Showing papers in "International Journal of Protein Research in 2009"

Study of hydrogen bonds in amino acids and peptides.

Abstract: An analysis of the various parameters associated with N-H… O type of hydrogen bonds has been made using data from reported crystal structures of amino acids and simple peptides. The different parameters at the donor and the acceptor ends have been suitably defined and evaluated. In some cases the analysis is done depending upon the chargedness or other characteristics of the donor and acceptor groups. Histograms giving the distribution of these parameters have been drawn and possible conclusions arrived at: 1. The distribution shows a maximum between 2.8 A and 2.9 A for the charged donor group and 2.9 A and 3.0 A for the uncharged donor group and is probably not dependent upon the charge on the acceptor group. 2. The angle between the directions CO and O. N tends to lie between two cones about C O with semi-vertical angles 40 and 70°. The orientation of the directions O. N and O. H with respect to the lone pair orbital directions on the acceptor oxygen atoms are analysed in detail using spherical polar coordinates. The analysis indicates that the group NH has a very strong tendency to point towards the acceptor oxygen atom. A general feature has been found in that the direction N-H tends to be closer to an orbital if the oxygen is an acceptor of two hydrogen bonds, while the direction tends to lie in between the orbitals when the acceptor oxygen is the receipient of only one hydrogen bond. The possible explanation of this on the basis of lone pair interaction is briefly discussed.

Thermophilic aminopeptidases from bacillus stearothermophilus. i. isolation, specificity, and general properties of the thermostable aminopeptidase i *

Abstract: A thermostable aminopeptidase (AP 1) from homogenates of B. stearothermo-philus was purified by filtration on Sephadex G-150, by heat treatment, by chromatography on DEAE Sephadex and Sephadex G-200, and by preparative polyacrylamide gel electrophoresis. The enzyme shows one band in polyacrylamide gel electrophoresis and has a pH optimum between 7.5 and 8 for LNA and between 9.2 and 9.4 for Gly-Leu-Tyr. One striking feature in the amino-acid composition of AP I in comparison with the data of leucinaminopeptidase is the larger proportion of hydrophobic amino-acid residues and of glycine. As regards its specificity, AP I differs from other amino-peptidases in certain important respects. The activation energy for the hydrolysis of Leu-Gly was calculated to be 16,300 cal/M-1. The enzyme remains stable for several hours at 80 C; after 30 min at this temperature the activity increases by about 20 per cent (Vm increases, while Km remains the same). The stability of the enzyme in urea, dodecyl sulphate, and various alcohols was studied. The activity of the enzyme even increases in 10 per cent tertiary butanol, in the same way as after heat treatment (Vm increases, while Km remains the same). The kinetics of the increase in activity following heat treatment or in 10 per cent tertiary butanol are discussed. At 20°C, AP I exhibits a sigmoidal dependence of reaction velocity on the concentration of LNA whereas at 40°C or 80°C the curves show a hyperbolic behaviour. In the metal-enzyme complex of AP I the Co2+ ions are strongly bound at pH 8 (up to 10-2MEDTA). At a pH lower than 6 the stable apo-enzyme is formed in 10-2M EDTA. The apo-enzyme can be reactivated to the holo-enzyme by adding Co-2. The metal-enzyme complexes of various metals show different substrate specificities.

Studies on the conformation of amino acids. VI. Conformation of the proline ring as observed in crystal structures of amino acids and peptides.

Abstract: The paper deals with an analysis of the available crystal structure data related to proline compounds so as to obtain information about bond lengths, bond angles, and the conformation of the pyrrolidine ring. The interesting results are: 1. The atoms Cβ, Cα, N, and Cs are nearly coplanar, with the torsion angle 0 about the Cα - N bond varying from about -15° to -15°. The Cγ atom is displaced from this plane, either up or down, so that the ring exists in one of the two puckered conformations, designated A and B. Conformation A is characterized by negative and may be termed Cγ-exo when referred to the displacement of the carbonyl carbon C. Conformation B has positive x l and is Cγ-endo; Cγ-exo is slightly preferred over C-endo, although both conformations occur simultaneously in some crystal structures with partial probabilities. In the other structures, the non-occurring position for C y is found to be disallowed by intermolecular contacts. The proline conformations observed correspond to the 'envelope' type of conformation of the cyclopentane ring. In peptides, the three bonds at N are nearly coplanar, and the torsion about N- Cα bond is nearly - 60°. 2. The observed ranges of (x 1 , x 2 , x 3 , x 4 ) are (0 to –30°, 15 to 50°, –15 to - -30°, 5 to 25°) for conformation A and (20 to 35 0 , -30 to - 40 0 , 20 to 35°, 5 to -20°) for conformation B; for θ and φ the ranges are -15° to -15°, -45 to -75°. The bond lengths and bond angles are not influenced by the conformation of the ring, unlike ribose.

The reduction of protein disulfide bonds in the absence of denaturants.

Abstract: The ability of dithiothreitol to effect the reduction of the disulfide bonds in five proteins (lysozyme, ribonuclease, bovine serum albumin, insulin, and ovine prolactin) under mild conditions and in the absence of denaturant has been examined In general, reduction was carried out for one hour at room temperature in 01 M KCl, pH 81, using dithiothreitol in a twenty molar excess over protein disulfide content The extent of reduction was evaluated both by alkylation of the reduced protein with iodoacetamide followed by amino-acid analysis and by direct assay of the sulfhydryl content of the reduced protein using 5,5′-dithiobis-(2-nitrobenzoic acid) Lysozyme, prolactin, and insulin appeared to be quantitatively reduced Fourteen of the eighteen disulfide bonds in bovine serum albumin were cleaved, whereas ribonuclease was not reduced to any significant extent under these conditions

Preparation of some new biphenylisopropyloxycarbonyl amino acids and their application to the solid phase synthesis of a tryptophan‐containing heptapeptide of bovine parathyroid hormone

Abstract: Several new crystalline 2-p-biphenyl-2-propyloxycarbonyl (Bpoc) amino acids were synthesized and characterized. They were found to he stable for months as free acids if kept cold and dry. The Bpoc group could be quantitatively removed by anhydrous 0.5% trifluoroacetic acid in methylene chloride within 10 minutes, under conditions where side chain N-benzyloxycarbonyl groups, O-benzyl ethers, or benzyl esters were completely stable. The tryptophan-containing peptide, H-Glu-Arg-Val-Glu-Trp-Leu-Arg-OH, of bovine parathyroid hormone, was synthesized in good yield by the automated solid phase method using the Bpoc-amino acids.

Studies on the denaturation of tropomyosin and light meromyosin

Abstract: Optical rotatory dispersion has been used to study the heat denaturation of tropomyosin and light meromyosin fraction 1 (LMMFr I) as a function of pH and salt concentration. The unfolding of these highly a-helical molecules takes place in stages indicating regions of varying stability along the molecule. At all pH values NaCl and KCl increase the stability and this is attributed to electrostatic screening of charged groups. There is a decrease in stability to heat at alkaline pH values and the molecules are most stable on the acid side of the iso-electric point. Guanidine hydrochloride is more effective than urea as a denaturant. In the case of tropomyosin almost complete reversibility of the optical rotation changes by these denaturants can be achieved provided the -SH groups are prevented from forming disulphide bonds. For LMMFr I the rotation changes and reversibility are complicated by depolymerization into smaller units. Molecular weight determinations by sedimentation equilibrium indicate a critical urea concentration for depolymerization which is not evident from the optical rotation changes. Although guanidine hydrochloride is more effective than urea in abolishing the helical structure of LMM Fr 1, it is not as effective as a depolymerizing agent.

The solid-phase synthesis of alpha-melanotropin

Abstract: The tridecapeptide amide, α-melanotropin, has been synthesized by the solid-phase method. The protected tridecapeptide was cleaved from a phenolic resin with ammonia, and the amide was reduced with sodium in liquid ammonia. Chromatographic purification gave the highly purified and fully active hormone.

Mouse insulins--separation and structures.

Abstract: Two major components were detected by disc electrophoresis in twice crystallized mouse insulin. Gel filtration experiments indicated that the two components were two insulins rather than proinsulin and insulin. The components were separated by ion-exchange chromatography, and amino acid analysis showed both components to be insulin, differing in two amino acids. The mouse insulins were named Mouse I and Mouse II insulin by analogy with Rat I and Rat II insulin in accordance with the electrophoretic mobilities. Performic acid oxidation followed by separation of the A and B chains, and amino acid analysis of the separated chains showed both differences to be present in the B chains whereas the A chains were identical. Trypsinization of the B chains followed by separation of the tryptic peptides and amino acid analysis of the peptides yielded the following results: B 1 to B 3 were identical. B 4 to B 22 differed in one amino acid, mouse I having one proline and no serine, mouse II having one serine and no proline. Finally, free serine B 30 plus a heptapeptide (B 23-B 29) containing one lysine and no methionine was obtained from mouse I, whereas mouse II yielded an octapeptide (B 23-B 30) containing one methionine and no lysine. Mouse II insulin displayed amino acid compositions identical with those of rat II insulin in regard to the A chain, the B chain, and the tryptic peptides from the B chain.

L-5-hydroxylysine as a constituent of the shell membranes of the hen's egg.

Abstract: Hydroxylysine was detected in hydrolysates of the inner and outer membranes of the shell of the hen's egg by ion-exchange and paper chromatography. N-ɛ-DNP-hydroxy-lysine was detected by paper chromatography of hydrolysates of dinitrophenylated membranes. Complete amino acid analysis showed hydroxylysine to be the least abundant of the known amino acids in the membranes (1-2 moles/1(fig protein).

Improvement to the esterification procedure used in solid phase peptide synthesis

Abstract: A new procedure of esterificalion of N-protected amino acids to chloromethyl polymers using tetramethyl ammonium hydroxide as a base has been developed. Elemental nitrogen and amino acid analysis of the polymers are in good agreement.

Hydrogen ion titration of ionizable side‐chains in native and denatured glyci.nin

Abstract: Electrometric and spectrophotometric titration curves of glycinin were obtained at 0.4 ionic strength and in the presence of 6M guanidine hydrochloride and 6M urea. The electrometric hydrogen ion titration curves were irreversible in the pH range 6.5 to 12.0 in 0.4M KCl, but exhibited reversibility in the pH 2.0 to pH 6.5 range. The forward and backward electrometric titration curves were entirely reversible in 6M guanidine hydrochloride. The spectrophotometric titration curves of tyrosine groups were also irreversible in 0.4M KCl, but reversible in 6M urea. Alkali-induced denaturation produced a different spectrophotometric titration curve than the curves obtained with glycinin in 0.4M KCl and 6M urea. The electrostatic factor w was lower in the pH 2.50 to 3.75 range than in the pH 3.75 to 6.50 range. This was interpreted to be due to swelling and dissociation into subunits of the glycinin molecule subjected to acid denaturation. The empirical value of w=0.045 derived from the titration of the carboxyl groups in the pH 3.75 to 6.50 range coincided with the theoretically calculated value of 0.040 for a swollen sphere. Group counting and pK values of carboxyl, imidazole, e-amino, and phenoxy groups under normal and denaturing conditions are also reported.

Dye-sensitized selective photo-oxidation of cysteine.

Abstract: The phtalein derivative cresol red and the fuchsin dye crystal violet have been found to sensitize the selective photo-oxidation of cysteine, both free and incorporated into a protein. The specificity for cysteine of these dyes was checked in aqueous solution over the pH range 2.5-9, as well as in aqueous acetic acid. In acidic solutions, cysteine is quantitatively converted to cysteic acid; on the contrary, in neutral or alkaline media, the photo-oxidative process is competed by a dark reaction, which causes the oxidation of cysteine to cystine. The irradiation of lysozyme, after reduction of a single disulphide bridge, in 16%acetic acid solution and in the presence of either sensitizer yielded a disulphonic derivative, which displayed about 40% enzymic activity. Moreover, fully reduced lysozyme, after photo-oxidation under the same conditions, appeared to contain eight cysteic acid residues per protein molecule and was devoid of enzymic activity. Therefore, this method appears to be suitable for the selective and quantitative modification of the cysteinyl residues in biologically active polypeptides and for investigating their importance for the biological function.

studies on the conformation of amino acids. V. Conformation of amino acids with δ-atoms

Abstract: Preferred conformations of the backbone and side-chain of the amino acids, norvaline, leucine, phenylalanine, tyrosine, tryptophan, and histidine obtained by calculating the energies of conformations are reported. The conformations of non-aromatic side groups are restricted to only certain values of χ1 and χ2, and the backbone conformation is the same as shown for aminobutyric acid having a single γ-methyl group. The aromatic side groups prefer a perpendicular orientation about the bond Cβ-Cγ and deviations from this orientation are characteristic of the position of the γ-atom. The experimentally observed conformations of amino acids with S-atoms and aromatic groups in the side-chain are compared with the theoretical predictions and the agreement is found to be excellent.

The destruction of serine and threonine during acid hydrolysis.

Abstract: Bovine submaxillary mucin (a glycoprotein) was hydrolyzed for varous times (1 to 300 hr) at 110° in 6 N HCl (0.3 mg protein per ml). The kinetics of destruction of serine were found to be first-order and that of threonine to be zero-order. The velocity constant for the destruction of serine was k – 4.3 X 10-3 hr-1 and of threonine was k – 5.6 X 10-4 mole hr1. The amounts of serine and threonine destroyed after 22 hours hydrolysis were 10.0 % and 2.7 %, respectively. Since the losses were the same in the absence of carbohydrates and since several prior extensive investigations reported similar corrections for serine, these corrections may be applicable to most proteins and glycoproteins.

Denaturation of tropomyosin by guanidine hydrochloride.

Abstract: The denaturation of tropomyosin by guanidine hydrochloride has been studied as a function of denaturant concentration and temperature. Denaturation involves both loss of a helix, which was followed by optical rotatory dispersion, and change in molecular weight. The transition between the native and unfolded states is reversible and the optical rotation results were shown to fulfil some of the criteria for a two-state process. When the molecule is more than half-unfolded, subunits of molecular weight 34,000 appear in solution. However the appearance of subunits does not parallel the unfolding as determined by optical rotation, indicating that the whole process cannot be considered as a two-state one. The molecular weight in the transition region is very dependent on temperature, with indications of a reversible equilibrium between partly unfolded monomer and subunits. The system in this region of guanidine hydrochloride concentration did not show dependence of molecular weight or optical rotation on protein concentration as might be expected for a conformation-linked association. Guanidine thiocyanate is a more effective denaturant than the chloride for tropomyosin whereas the carbonate and sulphate are relatively ineffective.

The role of disulfide bonds in the protein structure. Conformational studies on reduced ribonuclease and lysozyme.

Abstract: Circular dichroism spectra in different media of native, partially reduced, and fully reduced rihonuclease and lysozyme are reported and discussed with reference to the role of the disulfide linkages in maintaining the tertiary structure of the native enzymes. The results obtained indicate a large disorganization of the native molecules when all disulfide bridges are broken even if in both fully reduced proteins there appears to be some residual non-covalent structure. However, a tendency toward the partial recovery of the native structure is shown by the reduced proteins on decreasing the temperature or in the presence of methanol. In addition it has been found that some particular disulfide linkages are unessential for achieving the overall conformation of the molecules.

ISOLATION OF NON‐IDENTICAL POLYPEPTIDE CHAINS OF α‐CRYSTALLIN

Abstract: α-Crystallin–a water-soluble lens protein–was dissociated into subunits by means of urea treatment at pH 3. The dissociated protein was resolved into three distinct polypeptide species by chromatography on SE-Sephadex columns at pH 3.2 equilibrated with 7 M urea. In the presence of urea the isolated polypeptides, designated as I-a, I-b, and II, have different electrophoretic mobilities at acid and alkaline pH. It could be demonstrated that two cysteine residues are present in polypeptide I-a whereas cysteine is absent in polypeptide II. Moreover, amino-acid analysis reveals that both polypeptides have a different amino-acid composition. The third polypeptide has shown to be a dimer of one of the polypeptides. This dimer arises during isolation by disulphide bond formation. The results indicate that a-crystallin is composed of two distinctly different kinds of subunits. Evidence is presented that both types are built up by two different polypeptide chains.

The primary structure of the alpha and beta polypeptide chains of adult hemoglobin of the Rhesus monkey (Macaca mulatta). Biochemical studies on hemoglobins and myoglobins. IV.

Abstract: Countercurrent distribution separated rhesus monkey hemoglobin into α and β polypeptide chains, which were respectively oxidized with performic acid, dissolved in 8 M urea to be denatured at 60° for 45 min, and then digested with pepsin at 37° for 2 hours. Peptic peptides thus obtained from each polypeptide chain were isolated and purified by column- and by paper chromatography, and the amino acid composition of each peptide was analyzed. Sequence analysis was carried out by using PTC and DNP methods, if necessary. From the results thus obtained, the present authors established the primary structure of rhesus monkey hemoglobin, taking account of the amino acid sequence in all the tryptic peptides from the α and β polypeptide chains previously reported.

On the conformation of bovine serum albumin after alkaline or thermal denaturation

Abstract: An α-helix to β-form transition has been suggested in bovine serum albumin upon irreversible denaturation by alkali or heat. Since the optical properties of the β-con-formation are better known at present a renewed study was made of the structure of bovine serum albumin after alkaline or thermal denaturation by means of optical rotatory dispersion and infrared absorption measurements. Electrophoresis on poly-acrylamide gels and gelfihration on Sephadex G-100 or G-I50 were employed to investigate the extent of aggregation ami the kind of binding which stabilizes the aggregates formed after denaturation by heat. The results indicate that the α-helical conformation of bovine serum albumin is rather resistant to denaturation. Only a partial transition from α-helix to random coil was found without the introduction of a new conformation. This transition seemed to be at least partially reversible upon cooling after thermal denaturation or upon neutralization after alkaline denaturation.

Molecular forces in protein structure and crystallography

Abstract: The paper deals with the nature of the intermolecular forces and the laws governing these, which play an important part in the stability of molecular crystals and in the conformation of biological macromolecules. The method of contact criteria which give the disallowed ranges of conformations is briefly discussed and then the energy criteria are considered. Intermolecular potential functions for non-bonded interactions (both van der Waals and repulsive), electrostatic forces, hydrogen bonds, and hydrophobic interactions are discussed, followed by a treatment of intramolecular distortions of bond lengths, bond angles, and torsional angles. The last of these are important for the study of conformations. The possibility of non-planar distortion of peptide units is specially discussed. Application of these ideas to the calculation of the structures of molecular crystals is indicated.

Structural changes involved in the folding of proinsulin

Abstract: The circular dichroism spectra of reduced proinsulin (precursor of proinsulin), reduced insulin (A plus B chain), and C-peptide, all of bovine origin, were recorded The difference spectrum between reduced proinsulin and the sum of reduced insulin and C-peptide was constructed The difference spectrum indicates the presence of structures which may be those necessary to keep the proinsulin precursor in position for correct formation of the three cystine bridges of proinsulin and insulin The difference spectrum can be interpreted as being due to approximately 6 additional amino acids in theα-helix and 9 additional amino acids in theβ-structure in reduced proinsulin, all 15 being in random coil state in the separated chains

A study of the thermal denaturation of ribonuclease by differential scanning calorimetry.

Abstract: A study by differential scanning calorimetry (DSC) of ribonuclease thermal denaturation in water, 1.5, 2.5, and 4M aqueous urea, and in 1M and 2M aqueous hexame-thylenetetramine has been carried out. Application of the DSC technique to this type of investigation appears a useful one, in our experience. Our results indicate that whereas urea only slightly changes enthalpy variation accompanying denaturation but lowers the melting temperature of ribonuclease, hexamethylenetetramine does not effect either factor. According to these results, which are beyond experimental errors, the ability of hexamethylenetetramine in influencing hydrophobic-forces-driven phenomena (Barone, Crescenzi & Quadrifoglio 1967) has thus apparently no counterpart in any important action of this compound on the stability of a partially hydrophobic protein in aqueous solution.

The conformation of the high-sulphur proteins of wool. I. The preparation and properties of a water-soluble metakeratin.

Abstract: Whereas the S-cyanoethyl derivatives of the high-sulphur kerateines extracted from Merino wool were virtually insoluble in aqueous buffer solutions, the disulphide form of a high-sulphur fraction designated Metakeratin-B(5,0-8+4) (M-B(5.0-8+4)), obtained by dialysing the corresponding kerateine fraction in the presence of oxygen, was soluble in aqueous buffer solutions at pH 8.0. The material was highly aggregated in solution and could be partially disaggregated by 8 M urea. Gel filtration of the meta-keratin fraction in 8 M urea on Sephadex G-200 indicated that the molecular weights ranged from values exceeding 100,000 (excluded from column) to about 10,000. The latter value was confirmed by sedimentation measurements. When reduced and alkylated with iodoacetate the S-carboxymethylkerateine fraction so formed (SCMK-5(5.o 8.4)) contained chiefly components with molecular weights of about 10,000 as determined by gel filtration. M-B(5,0-8.4) showed a minimum solubility at about pH 4.4 in salt solutions, the pH of minimum solubility decreasing to about 3.0 with progressive S-carboxymethylation of the protein. No evidence could be obtained for the presence in M-B(5.0.8.4) of asymmetrical disulphide groups of the form CyS-S-CH2COOH. The incorporation of 8 M urea in solutions of the metakeratin at pH 8 gave rise to a positive difference spectrum, suggesting that few if any additional tyrosine residues become accessible in this solvent. Optical rotatory dispersion measurements gave a value of–50 for the parameter b0 in the Moffitt-Yang equation. In the presence of 8 M urea the value of b0 became zero. M-B(5,0-8.4) was digested relatively slowly by Pronase P but the initial rate of digestion increased greatly as the disulphide bonds were converted progressively to S-carboxy-methyl groups, the greatest change in rate occurring over the range 10 to 20% conversion of the cystine residues to S-carboxymethylcysteine residues. The wavelength of maximum fluorescence of M-B(5.0-8.4) was unaffected by the S-carboxymethylation but the intensity of the fluorescence was greatly increased by this modification. The ultraviolet absorption curve for the metakeratin showed absorption maxima at about 276 and 330 nm. With progressive S-carboxymethylation the ab-sorbance over the whole range of wavelengths decreased, the absorbance peak at about 330 nm disappeared, and an additional peak at about 280 nm appeared. The results suggest that the reduced chains of the high-sulphur protein fraction from wool, when allowed to oxidize to the disulphide form, tend to fold in a manner resembling native globular proteins. The unit structures so formed appear to be stabilized by intrachain disulphide bonds.

Correlations between amino acid sequence and conformation of immunoglobulin light chains. I. Hydrophobicity and fractional charge.

Abstract: The significance of nonpolar and of charged residues for the corrformation of light chains is discussed, based on the amino acid sequence of 15 light chains and on the amino acid composition of light chains from I1 species. The nonpolar residues contribute to the conformational stability by hydrophobic interactions with an average hydrophobicity of about 1000 caliresictue; this value is essentially independent of isotypic and ictiotypic sequence variability, and is similar to that for other globular proteins. While ihe total h.vdrophobicity of many globular proteins containing no SS bridges compensates afniost exclusively for their cotiforma- tional entropy, a significant discrepancy is observed in the case of light chains. This rnav rejlect the loss of conformational entropy clue to ihe two SS bridges forming independent covalent loops. The ioni7cd residue;, give a fractional charge close to 0.20 unitsiresirlue. This relatively low value, together with the relatively high average hydrophobicity, cxplains qualitatively many of the a5sociation and thermal properties of light chains. Variable and constant regions of light chains exhibit nearly identical average hydrophobicities and fracfional charges, suggesting that the molecules are folded into two distinct structural subunits of compact conformation. Some preliirrirrary results on heavy chains are also considered: The fructional charge of the Fdjragments are much lowcr than that of the Fc fragments. This may fucilitate the intimate contact oj the light chain with the former fragment in the intact antibody molecule.

Correlations between amino acid sequence and conformation of immunoglobulin light chains. II. Sequence comparison and the pattern of nonpolar residues.

Abstract: A detailed comparison of the primary structure of 15 individual immunoglobulin light chains including 6 human k chains, 2 mouse k chains, and 7 human. chains is given. Their chemical similarities and the possible conformational influence of the invariant residues are discussed. Some preliminary results on heavy chains are also considered. The most striking structural feature common to all L chains examined is the pattern of distribution of nonpolar, but not of polar or ionized residues along the sequences. Both variable (VL) and constant (CL) regions of L chains contain approximately 55 nonpolar residues. Out of these, 30 nonpolar residues are found in all VL, regions at structurally identical sequence sites and 33 in all CL regions. This frequency of consistently nonpolar residues is similar to that in globin chains and is probably due to thermodynamical and stereochemical requirements. The globular folding of L chains is essentially stabilized by hydrophobic interactions and most of the consistently nonpolar sequence positions are likely to represent intrachain hydrophobic contact sites. These, however, require well-defined distances between the interacting groups. There is no significant similarity between the pattern of nonpolar residues of the VL, and Ch regions. However, the rule of isopolar substitution suggests a similar folding of all VL, (and possibly Vrd regions, on the one hand, and a similar folding of all CL (and possibly CH) regions, on the other. The low a-helical content of L chains is explained, and some conclusions concerning future structural studies are drawn.

Human ceruloplasmin as a polymorphic glycoprotein. Tryptic glycopeptides from two forms of the protein.

Abstract: Reduced and cyanoethylated human ceruloplasmin form I was freed of sialic acid and digested by trypsin. A glycopeptide fraction isolated by gel filtration was separated into three carbohydrate-containing fractions by ion exchange chromatography on Dowex I. Each of these was further separated by column electrophoresis to yield a unique glycopeptide. With a stoichiometry of two chains per mole, these peptides account reasonably well for all the carbohydrate in the molecule. In a corresponding preparation from ceruloplasmin form II with smaller carbohydrate content, two of the glycopeptides were, present in greatly reduced amounts. The three glycopeptides were obtained in a pure state from sialic acid-containing ceruloplasmin form I by gel filtration, ion exchange chromatography, and column electrophoresis. Two different kinds of oligosaccharide chains were found attached to the purified peptides. The first contained 6 hexose, 5 glucosamine, 2 sialic acid, and 0.5 fucose residues; the other about 9 hexose, 8 glucosamine, 4 sialic acid, and 0.5 fucose residues. According to the composition of the protein, the larger chain accounts only for a small part of the carbohydrate content. On the basis of the results, it is proposed that human ceruloplasmin has six carbohydrate chains, three on each of two identical or nearly identical halves of the molecule. The carbohydrate chains are found in two different degrees of completion. Form II of the protein lacks 2–3 of these chains, having two particular sites partly unsubstituted.