California Association of Chemistry Teachers
John Leo Abernethyl
University of California 10s Angeles, 90024
Lyotropy With particular application to collagen
Since the reports of the original experiments by Franz Hofmeister and his students (1) concerning the behavior of salts toward proteins in 1888 to 1896, the definition of lyotropy ( 2 ) has undergone progressive revision. Lyotropy now means the observable effects on hydrophilic colloids caused by relatively small, water soluble, reagent molecules or ions that are usually in solution. When proteins are the observed hydrophilic colloids, lyotropy sometimes refers t o precipitation by the reagents; while a t other times, it means a dissolving action or tendency toward dissolving. It usually involves formation or cleavage of one or more of the three common types of non-covalent bonds, namely, ionic bonds, hydrogen bonds, or hydrophobic bonds. Hydrophobic bonds deal with van der Wads forces that result from non-covalent association of nonpolar portions of proteins in an aqueous environment. This environment often enhances the association of such protein portions. The special structural, configurational, and conformational features of collagen have made it a very useful substrate for studying lyotropic action of a variety of reagents. The things that happen depend on the reagent used and the structural portions or combined portions of collagen that are affected. The Esoteric Features of Collagen
Collagen is the chief protein of several soft tissues, such as hide, tendon, and cartilage, as well as hard tissues like bone and teeth. It is commercially important to the leather, gelatin, and glue industries, with widely diversified applications in areas like photography and medicine. Besides its obvious anatomical function in animals, it enters into special considerations, for reasons of calcification, of the formation of hones and teeth, and hone resorption. To chemists it has offered new insights into primary, secondary, tertiary, and quaternary protein composition, with unique facets concerning its conformation. While there are differences from one animal species to another, collagen shows these important amino acid residues in its primary composition: about 33% of the residues are glycine and about 25% are a combination of proline and hydroxyproline. This makes collagen distinctly different from other proteins. The presence of the basic residues, lysine and hydroxylysine, Address: Department of Medicine, UCLA School of Medicine, University of California, Los Angeles, Calif. 90024.
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while not large, is far greater than in other proteins. Histidine usually contributes somewhat less to the basic residue composition. Arginine is the principal basic residue. Both aspartic and glutamic acids are the important acidic residues. Alanine contributes about 10%. Serine, threonine, and hydroxyproline are hydroxy residues whose total percentage remains fairly constant, although the individual percentage varies from one species to another. While the sulfur-containing residue, methionine, is present to a very small extent, both cysteine and cystine are essentially absent. The content of phenylalanine is very low and the aromatic residue tyrosine is still lower, but it does supply important aggregational properties to soluble collagens. There is essentially no tryptophan to provide the normal aromatic residue absorption peak,, . ,X E 2800 A, that is shown by most proteins. The residue sequence gly-pro-hypro occurs t o an unusually large extent and exerts conformational restrictions on the collagen helix. Special significanceis attached t o the residue sequence, NHZ XPrA1A2PrY COOH, which apparently permits an attack by collagenase in causing hydrolysis between the A, and Az amino acid residues (3). Pr can be either proline or hydroxyproline. A1 is often hydroxyproline but can be a number of other amino acid residues. A? is usually glycine, although it can also be a residue with a small side chain. Hydrolysis between Al and A2 accounts for the preponderance of hydroxyproline as a carboxy terminal residue and glycine as an amino terminal residue in the peptide products of collagenase-catalyzed hydrolysis. When Pauling applied the stochastic method (4) in proposing the structure of cu-keratin, the a-helix (6) became one of the few hydrogen-bonded conformations, of which two natural ones are known t o be helical (6), that are now called the secondary structure of proteins. Theoretically the a-helix, which consists of L-amino acid residues except for glycine, can either be clockwise or counterclockwise. Calculations from optical rotations (7) have shown that the natural backbone polypeptide, stripped of the side chains at the asymmetric centers, has a specific rotation [ a ] C ~ 100". This backbone has a cloclrwise twist as it recedes down its axis away from the observer. An attempt to arrive a t the conformation of collagen through the stochastic method failed when Pauling (4, 8) and Corey suggested a triple helix with all three helices winding around a single, common, axis. Ramachandran and Kartha (9)
-
-
+
Figure 1. Collogen rnocromolecule of three coilsd coils, with major axis pitch 185.6 A) campressed for convenience of illurtrotion.
moved intuitively in the direction of three helices, each with an independent axis, and all three axes parallel to each other. Some features were still a t variance with known parameters of collagen. Ramachandran (10) progressively modified this conformation into a more satisfactory form (Fig. 1). The main improvement was to maintain the three helices in parallel counterclockwise directions as they receded away from the observer down their independent axes, and then twist them together in a clockwise direction about a common major axis as they receded from the observer. This provided reasonable atomic and residue relationships in the direction of the major axis and a dense packing of atoms. Shortly after this, Rich and Crick (11) proposed a similar structure but with a few variations concerning the conformation of peptide carbonyls with respect to atoms and their attached groups on either side of the carbonyl. In both models the macromolecule was considered to he composed of three coiled coils of the polypeptide chains bound in a rigid rod, with hydrogen bonding between coils and considerable rod flexibility due to its great length. The main differences between the two conformations were the following. The Rich and Crick model permitted a maximum of one interchain peptide hydrogen bond for every three non-pyrrolidinecontaining amino acid residues of a given chain, while the Ramachandran model permitted two such hydrogen bonds. In regions of occurrence of the proline and hydroxyproline residues, the heterocycles were projected inwaril within a given helix for the Rich and Crick model, and outward for the Ramachandran model. These pyrrolidine residues contain no hydrogens a t the peptide nitrogens for interchain hydrogen bonding. However, the hydroxyproline can provide hydrogen from the hydroxyl group for hydrogen bonding within the same helix for the Rich and Crick model, but only between chains for the Ramachandran model. The Ramachandran model appears to be more satisfactory because it is more densely packed and has certain stereochemical advantages because of less crowding in important zones. I t permits van der Waals or hydrophobic bonding between minor helices by non-polar overlapping a t appropriate parts of pyrrolidine rings and also a t other residue side chains. A certain amount of all three types of non-covalent bonding is possible intramacromolecularly and also between contiguous macromolecules. Some modifications of bond distances are necessary, when compared with amino acids or simple peptides, but to no greater extent, it is reported ( I t ) , than is involved in the a-helix of other proteins. It should be stressed that gly-pro-hypro sequences cannot have two hydrogen bonds from peptide nitrogens because two of the peptide nitrogens are in pyrrolidine rings and have no bonded hydrogens.
It is the non-pyrrolidine residues in sequence in other portions of the chains that allow the maximum of two interchain peptide hydrogen bonds per three residues in a staggered manner across the twisting major helices and provide strength for the structure. However, this seems to be assisted by a strong loclcing function of the carbonyl between pyrrolidine rings, which locking function accounts for the counterclockwise twist of the minor helix. Space filled models show that there is steric hindrance toward a clockwise twist due to pyrrolidin residues. Studies with gelatin and poly-kproline (IS) confirm this very special locking function from optical rotation considerations. The backbone polypeptide of collagen must have a counterclockwise minor helix. Crowding, due to the hydroxyproliue residue, accounts for the clockwise major helix about the major axis. The macromolecular unit is called a protofibril (14) in the natural, fibrous condition, hut is named tropocollagen (1.4) in solution. Tropocollagen appears to have non-helical extensions at each end of the coiledcoil portions of the macromolecule in solution (16). The molecular weight of the collagen macromolecule is approximately 350,000 (16). Each of the three minor helical strands should have a molecular weight of about 115,000. Actually, two strands, on, are alike for a given source of tropocollagen, with a molecular weight appreciably less than for the third strand, a p . Residue composition and sequence that comprise the primary structure are different for the a1 and 012 strands. The diameter of the macromolecule $ about 14 A, and its length is approximately 2600 A. The pitch of the major helix, or distance along the major axis for one complete major helical loop, is about 85.6 A. Formerly, integral relationships were supplied with exactly 30 residues per pitch or per single clockwise loop, and a corresponding nine turns for each counterclockwise minor helix in traversing the same major pitch distance in the macromolecule. Currently, it is felt that there is not quite an integral relationship, hut instead about 35.3 residues per major axis loop and 10.6 turns of each minor axis in making one loop. Hydrothermal Shrinkage Temperature ( 1 s )
Non-covalent forces account for the tertiary structure of collagen in holding the triple helices of the macromolecule together. With aging these forces extend increasingly by better alignment (17) between the rigid rods of the macromolecules which produce the fibrillar structure. Quaternary structure involves a polymerized, parallel, quarter length overlap of these macromolecules in the fibrils. Terminal amino group are always directed toward the same end of the fibril, in both an intra- and intermacromolecular sense, with terminal carboxyls oriented toward the opposite end. This provides identical vectorial polarity for the molecules. Three dimensional fibers have great resistance toward rupture perpendicular to the fiber axis. This can he increased somewhat by cross-linked covalent bonding on aging. When collagen fibrils, which are very small fiber segVolume 44, Number 6, June 1967
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ments, are teased out of the fiber and captured on a Formvar or Collodiou film, they can be chromium shadowed (Fig. 2). Electroil micrographs often proTUNCSTEN FILAMENT
C~ROMIIIU DEPOSIT
I
ECEWTCD FIBRIL SURFACE
Figure 2. Chromium shodow technique. Chromium vapor followr paths normal to heated filoment and condenses. Fibril portion is enormovrly enlarged relative to flloment size.
duce over 100,000 fold enlargements, when the beam of electrons is partly reflected from t,he relatively opaque chromium and partly transmitted through the largely transparent, exposed, fibril and supporting film onto a photographic film. Appropriate processing yields a typical banded picture of the fibril (Fig. 3A) that has the appearance of a pipe cleaner or striped s~lake. Distances between baud centers are 640-650 A. This is one quarter of the length of the protofibril macromolecule, which is 2600 A. If collagen fibrils are stained with phosphotungstic acid (PTA), uranyl acetate, or salts of cationic complex ions of trivalent chromium, then the electron micrographs produce a more detailed banded appearance (Fig. 3B). The hands lack a center of symmetry, as a consequence of vectoria! polarity. Patterns repeat a t about every 640-650 A. Phosphotungstat,e is held largely by 8-guanidinium side chains, -XH-C(KHs)=
of collagen cationic and anionic groups in these hand regions of the fibrils. The less stained zones are called the interband regions where nou-ionic groups are concentrated. I n solution, light scattering shows (15) ghat the macromolecules have lengths of about 3000 A, with chain ends that flap loosely beyond central coiled coils that are pliable because of their length. A natural collageu fiber can be tied a t each end with a thread and held taut under water by means of a small weight over a frictionless pulley (Fig. 4). The water serves as a heat transfer medium and an exchange for structural water of the collagen, particularly in a lubricating manner when the collagen shrinks. As t,he water is heated, mature mammaliau collagen - fibers usuFigure 4. Determinotion ally shrink over a tnwdegree temof hydrothermal shrinkoae perature range a t about 6 3 T to - temoerature. . . T.S.less than one-third their original leneth. This is called the hvdrothermal shrinkage tem" perature, T s . It involves a macromolecular phenomenon, quite similar t,o melting of organic or inorganic compounds. Increasing the weight over the pulley can cause an increase in T,. Thermal energy breaks the comhinations of non-covalent bonds in an intra- aud intermacromolecular fashion (Fig. 5 ) . Such bonds give rigidity aud thermal stability within each protofibril and to the fiber by intermacromolecular action. It is t,hought that ionic bonds contribute only slight stability to fiber strength (18). Aging of albino rat skin collagen shows a progressive increase in T,9from 51' a t birth to u
-
SH2, and e-ammonium side chains, -?\THa, of the collagen. Uranyl (U02+) or positive chromium cations .
are bound a t carboxylate side chains, -COO. Since nearly identical striped patterus are produced from all of these stains, there appears to be a high concentration Figure 5.
Figure 3. Electron m i ~ r o ~ r ~ ~ A. h s Chromium . rhodowed thick rottoil fibril showing 640 A bond repeat dirtonce (from o slide loaned by Dr. F. 5. Siortrand. Zoology Deportment, UCLA). 8. Phosphotungrtic acid stained thin rattail fibril with unrymmetricol band patterns ot 640 A repeot dimnce. Reproduced with permission of the author, C. E. Hall (Am. Ann. Photography, 61. 37, 19471.
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Alterations of protofibril$from ordered to random units.
5G0 a t 10 months and 61' a t two years of age (19). Int,raand i~itermacromolecularcovalent and non-covalent bonding are believed to be increased, creating greater thermal stability. Collagen chain crosslinliages of ester, amide, or acetal types could be formed by proper juxtaposition of prot,eiu carboxyls, amino groups, or hydroxyl groups. Hydroxy acids lilie cit,ric acid or chondroit,in sulfate, or hexopyranose units ( I G ) , might bridge protein chains by appropriate formatiou of ester and acetal groups. Synthetic cross-linkages of an intra- or intermacromolecular nature can be formed by an organic reagent lilie formaldehyde or by an inorganic reagent such as a coordination polymer of trivalent chromium. Formaldehyde IinBs c-amino groups of lysine. either with guandinium groups or with amide groups (20).
Collagen-NH.
+ CH?O+ N H ~ - C ( N H ~ ) = I ? H ~ - C O ~ I ~ ~ ~ ~ (or NI&CO-Collagen)
+
C o l l a g e n - ~ ~ - ~ ~ ~ - ~ ~ - ~ ( ~ ~ ~ ) = & ~ ~ - - ~ o &O lla~en
An increase of 15-20°C in T s has been observed by tanning with formaldehyde ($la). Cationic polymers of trivalent chromium are attracted to anionic carboxylates of collagen. On prolonged contact carboxylates displace ligands and enter the coordination shell.
ZM~KS.
M
,.
Crystal field stabilization results as the ligands take positions along the lobes of the d , ~and d,. - orbitals of the trivalent chromium. In the old "boil test" for chrome tanning of leather, portions of the leather were placed in boiling water. With insufficient tanning the leather shrank, but with proper tanning the strong cross-linkage raised the T, above 100°C and prevented shrinkage. An important correlation between ambient temperature, composition of fish collagen, and T , has been noted. The T s of cold-water fish ranges from about 36-43% For warm-water fish the T s range is 53-57"C, and for hot-water fish, or lungfish, the T , is 63'C (22). Furthermore, the table shows correlations between the Hydrothermal Shrinkage Temperotures of Typical Fish Skin Collagens
-Pyrrolidine-Containing Residuesin 1000 Total Residues Fish Skin Hvdroxvmoline Proline Total .. Halibut Shark Pike Carp Lungfish
63 79 TO 73 78
108 113 129 124 129
Shrinkage Temperature, T.v. "C
171 192 199
40" 53*
197
57" 63"
207
55"
content of either hydroxyproline or proline, or a combination of both, in fish skin collagen and the shrinkage temperature. A higher shrinkage temperature generally means a higher pyrrolidine residue content (23). When three separate straight line plots were tried for residue content versus T s , from a more extended listing of fish collagens, the total pyrrolidine residue versus T , plot was the best statistical one. The interpretation has been that pyrrolidine residues enhance internal and external protofibrillar stability. Carbonyl groups between co~isecutivepyrrolidine rings, coupled with steric preference of these rings, provide a very important locking function favoring the counterclockwise minor helix. Also, hydroxyl groups of hydroxyproline form hydrogen bonds with peptide carbonyls, while hydrophobic bonding between overlapping pyrrolidine rings adds further to the stability. Denaturation Temperature, TD, and Intrinsic Viscosity
The conversion of collagen to gelatin chiefly involves cleavage of non-covalent bonds, first between adjacent macromolecules and then between intramacromolecular helices. This yields a colloidal solution mainly of various combinations of uncoiled helical chains (Fig. 6), but with some intact and some partly uncoiled
E
zm
iKILY ~M~KICN
nACPOfiOLEC~ES s L l 6 m n
OF YOUNG COLLIGIN lRlslE HELICES
A ~ ~ o
ne9cmm CMLS oc m o s 0 ( 0 ~ I f f i E N
MararTrrr AGED
INSOLUBLE
znt KSCN
Exxanvrry AGED
INIOLWLE
.
(. .); NO*-EWLLEW BDllDS
(-1'
COWLEYr
mmos
Figure 6. Effects of dilute acetic acid and long time contoct of 2 M KSCN oncollagen after voriour degrees of aging.
tropocollagen. In practice, some covalent bonds are also broken by hydrolysis, either a t peptide chains or at covalent cross-linkages. By careful control of reagents (Fig. 6), intact tropocollagen macromolecules can be taken into solution. Often 0.01 M acetic acid or 0.6 M buffered citrate at pH 4.1 is effective when the macromolecules are not extensively non-covalently bonded. Short-time contact with strong lyotropic salts like 2 M KSCN also produces intact tropocollagen, even breaking extensive non-covalent bonding between protofibrils. A longer time of contact with a strong lyotropic salt changes tropocollagen to gelatin. Figure 6 summarizes cleavage of non-covalent bonds in dissolving collagen. The principle of forming @- or a-gelatin from single protofibrils or adjacent protofibrils is clearly given in Figure 6, with the 1 and 2 subscripts used in showing combinations of w, and arz strands. When solutions of tropocollagen are prepared from citrate buffer, then dialyzed and redissolved a t pH 3.7, intact tropocollagen rods result. For most collagens, this solution has a specific rotation of approximately [w]DROOm Temp = -350°, with a range from about -330 to -415", depending on the collagen used. When such a solution is heated for short periods of time a t fixed elevated temperatures, then cooled and the optical rotation measured, the rotation remains constant until just before the prior elevated temperature reaches the denaturation temperature, T,. Increasing the prior temperature near the T , and beyond causes the optical rotation to drop in absolute magnitude over a narrow temperature range until it reaches a value of about [a]DRoomTemP = -120°, with a variation depending on the collagen source of -110' to -135'. The temperature at mid-position of change in optical rotation is the denaturation temperature. For nearly all tropocollagens, the difference T s - T , is approximately 2025°C. For example, the T , for sharkskin (24) is 5 3 T while its T , is 29°C. Denaturation in this case refers to an unwinding of the triple helices of tropocollagen to form gelatin molecules. The conformational change yields molecules that are not quite random coil. Intrinsic viscosity of tropocollagen (18) undergoes a similar decrease in value. A set of solutions of tropocolVolume 44, Number 6, June 1967
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lagen of known progressive dilution is heated at a given fixed temperature (1) for 30 min, cooled to room temperature, and their respective viscosities are measured. The intrinsic viscosity [v,], freed from molecular interactions due to concentration effects, is determined by appropriate extrapolation t o zero concentration. Similar extrapolation is made for unheated solutions of tropocollagen to give the intrinsic viscosity [?I. As the temperature, t, is increased for new sets of solutions the ratio [7,]/[7]remains essentially equal t o unity until slightly before the T D is reached. Then, over a small temperature range this ratio drops to about 0.2 or less and levels off. The temperature a t mid-point value of [q,]/[q] is also called the T, and corresponds well with the value obtained from optical rotation. It is evident that tropocollagen has a higher intrinsic viscosity than does gelati11 because of the difference in conformation between the two. During hydrothermal shrinkage, hoth inter- and intra-protofibril non-covalent bonds are broken but a gelatin solution cannot be formed because the protofibrils are not separated into independent protofibril units. When the T, is measured, the protofihril units are already freed as independent tropocollageu macromolecules. Essentially only the intramacromolecular non-covalent bonds are broke11 and less thermal energy is necessary. Tropocollagen can be reconstituted to native type collagen with dilute NaCI, in which ease the macromolecules regain their quarter-length overlap. The X-ray patterns are similar to patterns for native collagen. When chromium shadowed or stained, this solid material gives electron micrographs similar to those displayed by collagen.
power series was S C N > I- > CIOI- > Br- > C1- for lowering the shriukage temperature of rattail tendon. Under slightly weighted tension in water, the T, was 68°C. I t was 52°C in XaSCN and fi5"C in KaC1. Shrinkage temperature decreased when ious penetrated protofibrils with water and weakened the structure. For 1 M N&OI the Tswas 83%. Sulfate is notedly a strong dehydrating ion, which can cause a decrease in the ability of protofibrils to glide past each other, compared to effectsof other salts. Withdrawal of water from tendon fibers by 1 M sucrose solution elevated the T, to 70°C and at 2 M it was 72°C. For 8 M ethanol it was 70°C, at 12 M it was Sl°C, and pure ethanol raised it to 86°C. Pure formamide lowered it to 15"C, due to disruption of protein-hydrogen bonds and ionic bonds (2lb). When 1M chlorides were used, the Hofmeister cationie power series for lowering the Tswas Ca2+> Mg2+> Li+ > S H I + > Na+.
Lyotropic Action
Figure 7. Differences of swelling oction of CoCI2 rolutianr on mesh bovine tendon.
A few examples can be cited to show the diversity of lyotropic action. When hydrothermal shrinkage temperature is studied in salt solution a t pH 7, there is often a progressive decrease in T swith increased salt concentration (25). Fibers that showed a T, of 6S°C, held taut by a given b e d weight in water, displayed a shrinkage temperature of 47'C in 1 M ICSCN and 39'C in 1.5 M IBCN. Similar lowering was showu in NaCIOn and K I solutions. Weakening of thermal stability was undoubtedly due to combined effects on interruption of polar bonds, hoth ionic bonds and hydrogen bonds, by ions that carry a certain amount of water with them. Chiefly, interprotofihrillar bonds would be broken. When urea was used, the T, was 64'C a t 0.5 M and 55°C a t 2.0 M, while 0.5 A[ guanidine gave a T, of 53% and a Tsof 49'C a t 0.75 M. Urea would break collagen hydrogen bonds when the urea carbonyl competes for peptide hydrogens. Amide groups of urea would compete for peptide carbonyls. It is thought that hydrophobic honds can also be broken because urea has a certain amount of hydrophobic character. Guanidine in aqueous solution is largely ionic because resonance stabilization, or electron delocalization, of the guanidinium ion produces a strongly basic solution. HCI would neutralize the solution t o pH 7. The guanidinium ion would compete for earboxylate ions of collagen and interrupt polar hydrogen bonds by its ionic character and also by its amide hydrogens which would seek out peptide carbonyls. When 1 M sodium salts were used, the Hofmeister 368
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Journal o f Chemicol Education
Lyotropie swelling is shown in Figure 7 for 1 g samples of lyophilized bovine tendon, 10-20 mesh particle size, shaken with 20 ml of 0.2 M, 0.5 d l , and 1.0 llf CaClz solutions for 90 hr a t 37°C. Solutions were then centrifuged for 10 min a t 10,000rpm. Strong lyotropic action occurred a t concentrations above 1 If. C&12 would first break protein polar bonds. With penetration of increasing amounts of water of hydration the hydrophobic bonds would finally be forced apart. Separated protofibrils would be slowly gelatinized by cleavage of intramacromolecular bonds, but the gelatin would remain chiefly undissolved unless larger volumes of solution were used. When hide powder is exposed to solutior~sof most salts, swelling occurs first. Then, over periods of time up to 100 days or more, increasing amounts of collagen are dissolved (26). Among the strongest lyotropic ions are CaZ+ and SCN-. Lyotropie cleavage of noncovalent bonds by CaCL solutions is shown in Figure 8. This is applicable to effects on swelling arid T,,also. Sodium sulfate has less dissolving power toward hide powder than water. This explains the strong precipit,ating action of sulfates. When gelatin is treated with sodium sulfate, fractionation of a-,8-, and y-gelatins can be brought about (16). For lower concentrations of sodium sulfate the y-gelatin precipitates first. Increasing the salt concentration precipitates p-gelatin; then with still higher salt concentrations the cr-gelatin,
proline: I has [a]i5 = +40° in water, and I1 has The levo-rotation of poly-L-proline [a]F = -540'. I1 drops to -250' in 12 M IiBr, showing the hydrated carbonyl lock to be broken with resultant cis-trans conformational changes. The severity of phenolic "burns" on skin collagen is a well known lyotropic behavior. Phenol has strong lyotropic power in cleavage of protein hydrogen bonds because the phenolic hydroxyl competes for peptide carbonyls. The hydroxyl can also form hydrogen bonds with protein carhoxylate groups and therefore cleave protein ionic bonds. Hydrophobic bonds can be broken by van der Waals action of the phenyl group of phenol, thus rounding out its destructive lyotropy. Factors Affecting Lyotropic Action Figure 8.
Compositeof some d the lyotropic action of CoC12solutionr.
which is the lowest molecular weight fraction, precipitates. Whereas urea, formamide, and guanidinium salts are known to be reagents that break peptide hydrogen bonds, concentrated aqueous solutions of LiBr have the opposite effect toward those proteins that can conform to an a-helii type of fold ( I S b ) . Serum albumin is known to be extensively hydrogen bonded in aqueous solution. In concentrated LiBr it showed essentially no change in specific rotation or in Drude rotatory dispersion constant, A,. This indicated that LiBr caused no solvent effect on rotation. Clnpeine from herring, which is unfolded in aqueous solution, exhibited [a]: = -104.5" and A, = 200 mp, but in 6 M LiBr [a]? = -48.9' and X, = 230 mp. Similarly, oxidized ribonuclease is unfolded in aqueous solution with [or]? = -91.1" and A, = 22.5 m, whereas in 9.9 M LiBr it showed [or]? = -49.5' and X, = 250 mp. The LiBr hound the water and reduced its activity toward keeping peptide hydrogen bonds separated. The protein folded with appreciable content of or-helix type of fold. It was previously demonstrated (87) that salts can change tropocollagen solutions to gelatin. I t has more recently been shown that LiBr has a special effect on gelatin (88). In citrate buffer a t pH 3.7, ichthyocol collagen displayed [a]:5 = -330' and X, = 204, while the gelatin showed [or]: = - l l O D and X, = 204. In 2 M KSCN this gelatin exhibited [a]: = -86' and A, = 204; and in 12 M LiBr, [or]: = -56' and A, = 202. The dispersion constant remained essentially unchanged. Furthermore, the intrinsic viscosity of bovine gelatin in the very weak lyotropic agent 0.2 M KC1 was the same as in 8.5 M LiBr. This meant that no extensive folded configuration developed through LiBr. The interpretation has been that gelatin in water retains a small amount of residual collagen helical conformation. Hydrated carbonyls between pro-hypro residues have a locking function that, coupled with steric factors, favors the counterclockwise helix. LiBr breaks the residual lock and permits further cis-trans isomerizations to occur about this carbonyl with a conformation more nearly random coil. For ichthyocal tropocollagen, the back bone counterclockwise helix has a calculated value of = -240'. This is called a collagen I1 helix on the basis of the two conformations of poly-L-
Some of the evident variables that effect lyotropy are concentration, particle size, time of contact, and pH. For 2 M sodium salts, the relative dissolving action on hide powder a t the end of 70 days exhibited I- > Br- > C1-, while for 3 M it was Br- > I- > CI- (86). The T, of sheepskin for 0.5 M NaC104 (8@ remained at 57.5"C from pH 3.5 to 10.5, hut it dropped sharply when the pH was lowered below 3 and when it was raised above 11. I t is obvious that particle size and time of contact can have an effect on dissolving action and on swelling of collagenous substances, as well as on other observable properties. Other important factors relative to cations and anions include unfree ions or coordination complexes. Variables that also must be considered are aspects of soft and hard acids and bases (ZQ),electronic confignration and orbital population, stability constants, ionic hydration, ionic charge, polarizahility with electron cloud deformation, ion mobility, and more intimate details centered about these variables. To cite one cationic example, the calcium ion is recognized as one of the strongest lyotropic cations when the events leading to dissolution of collagen are ohserved. These events include swelling and allied effects on Ts,T,, and gelatinization. This ion is rather small with its ionic charge of + 2 and electronic configuration IsZ,2s2, 2p6, 3sZ,3p6. For complex ion formation, the calcium ion can use its 4s, 3d, and 4 p unfilled orbitals in various degrees of hybridization to accommodate up to six pairs of ligand electrons. Since it does not have an incomplete d orbital, no crystal field stabilization is possible, and coordimation is usually rather weak. Under certain conditions complexing could tend toward linear (4s 4 p ) trigonal (4s 4p2), tetrahedral (4s 4p3), square planar (4s 4p2 3d), trigonal bipyramidal (4s 4p3 3 4 , square pyramidal (4s 4p33 4 , and octahedral (4s 4pa 3d3. The less likely hybrids like linear, trigonal, square planar, or square pyramidal can be encountered on occasions of unusual ligands or where hydrophobic protein groups hinder normal octadedral liganding. Any or all of these situations can have transitional involvement in the loose, rapid, dynamics of interchange. Weak bonding of water permits calcium ions to carry it in between protein strands and free it for maintaining protein cleavage. Under special conditions, polydentate ligands could clamp rather firmly onto calcium ions through appropriately spaced electron pairs from protein groups, as they squeeze the inhibiting electron cloud out of the way and penetrate closer to the nucleus. Volume 44, Number 6, June 1967
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G. N., AND KARTHA,G., Nature, 174. ( 9 ) KAMACHANDRAN, 269 (1954). G. N., "The Triple Helical Structure of (10) RAMACHANDRAN, Collagen," i n "Aspects of Protein Structure," (RAMACHANDRAN, G. N., Editor), Academic Press, Inc., New York, 1963, p. 39. (11) RICH.A., AND CRICK,F. H. C., Nature, 176, 915 (1955). N , E., "X-Ray Analysis and Protein Stmc(12) D I C ~ R S O R. ture," in "The Proteins," Val. 11, (2nd Ed., NEURATH, H.. Editor). Academic Press. Inc.. New York. 1964. ,' 997 '(1958); ( b ) (13) ( a ) HARRINGTON, W. F., N ~ ~ u w 181, HARRINGTON, W. F., AND SELA,M., Biochim. Biophys. Acta, 27,24 (1958). J. W., AND SCHMITT, F. O., PTOC. (14) GROSS,J., HIGHBERGER, Nat'l Acad. Sci., 40,679 (1954). Acknowledgments H., AND DOTY,P., J . Am. Chem. Soe., 78, (15) BROEDTKER, 4267 (1956). Many stimulating discussions with Dr. Marshall R. (16) YEIS, A,, '*The Macromolecular Chemistry of Gelatin," Urist, Director of the UCLA Bone Research LahoraAcademic Press, Inc., 1964. tory, were the challenge to integrate the facets of M. L., AND KATE,E. P., J . Ultrastructural (17) ( a ) GLIMCHER, lyotropy for this paper. Dr. Frank Galey of UCLA M. L., KATZ, Research, 12, 705 (1965); (b) GLIMCHER, E. P., A N D TRAVIS,D. F., ibid., 13,163 (1965). developed the photograph showing the exact hand (18) BWRGE,R. E., AND HYNES,R. D., J . Molec, Bwl., 1, 155 distance correlation between chromium shadowed and (1954). PTA stained collagen fibrils. The photograph of Figure n , T., AND BOSEN,S. M., "Biological Aging in (19) J o s ~ ~ K. 7 was modified from a report t o he published with Dr N., Editor), Collagen," i n "Collagen," (R-~MANATHAN, Interscience Publishers, Division of Jahn Wiley & Sons, Marshall R. Urist on calcium binding by bovine tendon Ine.. 1962. o. 377. H. S., J . Am. Chem. (20) F R A E N K E I ~ C ~ N R A T , H., AND OLCOTT, Literature Cited Soe., 70,2637 (1948). ( 1 ) ABERNETHY, J. L., J. CHEM.EDUC.,44, 177 (1967). K. H., "The Chemistry and Reactivity (21) ( a ) GWSTAVSON, E. H., Chem. Weekblad, 39, 402 (1942). ( 2 ) BUCHNER, of Collagen," Academic Press, New York, 1958, p. 228; I., "Collagenases and Elastases," i n Adv. Enzymol( 3 ) MANDL, (b)ibid.,p. 205. F.F., Editor). ogy, 23,161 (19611, (NORD, (22) LEACH,A. A., Biochern. J., 67, 83 (1957). ( 4 ) PAULING, L., American Scientist, 43,285 (1955). (23) (a) PIEZ, K. A., J . Am. Chem. Soc., 82, 247 (1960); (b) L., COREY,R. B., AND BRANSON, H. R., Pmc. ( 5 ) PAULING, PIEZ. K. A.. AND GROSS J. J . Bid. Chem... 235.. 995 Nat'l Acad. Sci., 37,205 (1951). (1960). V., Proe. Indian Nal'l Acad. Sei., 53, ( 6 ) SASISEKHARAN, W . F., A N D VONHIPPEL,P. H., Ad". Protein (24) HARRINGTON, 296 (1961). Chem., 16,74 (1961). G. N., AND VENKATARAMAN, S., ( 7 ) ( a ) RAMACH.~NDRAN, F. G., Biodim. Biophys. Acta, 3,170 (1949). (25) LENNOX, "Theory of Optical Rotation of the Alphe. Helix and its A. W., AND FOSTER,S. B., Ind. Eng. Chern., 17, (26) THOMAS, Absolute Configuration," i n "Collagen," (RAM.ARATH.AN, 1162 (1925). N., Editor), Interscience Publishers, Division of Jahn D. C., AND LOVELACE, F. E., J . Am. Chem. (27) C-LRPENTER, Wiley & Sons, Inc., New York, 1962, p. 199; (b) YANG, Soe., 57,2337 (1934). J . T., Tetrahedm, 13,143 (1961). W. F., Nature, 181,997 (1958). (28) HARRINGTON, L., AND COREY,R. B., Proe. Nat'l Acacl. Sci., ( 8 ) PAULING, R. G., J . Am. Chem. Soc., 22, 3533 (1963). (29) PEARSON, 37,272 (1951). (30) PECSOK,R. L., J . CHEM.EDUC.,29. 597 (1952).
This could give stable complexing, much as with EDTA ($0). Thus liganding can vary from facile to quite firm. An anion like sulfate has its -2 charge equally distributed over the four tetrahedrally arranged oxygens. I t readily hydrates through hydrogen bonding with water. I t is an excellent precipitating agent for many proteins in solution, partly because of this dehydrating strength. Special explanations are necessary, of course, for ions such as the strongly lyotropic guanidinium cation, previously cited, and the thiocyanate anion.
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