . if' n polypep I I rsw&c^* I’U'^ovl r rt •

linkages, the reactions and equilibria in cigur© 3 ®1<I

be ©rived un or certain conditions. Curtis and : pikes

(1962) have shown that carlolmi .es catalyse the oration

of peptide linkages without themselves bein involved., and

so th© glutsrimide ring system could be formed in this

fashion, fristram also re erred to work ty 1ovecs et.

al. (1953) on the Hofmann degradation o polyanhydro

aspartic acid, which showed that this polymer opens to

X - glutamyl linkages ere present in c

substantial numbers, rs ^ranablsu suggests, then the two methylene groups Introduced into the polypentide

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  1. Christie

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  2. Wajih

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  3. Taudal

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  4. Tab

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