THE p-RIMARY STRUCTURE OF A HlJMAN LAHBDA TYPE IMMUNOGLOBULIN LIGHT CHAIN CONTAINING CARBOHYDRATE: BENCE JONES PROTEIN WHITE
by
HENRY MURRIEL PATTON, JR.
Submitted to the F~culty of the School of Graduate Studies
of the Medical College of Georgia in Partial Fulfillment
of the Requirements for the Degree of
Master of Science ,
June
1980 THE PRIMARY STRUCTURE OF A HUMAN LAMBDA TYPE IMMUNOGLOBULIN LIGHT CHAIN CONTAINING CARBOHYDRATE: BENCE JONES PROTEIN WHITE
This thesis submitted by Hen~y Murriel Patton, Jr. has been examined and approved by an appoint,ed committee of the faculty of the
School of Graduate Studies of. the Medical College of Georgia.
The signatures 'tvhich appear below v~rify the fact that all required changes have been incorporated and that the thesis has received final approval with reference to content, form and accuracy of pres~ntation.
This thesis is therefore accepted in partial fulfillment of the requirements .for the degree of Master of Science.
Date ACKNOWLEDGEMENT
I would like to express my appreciation for the assistance and guidance received from Dr. Gary K. Best, Dr. Cheruhanta N. Nair,
Dr. George So Schuster and Dr. Linda L. Smith, and especially th~ patience and understanding of Dr. Frederick A. Garver. A special thanks goes to my wife, Vicky, for her patience and encouragement. TABLE OF CONTENTS
INTRODUCTION PAGE
I. Statement of Problem 1
IIQ Review of the Related Literature 4
MATERIALS AND METHODS
I. Isolation of Bence Jones proteln WHITE from urine 13
II. Determination of purity 13
IIIe Serological typing of protein WHITE 13
IV. Determination of molecular weight 14
V. End group analysis 14
VI. Amino acid·anal'ysis 15
VII. Reduction and aminoethylation 15
VIII. Tryptic digestion 16
IX. Isolation of tryptic peptides 16
X. Sequence determination 18
RESULTS 22
DISCUSSION 51
SUMMARY 55
REFERENCES 56 LIST. OF. FIGURES
FIGURES PAGE
1 Tiselius and Kabat's experiment 5
2 Porter .1 s four chain antibOdy model· 7
3 Migration of common amino acids on polyamid.e sheet 20
4 Lambda chain subgroups 25
5 Chromatogram of tryptic peptides 26
6 Rechromatogram of peak a 27
7 Rechromatogram of peak b 27
8 Rechromatogram of peak c 28
9 Rechromatogram of peak d 28
10 Rechromatogram of peak e 29
11 Rechromatogram of peak f 29
12 Rechromatogram of peak ~ 30
13 Rechromatogram of peak h 30
14 Rechromatogram of peak i 31
15 Rechrbmatogram of peak l 31
16 Rechromatogram of peak k 32
17 Rechromatogram of peak 1 32
18 Rechromatogtam of peak m 33
19 Rechtomatogram of peak n 33
20 Rechromatogram of peak o 34
21 Chromatogram of thermolysin peptides of -T-3 39
22 Chromatogram of thermolysin peptides of T-15+6 42
23 Chromatogram of thermolysin peptides of T-18b+c 46
24 Primary structure of protein WHITE 52 LIST OF TABLES '
TABLE PAGE
1 Isotypic variants of lambda chains 11
2 Amino acid composition of WHITE 24
3 Amino acid composition of tryptic pep tides 35
4 Amino acid symbols 38
5 Amino acid composition of thermo lysin peptides of T-3 40
6 Amino-acid composition of thermo lysin peptides of 43 T-15+6
7 Amino acid composition of thermo lysin pep~ides of 47 T-18b+c INTRODUCTION
I. Statement 6f Problem
In the pa~t twenty-five years immunology has $rown into one of the most important disciplines of biological sciencee Researchers now
realize that an understanding of the body's defense system may be the key to such important problems as gene regulation in higher organisms, molecular recognition and· interaction, graft acceptance or reject~~on, and cancer origination and proliferation. Essential to the clarifica
tion of how the immune system_functions is a complete understanding of its key molecule the antibody.· Since normal individuals have heterogeneous mixtures of immunoglobulins, it is difficult to separate and purify them in sufficient quantities for investigation. Most of the progress in our understanding of the structural, physiological, and genetic properties of antibodies has come from t~e study of the
Bence Jones and other monoclonal proteins produced by neoplastic lympho- plasmacytes in patients with multiple myeloma and related plasma cell dyscrasias.
Recently a patient with multiple myeloma in Talmadge M·emoria 1 Hospi- tal was found to produce a Bence Jones protein. There are three main reasons why this particular protein, Bence Jones protein W~ITE, is of special significance. ~irst, it is important genetically. In contrast to all other protein families, immunoglobulins have a separate genetic con t ro 1 for both the co n.s tan t and v a r i ab 1 e regions • Wh i 1 e it is generally accepted that there is one gene responsible for each constant
region, there are several theories as to the ~enetic control of the variable part. The twO current theories are the somatic mutation theory and the multigene germ line theory. The Somatic mutation theor~ states
l 2 that there are a few stable germ lines and as the individual is exposed to the exogenous antigen, this antigen acts as a selective agent which stimulates the expansion of clones of cells synthesizing light-heavy pairs that react effectively with ~hat antigen. As a person's lympho cytes are stimulated they undergo s6matic mutation through deletion, insertion, or recombination and beneficial mutations are further
stimulated (1,2,3,4,5 1 6). The multigene germ line theory states that individuals have a separate germ line for each variable region that the person is capable of producing and that these multiple genes arose by normal processes of g~ne duplicati6n followed by mutation and selection
(7,8,9). On the basis of sequence homologies of the light chain variable regions that have been investigated so far., attempts have been made to group hum~n lambda ~ype light chains into four or five basic subgroups
(10, 11) •. As more human lambda type light chains such as WHITE are sequenced, they might shed light into the evolution of the greater variability of the human Lambda chain family. Protein WHITE is also important because it is onE; ·of the few lambda type Bence Jones proteins to be found that has carbohydrate attached to it. In 1970 Sox and Hood studied the amino acid sequence a_t the point· of ·attachment of carbohy drate to four kappa type light chains and one lambda type light chain
(12). In all five light chains thei found the same sequence of Asx
X-Ser/Thr, where X represents any amino acid. They postulated that this sequence was the recognition site for the transglyc~sylase which attaches the carbohydrate to the asparagyl residue. It has been suggested that the carbohydrate has something to do with the secretion of the antibody, but the actual function of the carbohydrate and its true influence on the antibody specificity is unknown at this time. Another interesting characteristic of protein WHITE is that it is a lambda type light chain. 3 Although extensive sequence and immunochemical data on kappa chains have been obtained, the data on human lambda chains are much more limited. It is the purpose of this thesis to define the primary structure of Benc.e Jones protein WHITE. 4 II. -Review of the Related Literature
It has been known for centuries that a person who survives a disease such as plague or smallpox is usually "immune" to a second infection, but a general understanding of how the immune system works is a relatively recent development (13)~ A key step was taken in 1890 when Emil Von Behring and Shibasaburo Kitasato demonstrated the develop ment of a specific, tra~sferable, neutralizing substance in the blood of animals immunized by injection .with tetanus toxin ( 14). In 1939
Tiselius and Kabat localized-this antibody activity of the blood to the gamma globulin ·el"ectrophoretic fraction of the plasma proteins (15)
[Figure 1]. The~ in 1955 Williams, Grabar, and Slater, using the immuno~lectrophotesis ·technique Poulik had introduced in 1952, showed that the principal gamma globt1lin.arc also extended over the alpha and beta globulin regions (16,17). This maJor gamma globulin arc is now known are Immunoglobulin G (IgG). Since that time the immuno chemical techniques that were pioneete~ by these men have been used to distinguish four other classes of immunoglobulins designated immuno globulin A (IgA), immunoglobulin M (IgM), immunoglobulin D (IgD), ··and immunoglobulin E (IgE) (18,19).
Human IgG is the major immunoglobulin class, comprisi.ng seventy per cent of serum immunoglobulin. Throughout early research the struc ture of IgG has been considered the prototype for the other forms of immunoglobulin. In 1959 R.R. Porter isolated IgG from the serum of rabbits immunized with egg albumin and digested the immunoglobulins with a plant enzyme, papain, hoping to fihd a smaller unit of mole~ule containing ant~body activity (20). He found that papain splits the molecule into three fragments: a fragment that forms crysta,ls (Fe) FIGURE 1: Tiselius and Kabat's ~1939 experiment showing that the anti body activity of blood· is located in the .gamma globulin electrophoretic fraction of the plasma proteins. El~ctrophoresis scale diagram of anti-egg albumin rabbit serum 431-5 1:4 before (a) and aeter (b) absorption of the antibody [From Tiselius and Kabat (15)]. 5
300 }J.
250 )J y
200 ).J .
150 )J
100 )J
-50 )J
0 ..__.,5~.~10--15_2..... 0~ .. ---25_3_0~. Scale reading (mm) · Z b --~---~----~--~
300 }J
2'50 )J
:. 2·00 }J
150 )J
I 00 }-J
50 }J
o~-5~~,o~~,s~.-.~2~0~2~5~3o~.~ Scale reading ( m m)
Figure l 6 and two fragments (Fab) that do not crystallize but contain the antigen binding sites. When IgG is split by the enzyme pepsin, the cleavage occurs on the other side of the interheavy chain disulfide bonds and
the Fab fragments remain joined together. Pep'sin cleavage results in
(Fab)2 fragments, peptides, and component II (F'c)o Combining his own data and the data Edelman and Paulik had accumulated while working with myeloma proteins, Porter in 1962 proposed a four chain model of the structure of IgG c?nsisting of t~o identical heavy chains (M.W.
\ 50,000) and two identical light chains (M.W. 23,000) (20,21). Porter's model is shown in Figure 2.
Sequence analyses of myeloma proteins and Waldenstrom macroglobulins have shown that each heavy polypeptide chain is divided into two regions, the variable (V) region and the constant (C) region (22,23,24,25). The variable region of y chains consists of the .first 121 amino acids and contributes significantly to the antibody diversity. The constant region consists of the remaining 325 amino acid residues and determine$ the class, subclass, and biological activities of each antibody (26,27,28,
29). Heavy chains have been distinguished antigenically as y, a, ~' 1, and € chains for IgG, IgA, IgM, IgD, and IgE, respectively (30,31,32).
In addition, four subclasses of immunoglobulin G (IgGl, IgG2, IgG3,
IgG4) and two subclasses of immunoglobulin A (IgAl, IgA2 ) have been detected irnmunochernically (26,27,28,29). There is no conclusive evidence forth~ existence of subclasses of human IgM (33). Each heavy chain has· four homologous r~gions, consisting of between 110 to 120 amino acid residues, called do~ains. The constant region of the heavy chain is compose~ of three domains (CHl, CH2, CH3) (34,35,36,37). The variable region of the heavy chain is composed of one domain (either VHl, VH2, ~IGURE 2: Porter's hypothetical four chain antibody molecule showing sites of cleavage with papain and pepsin. [From: Principles in Hodern Immunobiology. (Edited by B.H. Park and R..A. Good), Lea and .Febiger, Philadelphia 1974, p. 92]. 7
MAJOR PAPAIN CLEAVAGES Fab+Fc+F'c Fob + ++I I. I. s,.._-...... ,. s sI sI. sI · F'c Fd I Fe s, I . I I I I I II , I I I s-,.,;.,....;-s s s ss 1 s s .S N -terminal _..___. C-terminal 5 s s ~ s s I I T I I' I I ?· f I . I sI I s s Component II I sI I s .I ... :I
MAJOR PEPSIN CLEAVAGES (Fab)2 + peptides +Component II
Figure 2 8 or VH3) (38). The first complete amino acid sequence of a human
IgG heavy chain was reported by Edelman, et al. in 1969 (38).
In 1848 Henry Bence Jones, a physician at St. George's Hospital in London, described a protein occu~ring in the urine of a patient with mollities ossium (multiple myeloma) that precipitated on being heated to 45 0 C, redissolved on boiling, and reprecipitated on cooling (39).
In the succeeding years some 700 papers were published concerning
Bence Jones proteins, but their relationship to the immune system remained obscure. The significance of this unusual urinary protein became apparent only fifteen years ago with Edelman and Gally's discoveri that Bence Jones proteins represent the free light polypeptide chains of immunoglobulins (40). Shortly afterwards, Hilschmann and Craig used two Bence Jones proteins to demonstrate that a light chain is composed of -two parts of approximately 107 amino acids each, the C-terminal half being identical in sequence in different light chains of the same type
(constant part), and theN-terminal half being different in length and seque~ce, but homologous (variable or va~iant ~art) (41). Light chains are now known to consist of two types, K (kappa) and L (lambda), named after Dr~. Korngold and Lipari who first distinguished them on the basis_of antigenic determinants (42). Both types of light chains are common to immunoglobulins of all five classes and each immuno- globulin molecule contains either two kappa light chains or two lambda light chains but never one of each. The ratio of kappa to lambda Bence
Jones pioteins in humans is about three to two (43). The same ratio is found in normal human immunoglobulins (44).
The variable region of immunoglobulin light and heavy chains may be divided into four framework (FR) and three complementarity-determining
(hypervariable) regions (CDR). In the variable r·egion of light chains, 9
FRl, FR2, FR3, and FR4 comprise residues 1~23, 35-49, 57-88, and 98-107, respectively, and CDRl, CDR2, and CDR3 comprise residues 24-34, 50-56, and 89-97, respectively (45). Based on sequence comparisons iri FRl, kappa light chains are subgrouped as either Kr, K11 , K111 , or Krv
(46-47). The frequency of occurrence of K1 , Krr' K111 , and Krv Bence
Jones proteins is approximately 60, 10, 28, and 2%, respectively, and reflects the proportion of these light chains among immun0globulins of normal serum (48). Even though fo~t distinct groups have been confirmed immunochernica1ly, intergroup and intragroup antigenic heterogeneity is evident (49,50,51,52,53). McLaughlin and Solomon, using imrnunochemi cal techniques that rapidly detect and localize differences in the vari able region of kappa light chains, have. found three distinct regions of the kappa variable region to be associated with specific antigenic sites (54). The residue at position 9 is associated with a group- related antigenic site. The residues at position 45 and positions 94 through 96 are associated with two distinct antigenic sites which are responsible for certain intergroup similarities and intragroup differences
(54). In addit~on to variable region determinants, the kappa chain constaht region exhibits an amino acid sequence variability at residue ,
191. This determinant is inherited in Mendelian \(allelic) fashion and thus falls into the category of genetic variation that Oudin termed
"allotypy" (55). Kappa light chains with leucine at position 191 are called Inv(l,2) and those with valine at posit{on 191 are Inv(3)
(56,57,58,59,60) .•
Sequence and immunochemical data on human lambda lig~t chains are much more limited than that for kappa chains. Just as kappa chains can be subgrouped, lambda chains fall into one of four basic categories 10 designated Ar, A11 , A111 , Arv' and, according to the World Health
Organization, Av (61,62). Although allotypic variants of human lambda chains have not yet been definitely identified, structural and immuno chemical intragroup and intergroup similarities have been demonstrated
(10,62,63,64,65,66). Seven variations in the amino acid sequence·of the lambda chain constant region have been noted and are presented in Table
1. These seven variants, Oz, Kern, Meg, Mz, Ev, Weir, and Way, are presumably present among the lambda chains of all ~ersons and thus are termed "isotypes" (67,68,69,70,71,72,73,74,75,76). Putnam, et al. have postulated that these variarits arose from gene duplication followed by mutation and represent separate gene products (77).
The most remarkable aspect of the immunological system is its ability to produce a vast number of diverse antibody molecules. Burnet, in his clonal selection theory, postulated that antibody heterogeneity is the result of diversity in the cells that produce the antibodies
(78). Several different hypDtheses have been developed to explain how this antibody variability takes place. Some have proposed a somatic hypermutation process of one, or a limited number of genes, while others have postulated separate genes ih the germ line"(2,79,80,81,82,83).
There are many arguments for and against all of these hypotheses (84,85,
86). The finding that chains of the same type ha~e identical sequences in the constant region but different sequences in the variable region seemed difficult to reconcile with the .doctrine that every polypeptide chain is coded for by one gene, and led Bennett and Dreyer to propose the "two gene-one polypeptide" hypothesis (64,83). This hypothesis suggests that there are separate structural genes coding for the variable and constant regions, and these two regions are joined togeth~r TABLE 1: Isotypic variants in the primary structure of the constant region of the lambda light chain. 11 '
factor Position Common Residue Variant Residue
MCG 114 Ala Asn
MCG 116 Ser Thr
MZ 145 Ala Val
EV 153 Asp Glu
KERN 154 Ser Gly
WEIR 158 Lys Glu
WAY 159 Ala Val
MCG 165 Thr Lys
MZ 173 Lys Asn
oz 191 Arg Lys
Table 1 12 . at some stage of synthesis. Several models have been proposed to
explain how the variable gene and the constant g.ene might be joined
at the DNA level (I,87,88,89,90,91)~ In the copy-inserti6n model,
a specific variable gene is copied and the copy is ~nserted at a site
next to the constant gene. In the somatic-translocat~on model, one
specific variable gene out of many closely associated var~able genes
is excised and then inserted adjacent to a constant gene. In the
deletion model, the DNA between a specific variable gene and a constant
gene is excised and removed so that previously distinct variable and
constant genes are united. In the inversion model, variable gertes and constant genes are arranged in opposite directions on a chromosome while a segment of chromosome between a specific variable gene and a
constant gene is then inverted. Although the current evidence supports
the somatic-translocation and invers~on models, Kabat has recently proposed a more complex system. He has suggested that the variable
region is not coded for by one gene but by several "minigenes" which
correspond to the various FR and CDR regions and operate through a random copy-insertion mechanism to produce a complete variable region
(45). There is a growing consensus that antibody diversity may actually
be a combination of several processes (111). Individual investigators, however, differ in their views of which of these processes is primary
and which is secondary. As more Bence Jones proteins are sequenced,
they should provide f,urther insight into the specific genetic mechamisms which account for the diversity of antibodies. MATERIALS AND METHODS
I. Isolation of Be·nce Jones Protein WHITE from Urine
Inez White was a patient in Talmadge Memorial Hospital with
multiple myelome. The Bence Jones protein was isolated from her
urine by salt precipitation with 65% ammonium sulfate. This precipi-
tate was dialyzed against distilled water for three days to remove
excess ammonium sulfate and then lyophilizedo
II. Determination of Purity
The purity of the lyophilized protein was ascertained by cellulose
acetate· electrophoresis according tb the procedure given in the Beckman
Model R-101 Microzone Electrophoresis Cell Instruction Manual. A
solution consisting of 2 mg. of protein in 0~4 ml. of 0.025M diethyl-
barbiturate-acetate buffer was applied to a cellulose acetate membrane
that had been pre-soaked in buffer. The protein-treated membrane was
mounted on a Beckman Model R-101 Microzone Electrophoresis apparatus
( and subjected to electrophoresis at 250 volts for 20 minutes. The
membrane was then stained, washed, and dried. at l00°C for 15 minutes •
. III. Serological Typing of Protein WHITE
The protein was serologically typed by the Ouchterlony method of
double diffusion precipitin reaction. Microscopic slides (Clay
Adams 50x75mm) were coated with a solution of 0.2io agarose in 0.025H
sodium diethylbarbiturate-acetate buffer. Four milliliters of a
heated solution of 1% agarose in 0.025M sodium diethylbarbiturate-acetate
buffer was then pipetted onto each slide and allowed to solidify.
Wells were cut into the a-garose with a Gelman Ouchterlony cutter. The
anti~erum (either anti-kappa or anti~lambda) was placed in the center
well and the samples (Bence Jones protein WHITE, lambda light chain,
13 14 and kappa light chain) were placed in the outer wells. The slidss
were placed in a closed container that was saturated with 0.025M
sodium diethylbarbiturate-acetate buffer and allowed to react for
twenty-four hours. After precipitin bands had formed, the slides were
washed in a 0.9% saline solution for three days to remove unpreci
pitated proteinso The slides were then placed in distilled water for
one hour, covered with a dampened absorbant wick, and dried at 37°C.
The slides were stained by placing them in 0.1% Amido-Schwarz dye for
_10 minutes, and finally destained with two 15 minute rinses·of 1%
acetic acid.
IV. Determination of the molecular Weight
The approximate molecular weight of Bence Jones protein White was determined by sedimintation velocity studies using a Spinco Model
E an~lytical ultracentrifuge and the Schlieran optical system.
Sedimintation velocity studies we~e performed at 25°C and pH 7.3 with 5 mg/ml of native protein in 0.1 M phosphate buffer, in 6 M
guanidine hydrochloride and in 6 M guanidine hydrochloride with 0.1 M
8-mercaptoethanol. Sedimentation coefficients were calculated from
the peak po·sitions and corrected to 20° in water. The approximate molecular weight of the reduc~d protein was determined by SDS
polyacrylamide gel electrophoresis using 10% gels in the presence
of 0.1% SDS as described by Weber and Osborn (92).
V. End Group Analysis
Analysis of the N-terminal end group was carried out according
to the dansylation procedure with subsequent identification of the dansyl
amino acid derivative by thepolyamide thin layer chromatography method
of Ponstingl, Hess, ~nd Hil~chmann (93). 15 The C-terminal end group was analyzed according to the procedure of Garver and Hilschmann, using carboxypeptidases A and B (94).
0.3~M of lyophilized peptide was dissolved in 2 ml. of O.OSM sodium phosphate buffer at pH 8.0o To this was added 500 A of carboxypep tidase A in phosphate buffer (0.25 mg. enzyme/ml.) and 25 A of carboxy peptidase B (6.1 mg./ml.)e This mixture was incubated in a 37°C water bath for 2 hours, stopped by lyophilization, and analyzed.
VI. Amino Acid Analysis
O.l~M of protein was hydrolyzed in constant boiling 6N hydrochloric acid for twe~ty-four hours at 110°C ... The hydrolys·ates were evaporated completely on a Buchler rotary evaporator and dried in a dessicator over sodium.hydroxide and calcium chloride. Amino acid analysis was then performed on a Beckman model 120-B au.tomatic amino acid analyzer.
To measure tryptophan, 2mg. of prot~in was dried under a vacuum for one hour at l00°C, cooled, weighed, and placed in a 16xl50 mm. test tube. 1.0 ml. of 3M p-toluene-sulphbnic acid containing 0.2% of 3-(2-aminoethyl)indole was added and the tube was evacuated and sealed by flame. After hydrolysis for twenty-four hours at 100°C, the tubes were opened and 2.0 ml. of 1M sodium hydroxide was added'! The solution was transferred to a 5 ml. volumetric flask and made up to 5.0 ml. with aqueous washings of the hydrolysis tube. The mixture was filtered through a 0.22~ Millipore filter and analyzed directly on the amino acid analyzer.
VII. Reduction and Aminoethylation
Protein WHITE was reduced and aminoethylated according to the method of Raftery and Cole (95). 2.0 grams of protein were dissolved in 100 ml. of 8M urea. 40 ml. of 4M propandiol buffer was added under a nitrogen atmosphere with constant stirring. The solution was then 16
reduced with 2.0 ml. of 2-mercaptoethanol by allowing the reaction to
continue for four hours at pH 8.6o At the end of the reaction the pro
tein was alkylated by adding 2.5 ml. of ethylenimine three times at
ten minute intervals. Ten minutes after the last addition, the reaction
was stopped by bringing the pH to 5.0 with concentrated glacial acetic
acid. The sample was then dialyzed against distilled water for three
days and lyophilized.
VIII. Tryptic Digestion
The r~duced and aminoethylated lyophilized protein was suspended
in 100 ml. of distilled water in a digestion vessel enclosed in a
circulating water jacket maintained at 37°C. The pH was adjusted to and
maintained at 8.0 with 0.5M sodium hydroxide throughout the reaction
while nitrogen was continuously·flushed through the vessel. 30 mg. of
TPCK-treated trypsin was added and the reaction was allowed to proceed
for four hours. The reaction was stopped by raising the pH to 9.5
with dilute sodium hydroxide.
IX. Isolation of Tryptic Peptides
The digested protein was put on a Dowex 1X2 anion exchange column
(25xl50cm) that had been previously equilibrated by washing for twenty
four hours with an initial buffer of 1% pyridine, 1% 2-picoline, and
1% 2-4 lutidine, adjusted to pH 8.5 with glacial acetic acid.
Fractions were collected at the rate of 10 ml./tube over a pH gradient
from 8.5 to 0.5. Aliquots of 100 lambda from each fraction were pipetted
into Nalgene tubes and subjected to alkaline hydrolysis by adding
0.5 ml. of 2.5N sodium hydroxide and heating to 100°C for two hours.
The cooled samples were neutralized with 0.5 ml. of 30% acetic acid •
. Ninhydrin (0.3 ml./tube) was added and then developed by heating the 17 tubes in a boiling water bath for 15 minutes (96). After cooling,
1.0 mle of 50% ethanol was added to each tube. Optical density was
read at a wavelength of 570 namometers on a Beckman model 24 spectro
photometer, and absorbance peaks were plotted on graph paper. The
fractions were pooled according to their peaks, dried on a Buchler
rotary evaporator, and stored in the freezer at -20°C. Pooled
fractions were then pipetted onto chromatographic paper in 15 A
aliquots. The spotted paper was placed into a chromatography tank with a solvent system of 4 butanol: 1 glacial acetic acid: 5
distilled water and allowed to develop for approximately twenty-four
hours. After drying, the developed chromatograms were sprayed with
ninhydrin and other sprays to detect arginine, tryptophan, tyrosine,
histidine, and sulfur compounds. Peptides containing arginine appeared
as red spots after sprayin~ the chromatogram with 0.1% 8-hydroxy
quinoline in acetone, drying at room temperature, and spraying with
sodium hypobromite (97). Tryptophan was indicated by purple spots
that appeared after sp~aying the chromatogram with a solution of 1% -
dimethylaminobenzalde'hyde and allowing it to dry at room temperature
(98). Tyrosine peptides were detected by the presence of red spots
after spraying the chromatogram with 0.1% alcoholic alpha-nitroso-
beta nephthol solution, drying the room temperature, spray~ng with
10% nitric acid, and drying at l00°C (99). His~idine peptides were
located by spraying the chromatogram with a mixture of 1% amylnitrite
in ethanol, allowing the sprayed paper to dry at room temperature, and
spraying with 1% pdtass~um hydroxide in ethanol. Red spots indicated histidine (100). Peptides with sulfur cofupounds were found by spraying with a solution containing 4 ml. of 0.002M hexachloro-platinic acid,
0.25 ml. of O.lN potassium iodide, 0.4 ml. of 2N hydrochloric acid, 18 and 76 rnl~ of acetone and then drying at room temperature. White spots on a pink background indicated sulfur containing peptides (101).
Pooled fractions that gave single spots with ninhydrin spray were considered pure and subjected to ~rnino acid analysise The impure pooled fractions that had· eluted from the original Dowex 1X2 column above pH
4 were rechrornatographed on Dowex 50X2 colu~ns (Oe9 x 25 ern). The
Dowex 50X2 resin was equilibrated by washing for twenty-four hours with an initial buffer of pH 2.2 (1292 rnl. distilled water, 8 rnl. pyridine,
100 rnl. formic acid, 600 rnl. propanol). Before the sample was applied, the Dowex 50X2 column was washed with 10 rnl. of 50% formic acid. The sample was dissolved in 50% formic acid and then put on the column and a pH gradient from 2o2 to 5.5 was started. After a pH of 5.5 was main tained for one hour, the column was washed with a 0.2N sodium hydroxide solution at pH 12Q .Fractions were again subjected to ninhydrin, plotted and pooled. Impure pool~d f~actions that had eluted from the original
Dowex 1X2. column below pH 4 were rechrornatogra,phed on a smaller Dowex
1X2 column (0.9 x 25 ern). The procedure was the same as for the original
Dowex 1X2 column except that the buffers contained 30% propanol for better ieparation and increased solubility of acidic peptides. Pooled rechrornatographed fractions that still were not pure after Dowex 50X2 or Dowex 1X2 with 30% propanol buffers were then put on a Sephadex column and separated by gel filtration. Pure peptides were applied to the amino acid analyzer and their analysis results were used to identify each tryptic peptide.
X. Sequence Determination
Sequential Edman degradation of the peptide followed by dansyla tion of the newly exposed N-term~nal residue was performed (102).
0.2~M of purified peptide was dried in the dessicator over sodium 19 hydroxide and sulfuric acid under a vacuum and an infrared heat lamp.
150 ~ of distilled water was added and 10 ~ was removed for dansylation.
Edman degradation was performed on the remaining 140 A by adding 150 ~ of 5% phenylisothiocyanate tn sequenol grade pyridine. The phenyl isothiocyanate was then coupled with the N-terminal amino acid by incubating the mixture at 45°C under nit~ogen for 90 minutes. The mixture was dried in the dessicator under vacuum and infrared light over sodium hydroxide and sulfuric acid. The phenylthiohydantoin amino acid derivative was then cleaved from the peptide by adding 200
A of trifluoroacetic acid and incub~ting at 45°C for 30 minutes under a nitrogen atmosphere. The cleaved phenylthiohydantoin amino acid derivative was extracted with 500 A of N-butyl acetate and discarded.
The remaining peptide was then ready for dansylation of the newly exposed N-terminus.
Dansylation was performed by placing 10 A of peptide in a pyrex tube (5X50mrn) and drying in the dessicator under vacuum and infared light over sodium hydroxide and sulfuric acid. The pH was adjusted to 10 with
0.2M sodium bicarbonate, and the peptide wa~ dried again. After adding
10 A of distilled water and 10 A of dansyl chloride (2.5 mg./ml. of ace tone), the mixture was incubated at 37°C for one hour and dried. The dansyl peptide was then hydrolyzed by adding 50 A of 6N hydrochloric acid and heating to 110°C for 18 hours under vacuum. Hydrolysis time was shortened to 5 hours when serine or proline were expected. After hydrolysis, the sample was dried and the dansyl amino acids were identified by thin layer chromatography on polyamide sheets as described by Woods and Wang [Figure 3] (103). Those peptides too long to be sequenced entirely were subdigested with thermolysin, chrornatographed, FIGUR~ 3: Separation of common amino acids by thin layer chromato graphy on polyamide sheets as described by Woods and Wang (103). 20
<::::::> E-Lys -:2 '0~ (J . Arg <( Gin HIS (J oOAsn ·-E ThrOOser c: ... Ala ..... Q~ U- Pro Val OQ fJQTyr ~E DNS-NH2 Gly QQ Asp Qrt) 0 () Glu .... lie Leu \) Met Q ~0 DNS-OH iL :I:C\J. Q 0 '\)Phe QHis HisQ E C) Q Cys 0 di-Lys Q 0 Trp 2 bis-Tyr -
Second Direction (90 ml Benzene: 10 ml Glacial Acetic Acid) . . . Third Direction. . . . _ . . . (100 ml Ethyl Acetate: 5 ml Methanol: 5 mf Glacial Acetic Acid) Fourth Direction (50ml I M Sodium Hydroxide:50ml Methanol)
Figure 3 21 reanalyzed, and then sequenced. The carboxyl end of each peptide was determined by digesting the peptide with carboxypeptidases A and
B, and then analyzing. Subtractive Edman degradation followed by high voltage electrophoresis was used to distinguish between aspartic acid
or ~sparagine and glutamic acid or gluta~ine. Since the N-terminal
peptide was blocked by alpha-pyrr~lidone carboxylic acid, hydrazinoly
sis was used to determine the sequence of this peptide. Approximqtely
3.0~~ of theN-terminal peptide was dried in the dessicator'under a vacuum. 5 ml. of anhydrous hydrazine was layered over the dried peptide and the tubes were frozen and sealede The hydrazinolysate was heated in an enclos~d gl~ss water bath for 8 hours, evaporated on a Buchler rotary evaporator, and then dried in the dessicator under a vacuuin for 18 hours •. 0.5 ml. of distilled water was added to the dried residue and then the hydrazides were, removed by adding 4 drops
of isovalerylaldehyde and extracting with three 2 ml. portions of ethyl
ether. The water phase was evaporated, and th~ remaining amino acid was detected on the amfno acid ana;yzer. RESULTS
Cellulose acetate electrophoresis of Bence Jones protein WHITE produced one major anionic migrating band, so the ammonium sulfate precipitate was homogenous and not contaminated with bth~r serum proteins.
Since Bence Jones proteins sometimes aggregate to form dimers and polymers, the molecular heterogeneity of protein WHITE was also tested. Sedimentation velocity studies on the native form of protein,
White, on the unreduced, partially unfolded protein, and on the reduced, unfolded protein save sedimentation values of 3.62 Svedberg units, 2.36 Svedberg units~ and 1.49 Svedberg units, respectively.
These data indicate that protein White in its native state is a dimer with a molecular weight of approximately 46,000 daltons that is covalently linked by disulfide bonds. SDS polyarylamide gel eLectro phoresis of the reduced protein produced a single band equivalent to a molecular weight of approximately 23,000 daltons.
Serological typing showed protein WHITE to be a lambda type light chain. It produced a single precipitin band against anti-lambda sera and was negative against anti-kappa sera when subjected to the
Ouchterlony method of double diffusion precipitin reaction.
Dansylation of the whole protein produced no dansyl amino acid derivative. This meant that the N-terminus presumably was blocked and probably began with alpha-pyrrolidone carboxylic acid. Alpha pyrrolidone carboxylic acid is characteristic of the N-terminus of lambda chain subgroups one and two.
Carboxypeptidase digestion of the whole protein revealed serine to be the C-terminal amino acid. This is consistent with the fact that all human lambda chains have serine as the C-terminal residue.
22 23 Results from N-terminal and C-terminal analyses are further
proof of the purity of the WHITE protein.
Amino acid analysis and tryptophan determination of the whole
protein showed that Bence Jones protein WHITE consisted ·of 216 amino
acids (Table 2)o Theoretically, the reduced and aminoethylated
protein should have been cleaved by trypsin at 22 sites (11
lysines, 6 arginines, and 5 aminoethylcysteines) producing 23
peptideso Actual tryptic diiestion, however, produced only 21
I tryptic peptides since one arginyl-proline bond and the penulti-
mate aminoe-thylcysteine were not split by trypsin. The alignment
of the tryptic peptides was deduced by comparison with the sequences
of lambda proteins BO, VIL, and NEI [Figure 4] (94,104,105).
As shown in Figure 5, the trypsin-digested protein eluted
from the Dowex 1X2 column in 15 main peaks. The purity of each
peak was tested by subjecting 30 ~ of sample to paper chiomato-
graphy and spraying with ninhydrin. All Dowex 1X2 peaks were
· found to be impure, so basic and neutral fractions were rechromato-
graphed on Dowex 50X2 columns and acidic fractions were rechromato-
graphed on columris of Dowex 1X2 with 30% propanol buffers. The
rechromatograms of the peaks are shown in Figures 6 through 20.
The tryptic peptides are designated T-1 to T-18 in accordance with
the analogous peptides of Bence Jones protein NEI (94). The amino
a~id compositions of the tryptic peptides are p~esented in Table 3.
Sequence determinations were carried out with the Edman degradation-
dansylation procedure as much as possible; however, hydrazinolysis
and carboxypeptidase digestion were also employed when necessary.
To facilitate communication and avoid confusion, the conventional
one-letter amino acid symbols will be used.in the text and the TABLE 2: Amino acid composition of protein WHITE. 24
Amino ,Acid Composition
Aminoethylcysteine 5 -Lysine 11 Histidine 3 Arginine 6 Aspartic acid 12 Threonine 21 Serine 38 Glutamic acid 19 Proline 14 Glycine 17 Alanine .17 Valine 15 Isoleucine 6 Leucine 13 Tyrosine 11 Phenylala~ine 5 Tryptophan·· 3
Sum 216
Table 2 FIGURE 4: Lambda chain subgroups. 25
ro 11 u , u t4 u 16 11 u '' zo 21 zz n zc zs u 21 a b " ze a J'l 11' ll JJ 14 •~ 16 n u u
t IIUMAH Ntv Glp Serffi]J•l t.0v Thr Gl.n .Pro Pro s,r Vel Set Alol AI• Pro Cly Cl·nfllil.a Val Thr tle S.r Cy• Ser Cly Cly ~r· T A~n I 1·~ ... ~ C.ly;sn·AM T.yr V.1l ~Cf' Ttrf11T'nCln~.t.41Y ~flo) I HCKA!oi HA Clp S~r Val .I.e· ii Thr Cln Pro Pro Sor V•l Ser y \t Pro Cly Cln rq. Y&l Thr U.~ Ser Cya Ser C.ly Cly ~C!If Scr 1\an C': 1t C:l\' f\,sn Asn.txL,.. V.Jl r!i!J.. 1'rp ~r.lft '• ·n :,"''.· rrn I ·JIU!Ioi'N VOR Cll" ~er V•l Lou Thr Cln rro Pro ;erf!l!JS•r Gl~ Thr Pro ely Cln Ar9 V•l 'f'hr U• Ser Cy• ser Cly ely A'~>n 1c sp ~ -·· Ch ''' Asn~Val~Trp Tyr Cln ~ . h rro
I! IIUAA.~ TAO Clp S~r Ala. ~\1 'thr Cln Pro ArC) Ser VAl Ser Cly Ser Pro Gly Cln $er Vel 1'h.r U~ Ser Cy• Thr Cly rhr Ser Ser Asp Ya Cly o1 .T c Asn . Ur V•l Ser Trp Tyr Cln Cln UU l'rn tl HU~~ SOtl Clp ser. Ala .C..u Thr Cln Pro Ar' Ser Val Ser Cly Ser Pro Cly Cln Ser V.tl Tftr lle Ser Cyl a Cly Tl\r S~r ·Ser A.ap V.al .Cly sn " e V.1l Sor Trf' Tyr Clft Cln. JUs. rro II IIUM/oN NEI Cl sor Al• Leu 'rhr ~.ln Pra a Ser Val ser Cly ser Pro Cly Cln ser e T.hr Ue Set C:ya r Cly Thr r Ser Aap Val Cly . cr yr sn e V11l s~r Trr ~ Cln Cln -..n rro II IIC'III~ VIL rt IIUJQ~t 00 ~ ~:~ ~~: ~:: ~~~ ~~: :~: A~: ~;~IEJ~:~ ~~~ ;~~·~~~·~ ~t: ~:: r;.• ~: i~: .::.~ ~~= ~:! g~~ ~:! 5:~ ~=~ ::: ~=~ ~~~. IP T ~~ h~: T~~ ~=~ ~:~ ~~~~~~: ~t~ Ill: ~~~ Ill IIUMJI~ .SII --- srr@:@ Leu Thr .Cln[!!f) Proi!EJYol ser vait.u•! t.eu!cly Cln Thr Vol S II•(!EJ Cy1 [§E) ely loop sn!EJ·-· ... ·--tArqfC&y!'rvrfAsrtAla'f"iofTrr Tyr cln ~In !E!Jrro V IICMAN 0£1. --·JTvrlval(LeliiE:Jctn rra Pro se'r Yol ser vo1(BPra ely Cln Thrf"AhjA~qJ Ile{!§cy•@!Jcly Aapjctyjno!··· --- ••• ClyJCiyjLytjsuJYalfE!!jTrP Tyr Cln Cln~rra
IV IIUMAN KtA:t ··~ Tyr 11. ,, lA.u ;hr Gln rro Pro S•r ·val Set' Vel r.~r Pr.o Cly Cln Thr .Ala. VII· tle Thr Cye Ser C. ly Alp Asn l.f:u. ••• ·•• ...... G u L s Thr Ph..- Y..1Jl Ser 'rrJ'I~Cln Clnr;Ar'l Pro tV HUMAN X ••• Tyi' !II, t.eu 'l'hr Cln Pro Pro Sdr Val Sol' Vo~l sei- Pro c;ly Cln Thr Ala or Ue Thr C'ye Ser Cly Alp ye ~u ••• ...... • y A1 1 ' I V.al p:"YilTrr~Cln Cln Arq Pro Ill IIUMN H.\IJ ...... Tyr • y Lcu.a Thr (iln Pro Pro SurEJsor Val Ser Pro Cly Cln Tl'\r At• S.r tle Thr C:y• Ser ctv Aap E.'l• l.u" ...... ••• ••• Cly • u . " yr '-~•1 ~Trt• T'fl' Gln Gll\ Y• rro
I IIUKI\N NI:V I IIUM.t\~ UA J UUMAN VOA
It IIUMN TJIO 11 IIUMAH DOH II IIUMAN N£1 II HUOV..~ YIL lt IIUKA~ 90
Ill IICIWl S,H
V IIUMAH DEt.
IV IIUMAN ICERN tV 11\JM~ X IV IIUMAN AAU
IU Uf Ill 191 H4 Ala Set Lyl Ar'l ... Ala Ser Lyl s~r Ala ... Lyo !§ Svr II ILUP'Afl TUtJ Ale !t•r Lyo II lllllltAI~ JWIU "'''' Jl UUM.\.:1 Ul:l Ala ~:., r• .,. Ar•l II UUMAN Vrt. :.; .. , l.y. ..~ II IIUMAN A0 ...... ~••r t.v• Ar•l rtr HUiw.su. Alo Ser Lyo Ar'l V IIUMAN 111:1.
IY tlUMft atiiiN Ale ..,. Ar'l IV llli"'AN X Ate ~ Lye Ar•r IV IIUAAH n-.u Ala Lya Arq m~··~··-· ($!) .... ~
Figure 4 FIGURE 5: Ion exchange chromatography of 2.0 grams of tryptic hydro lysate of reduced and aminoethylated protein WHITE on a column (2.5Xl50 em.) of Dowex 1X2 at 37°C. The solid line represents absorbance at 570 nm. after reacting 100 A with ninhydrin. The closed circles represent pH. Fractions consisted of 10 ml./10 min. The peaks are lettered from a to o according to their order of elution. 26
k n 0 a b f h E 3.0 c::: 0,_..._ 2.5 10 lO g. ..c 2.0 8 Q) 7 (.) • c::: 1.5 g 6 0 J: .0 5 Q. .. 4 0 1.0 (/) ..Q • • 3: <[ ·0.5 • 2 I 0 .. 50 100 150 200 250 300 350 400 450 500 550 600 650 Fraction Number
Figure 5 FIGURE 6: Rechromatography of peak a from Figure 7 on Dowex 50X2.
FIGURE 7: Rechrornat~graphy of peak b from Figure 7 on Dowex 50X2. 27
Abs. Abs. 570 570
pH pH T-3 2 • • 2 T-18a • T-2a • a- - T-3 " 8 I • • • • 4 • • 4
0 50 100 150 0 100 . ISO Fraction Number Fraction Number
Figure 6 . Figure 7 _ FIGURE 8: Rechromatography of peak c from Figure 7 on Dowex 50X2.
FIGURE 9: Rechromatography of ~eak d from Figure 7 on Dowex 50X2. 28
Abs. · Abs. 570 570 pH pH 2 • 2 • • 8 8 T-lla T-2b · • I· T-4 • 4 • • • 4
0 50 100 150 0 50 100 150 Fraction Number. . Fraction Number
Figure 8 Figure 9 FIGURE 10: Rechromatography of peak e from Figure 7 on Dowex SOX2.
FIGURE l~i Rechromatography of peak f from Figure 7 on Dowex 50X2. 29
Abs. Abs. 570 570 pH pH 2 • • 2 8 • 8 • • • • 4 4
0 50 100 .150 0 50 100 150 Fraction Number Fraction Number
Figure 10 Figure 11 FIGURE 12: Rechrornatography of peak~ from Figure 7 on Dowex 50X2.
FIGURE 13: Rechrornatography of peak h frorn.Figure 7 on Dowex 50X2. 30.
-· Abs. Abs. 570 570
pH 2 • 8 8
I 4
0 50 100 150 0 50 100 150 Fraction Number Fraction Number
Figure 12 Figure 13 FIGURE 14: Rechromatography of peak i from Figure 7 on Dowex 50X2.
FIGURE 15: Rechromatography of peak i from Figure 7 on Dowex 50X2. 31
Abs . .------~--lllillt Abs. ,.....-~...... _~------..... 570 570 pH - pH 2 • • T-10 8 .. -8 - I ~ • 4 • • • • -4 • -
0 50 100 150 0 50 100 150 Fract-ion Number Fraction Number
Figure 14 Figure 15 FIGURE 16: Rechromatography of peak k from Figure ·7 on Dowex 50X2.
FIGURE 17: Rechromatography of peak 1 from Figure 7 on Dowex 50X2. 32
Abs. Abs. 570 570 ·pH pH 2 2 T-12 • • 8 8
I . • • • • • • • 4 • 4
0 50 100 150 0 50 100 150 Fraction Number Fraction Number
Figure 16 Figure 17 FIGURE 18: Rechromatography of peak m from Figure 7 ort Dowex 50X2.
FIGURE 19: Rechromatography of peak n from Figure 7 on Dowex 50X2. 33
0 50 IQQ I'· 150 0 50 100 150 Fraction Number Fraction Number
Figure 18 Figure 19 FIGURE 20: Rechrornatography of peak o from Figure 7 on Dowex 1X2 with 30% propanol buffers. 34
I
Abso 570 T-16 pH 2 • T-18b+c 8
• 4 • • • 0 50 100 150. Fraction Number
Figure 20 TABLE 3: Amino acid composition of the tryptic peptides of protein WHITE. . \ 35
Amino Acid T-3 T-15+6 T-lla T-llb+C T-18a
Aminoethylcysteine 0.88 (l) Lysine 1.30 (1) 1.07 ( 1) Histidine 0.76 (1) Arginine 1.39 (2) 0.98 (1) Aspartic acid 2.31 (2) 0.95 (1) Threonine 1.80 (2) 2.30 (2) 0.86 (1) Serine 6.16 (6) 4.10 (4) 2.28 (3) 1.93 (2) Glutamic acid 3.23 (3) 2.40 (2) Proline 2.00 (2) 1.38 (1) 0.98 (1) 0.98 (1) Glycine 2.12 (2) 3.03 (3) 1.01 (l) 1.11 (1) Alanine 2.11 (4) 0.50 (0) 0.95 ( 1) Valine 0.99 (l) 2.02 (2) 0.91 (1) Isoleucine 1.97 (2) 2.87 (3) Leucine 0.94 ( 1) 1.27 (1) Tyrosine 2.00 (2) 1.64' (2.) Phenylalanine 0.78 (1) 0.93 (l) Tryptop~an + (1)
Sum 22 22 3 16
Amino acid T-18b+C T-17a+b T-17c T-2a T-2b
Aminoethylcysteine · 0.83 ( 1) Lysine 0.95 (1) 1.01 (1) Histidine Arginine 0.87 (1) 1.07 (1) Aspartic acid 3.22 (3) 1.01 (1) Threonine 2.06 (2) 2.21 (2) 1.02 (1) 1.05 (1) Serine 3.12 (3) 4.01 (4) Glutamic acid 3.28 (3) 'Proline 0.94 (1) Glycine 2.14 (2) 3.01 (3) 1.08 ( 1) Alanine 3.18 (3) Valine 0.99 (1) Isoleucine 0.85 (1) Leucine 1.99 (2) 1.00 (1) 1.89 (2) Tyrosine 2.09 (2) 1.01 (1) Phenylalanine 1.01 (1) Tryptophan
Sum 22 9
Table 3 TABLE 3: Amino acid -compsotion of the tryptic peptides of protein WHITE. 36
Amino acid T:-12 T-5 T-14 T-9 T-7 T-4
Aminoethylcysteine 0.98 (l) Lysine 0.99 (l) 0.98 (1) 0.91 (l) 0.94 (1) 1.10 (1) Histidine Arginine Aspartic acid 1.08 (l) 1.31 (l) 1.07 (l) 2.33 (2) Threonine 1.01 (1) 1.05 (l) 0.84 (l) 2.94 (3) Serine 2.87 (3) 1.30 (1) 1.99 (2) 1.09 U> 0.80 (1) Glutamic acid_ 3.12 (3) 0.94 '(l) 0.89 (l) Proline 3.09 (3) 0.90 (1) 1.22 (1) 1.05 (1) Glycine 1.20 ( 1) 1.08 (1) Alanine 3.01 (3) 1.10 (l) 2.31 (2) 1.10 ( 1) 1.08 (1) Valine 0 .• 91 (1) 0.83 (1) 1.79 (2) 0.88 (l) 0.88 (l) Isoleucine 0.99 (1) Leucine 1.92 (2) 1.04 (1) 0.96 (l) Tyrosine 1.05 (l) Phenylalanine 0.99 (1) 0.85 ( 1) Tryptophan + (1)
Sum 19 5 15 10 5
Amino acid T-16 T-1 T-8 T.,.13 T-10
Aminoethylcysteine '0.14 (0) 0.78 (1) 0.91 (1) Lysine 0.94 (l) 1.09 (1) Histidine 1.04 (1) 1.03 (1) Arginine 0.99 (1) Aspartic acid Threonine 1.01 (1) 1.95 (2) 1.98 (2) Serine 2.89 (3) 0.96 (l) 1.97 (2) 1.00 ( 1) 0.94 ( 1) Glutamic acid 2.06 (2) 3.13 (3) 1.09 (1) Proline 1.25 (1) 1.05 (1) Glycine 1.06 (1) Alanine 1.97 (2) 1.06 (1) Valine 1.80 (2) 1.05 (1) Isoleucine Leucine 1.88 (2) Tyrosine 1.98 (2) 1.05 (l) Phenylalanine Tryptophan + (1)
Sum 15 3 4 11 8
Table 3 (Continued) 37 conventional three-letter amino acid symbols will be used in the figures (Table 4).
The Variant Region:
T-3 (Positions 1-22)
l 5 10 15 20 22 J-S-A-1-T-Q-P-A-S-V-S-G-S-P-G-Q-S-I-T-I-S-C LTh5..J I Thl LJ'h2_j r~-~~~~~~ ~~~
~------~Th3.~------~
T-3 is a very basic peptide that represents the N-terminus of the light chain. Attempts at dansylation revealed that the alpha-amino group was. blocked, and further analysis showed that this was the iesult of an N-terminal ~lpha-pyrrolidone carboxylic acid. Carboxypeptidase hydrolysis showed that aminoethylcyste~ne was the C-terminal residue. Since t~e N-terminus of this peptide was blocked, it was necessary to subdigest it and expose new alpha- amino groups bef6re sequence determination could be started. After digestion of T-3 with thermolysin for 5 hours at pH 8 and 37 0 C, 5 main fractions (Th-1 through Th~5) ware isolated from a Dowex
1X2 column with 30% propanol buffers [Fig~~~-21]. The amino acid composition of each peak is given in Table 5. The first three amino acids of the N-terminus were contained in Th-5. The amino acid sequence of this tripeptide was determined by hydrazinoly- sis ( ~) and carboxypeptidase digestion ( o;:-). Thermolysin peptides one (positions 4-10), two (positions 20-22), three
(positions 4-17), and four (positions 13-22) overlapped nicely · and allowed sequence determination of the remainder of T-3 by the Edman degradation-dansylation procedure ( ~ ). Peptides TABLE 4: Amino acid symbols. 38
Amino acid 3-letter symbol 1-letter symbol
Alanine Ala A Arginine Arg R Asparagine Asn N Asparti·c acid Asp D Asn + Asp Asx B Cysteine Cys c Glutamine Gln Q Glutamic acid Glu E Gln + Glu Glx z Glycine Gly G Histidine His H Isoleucine Ile I Leucine Leu L Lysine Lys K Methionine Met M Phenylalanine Phe F Proline Pro p Pyrr. Carb. acid PCA J Serine Ser s Threonine Thr T Tryptophan Trp w Tyrosine Tyr y Valine Val v
Table 4 FIGURE 21: Chromatogram of the thermolysin peptides of T-3 after separation on Dowex 1X2 with 30% propanol buffers. 39
Abs. · 570
pH 2 Th-3 • 8 • • Th-4 • • 4 Th-5 • • • 0 50 100 150 Fraction Number
Figure 21 TABLE 5: Amino acid composition of the thermolysin peptides of T-3. 40
Amino acid Th-1 Th-2 Th-3 Th-4 Th-5
Aminoethylcysteine 0.91 (1) 0.82 (1) Lysine Histidine Arginine Aspartic acid Threonine lel3 (1) 0 .86_ ( 1) 1.13 ( 1) Serine 1.20 ( 1) 1.07 (1) 3.86 (4) 3.02 .(3) 0.97 (1) Glutamic,: acid 0.79 (1) 2.20 (2) 0.79 (1) 1.04 (1) Proline 0.85 (1) 1.71 (2) 0.76 (1) Glycine 2.12 (2) 0.81 (1) Alanine 1.31 (1) 1.01 (1) 0.95 (1) Valine 1.• 01 (1) 1.11 (1) Isoleucine 1.02 (1) 1.85 (2) Leucine 1.15 (1) 0.95 (1) Tyrosine Phenylalanine Tryptophan
Sum 7 3 14 10 3
Table 5 41 Th-1 and Th-3 were electrophoretically neutral so residues 6 and 16 are glutamines.
T-15+6 (Positions 23-41)
23 27 a b c28 30 35 41 T-G-T-S-8-D-V-G-S-Y-N-F-V-S(W,X,Y)Q-H-P-G-K ~~~~~~~~ ~~~
t__ Th 1---' I__ Th3____j ..______;;Th 2-'-. _____.
Carboxypeptidase hy~rolysis of T-15+6 showed that lysine was the C-terminal amino acid. Dansylation followed by Edman degradation revealed the sequence of the first 8 residues, but the extended length of the peptide required subdigestion before the complete sequence could be determined. Digestion with ther- molysin for 5 hours ·at pH 8 and 37°C split T-15+6 into three major fragments (Th-1, Th-2, Th-3) that were isoLa-ted after chromatography on Dowex 50X2 [Figure 22]. The amino acid composi- tion of each fragment is presented in Table 6. Peptides Th-1 and
Th~3 were sequenced entirely by Edman degradation and dansylation.
The placement of these two peptides in T-15+6 was confirmed by comparing their sequences with the sequence of the first 8 amino acids of the whole peptide. Peptide Th-2 contains tryptophan at position 3 so sequence determination by dansylation and Edman degradation was limited to the first two amino acids. The rest of the sequence of Th-2.was found by carboxypeptidase digestion, amino acid analysis and structural analogies to other lambda chains. Peptide Th-1 was acidicon electrophoresis so the residu~ at position 27a is aspartic acid. Peptide Th-3, on the other hand, was neutral when subjected to high voltage electrophoresis so the amino acid at position 30 is asparagine. Since Th-2 was strongly FIGURE 22: Chromatogram of the thermolysin peptides of T-15+6 after separation with Dowex 50X2. 42
Abs. 570 pH • 2 • • Th-1 • 8 Th-3 I c Th-2 • •• 4
0 50 100 150 Fraction Number
Figure 22 TABLE 6: Amino acid composition of the therrnolysin peptides of T-15+6.
\ 43
Amino acid Th-1 Th-2 Th-3
Aminoethylcysteine Lysine 0.76 (1) Histidine 0.75 (1) Arginine Aspartic acid 1.33 (1) 1.18 (1) Threonine 1.78 (2) Serine 1.93 (2) 1.21 (1) 1.06 (1) Glutamic acid 1.92 (2) Proline 0.77 (1) Glycine 1.08 (1) 1.26 (1) 1.42 (1) Alanine Valine 1.43 (1) 0.79 ( 1 )' Isoleucine Leucine Tyrosine 0.92 (1) 1.33 (1) Phenylalanine 0.78 (1) Tryptophan ( +) {1)
Sum 6 10 6
Table 6 44 basic.on electrophoresis, the residues at positions 36 and 37 are glutamines.
T-lla (Positions 42-44)
43
...... A-P-K -.l......
The amino acid composition of this peptide is presented in Table 3. T-lla was easily sequenced by the dansylation-
Edman degradation procedure.
T-llb+c (Positions 45-60)
45 so 55 60 1-I-I-Y-D-V-T-Y-R-P-S-G-I-S-S-R ...:.-:.:.....:;.~_:.~~~~-J.~-\-..\.-_..~~ This peptide was sequenced by the dansylation-Edman procedure using two samples which were degraded in pa.rallel. One sample was degraded and dansylated to yield the first ten steps, and the other sample was degraded for the first ten steps and then sequenced from steps ten through sixteen. The sequence was confirmed by automatic degradation on a Beckman model 890-C automatic sequenator.
T-18a (Positions 61-65)
61 65 F-S-G-R -l._.~~
The amino acid composition of peptide T-18a is presented in
_Table 3 •.
T-18b+c (Positions 66-87)
66 70 75 80 85 87 S-G--N-T-A-S-1-T-I-S-G-L-Q-A-E-D-E-A-D-Y-Y-C _.....:.~_:a~~ ~~.~....!1. I "'"'~r-
L.-.Jh L._j L-.-Jh 1__ 1 i Th3=-____,
...:. ~ ._..:.. ~ '" ~ ~ ~. ~ _.:. __.a. -&"....:a. ~
~
This peptide is very acidic and therefore eluted very late from the Dowex 1X4 column [Figure 5]. Carboxypeptidase hydrolysis showed that aminoethylcysteine was the C-terminal amino acid. 45 Thermolysin digestion of T-l8b+c for 5~ hours at pH 8 and 37°C followed by chromatography on Dowex lX2 with 30% propanol buffers revealed 3 main peaks [Figure 23]. The amino acid composi_tion of each thermolysin peptide is given in Table 7. The positions of peptides Th-2 (Positions 66-69) .and Th-1 (Positions 72-76) were located by comparison with the Edman degradation-dansylation sequence of the first 10 amino ~cids of the intact peptide.
Peptide Th-3 (Positions 77-85) was positioned by comparing its
C-terminal amino acids with the carboxypeptidase determined sequence of the last four amino acids of the intact tryptic peptide. Peptide Th-2 was neutral on electrophoresis so position
68 is occupied by asparagine. Sequential Edman degradation of peptide Th-3 followed by high voltage electrophoretic analysis of the remaining peptide showed positions 78, 80, 81, 82, and 84 to be glutamine, glutamic acid, aspartic acid, glutamic acid, and aspartic acid, respectively.
T-l7a+b (Positions 88-97) CHO 9o 1 95 97 S-S-Y-T-8-B-S-T---R . ._::....:..__;;,~...,:).~~.~' ;;:-
Peptide T-l7a+b is very important because amino acid analysis of thi~ peptide revealed the presence of hexosamine at position 93. The C-terminus was found to be arginine by carboxypeptidase hydrolysis. Due to the presence of carbohydrate at position 93, the dansylation-Edman degradation procedure had to be repeated many times before the sequence of the entire peptide wa.s determined. Position 96 was left blank to allow structural homology with other lambda chains. FIGURE 23: Chromatogram of the thermolysin peptides of T-18b+c after separation on Dowex 1X2 with 30% propanol buffers. 46
Abs·. 570 pH 2 • 8 •
Th-1 • 4 Th-2
0 50 100 150 · Fraction· Number
Figure 23 TABLE 7: Amino acid composition of the thermolysin peptides of T-18b+c. 47
Amino acid Th-1 Th-2 Th-3
Aminoethylcysteine Lysine Histidine Arginine Aspartic acid 0.88 (1) 2.06 (2) Threonine 0.96 (1) 1.25 (1) Serine 1.08 (1) 1.41 (1) Glutamic acid 2.93 (3) Proline Glycine 1.15 (1) 0.91 (1) Alanine 1.88 (2) Valine Isoleucine 0.95 (1) Leucine 0.86 (1) 0.95 (1) Tyrosine 1.19 (1) Phenylalanine Tryptophan
Sum 5 4 9
Table 7 48 T-17c (Positions 98-104)
100 104 1-F-G-G-G-T-K
The amino acid composition of T~l7c is given in Table 3.
T-2a (Positions 105-109)
105 109 1-T-V-1-R_.....;.._....:._, ' Peptide T...,2a is called the "switch" peptide because position
109 divides the light chain into variable and constant regions.
Carboxypeptidase hydrolysis showed that position 109 was occupied by arginine.
The Constant Region:
Since the constant regions of all lambda chains are almost identical, the sequence of the WHITE constant region was determined by c~mparing the compositions of the WHITE tryptic peptides with t~e tryptic peptides of other lambda chains. Those regions of the
WHITE constant region that have shown isotypic variation in other lambda chains were completely sequenced by the dansylation-Edman degradation procedure.
T-2b (Positions 110-112)
111 (Q,"P ,K)
t-12 (Positions 113-131)
115 120 125 130 A-A-P-S-V-(T,1,F,P,P,S,S,E,E,1,Q,A,N,K) --~~- .
Since positions 114 and 116 are associated with the MCG isotypic marker, the first part,of this peptide was sequenced by the dansy1ation-Edman degradation procedure. 49 \ T-5 (Positions 132-136)
13 2 13 6 (A,T,L,V,C)
T-14 (Positions 137-151)
140 145 150 1-I-S-D-F-Y-P-G-A(V,T,V,A,W,K) __.~~~~ ...... ~~
Peptide T-14 includes the'MZ isotypic marker at position
145, so the sequence of the first 9 residues was determined.
T-9 (Posit ions 15·2-158)
155 158 A-0-S-S-P-V--K
Since this peptide contains isotypic markers at positions
153 (EV), 154 (KERN), and 158 (WEI~), the sequence of the entire peptide was determined.
_T-7 (Positions 159-168)
"160 165 168 A-G-V-E-T-T-T(P,S,K) _.!,.~~~~...::...~
Residues 159 and 165 are associated with the WAY and MCG
isotypes, so the first 7 ami9o' acids of T-7 were sequenced.
T-4 (Positions 169-173)
170 173 (Q,S,N,N)~
Peptide T-4 was subjected to carboxypeptidase hydrolysis
to establish the status of the MZ isotypic marker at residue 173. ,
T-16 (Positions 174-188)
175 180 185 188 (Y,A;A,S,S,Y,L,S,L,T,P,E,Q,W,K) 50 T-1 (Positions 189-191)
190 ( S ,--H) R '\
Carboxypeptidase hydrolysis of peptide T-1 showed that arginine occupied position 191 and hence is Oz-.
T-8 (Positions 192-195)
192 195 (s,.Y,s,.c)
T-13 (Positions 196-206)
196 200 205 (Q,V,T,H,E,G,S,T,V,E,K)
T-10 (Positions 207-214)
207 210 214 (T-V-A-P-T-E-C) S ' End group analysis showed serine to be the C-terrninal residue. DISCUSSION
Bence Jones protein WHITE was isolated from the urine of a patient in Talmadge Memorial Hospital who had multiple myeloma.
An extensive investigation of this protein using amino acid analysis, end group analysis, tryptic digestion, ion exchange ) chromatography, paper chromatography, carboxypeptidase hydro~ lysis, dansylation, Edman degradation, hydrazinolysi~, and high voltage electrophoresis revealed the primary structure that is presented .in Figure 24. ~he sequence of the constant region of protein WHITE (Positions 110-214) appears to be identical to that of other lambda chains. Isotypically protein WHITE may be classified as OZ-, KERN-, MCG-, MZ , EV , WAY-, and \~EIR-, because it has the common residues rather than the variant residues at positions 191, 154, 114/116/165, 145/173, 153, 159, and 158, respectively.
In contra.st to the constant region, positions 1 through
109 of lambda chains are highly variable. Because the varia~ility occurs in a highly restricted and nonrandom fashion, lambda chains may be divided into four subgroups. When the primary sequence of protein WHITE is compared to the sequence of other lambda chains it becomes evident that protein WHITE is a member of the lambda chain subgroup II (Fig~r~ 4). Protein WHITE consists of 216 amino acids (M.W. 23,000) as db all lambda chains of subgroup II. In contrast, proteins of subgroups I,
III, and IV contain 217 (sometimes 216), 213, and 211 amino acids, respectively. In order to maximize sequence homology between all lambda chains, gaps and insertions are included in sequence alignments ~herever they differ from the fi~st lambda chain
51 FIGURE 24: The primary structure of Bence Jones protein WHITE. 52
PCA-Ser-Ala-Leu-Thr-Gln-Pro-A1a-Ser-Va1-Ser-G1y-Ser-Pro-G1y- : 1 . 10. . T-3 G1n-Ser-11e-Thr-Ile-Ser-Cys-Thr-G1y-Thr-Ser-Ser-Asp-Va1-G1y- 20 T-15+6 Ser-Tyr-Asn-Phe-Val ... Ser-Trp-Tyr-G1n-G1n-His-Pro~G1y-Lys-Ala- ~ ~ T-11a Pro-Lys-Leu-Ile-Ile-Tyr-Asp-Va1-Thr-Tyr-:-Arg-Pro-Ser-Gl.y-I1e- 50 - T-llb+c T-18a Ser-Ser-Arg-Phe-Ser-G1y-Ser-Arg-Ser-Gly-Asn-Thr-Ala-Ser-Leu- 60 70 T-18b+C Thr-Ile-Ser-Gly-Leu-Gln-Ala-Glu-Asp-Glu-Ala-Asp-Tyr-Tyr-Cys- 80 CHO T-17a+b Ser-Ser-Tyr-Thr-Ser-Asx-Ser-Thr-'l'nb'c'.-Arg-Leu-Phe-Gly-Gly-Gly- 90 96 100 T:.,;,17c T-2a T-2b Thr-Lys-Leu-Thr-Va1-Leu-Arg-Gln~Pro-Lys-Ala-A1a-Pro-Ser-Va1- 110 T-12 Thr-Leu-Phe-Pro-Pro-Ser-Ser-Glu-G1u-Leu-G1n-A1a-Asn-Lys-Ala- 120 130 T-5 Thr-Leu-Va1-Cys-Leu-Ile-Ser-Asp-Phe-Tyr-Pro-Gly--A1a-Val-Thr- 140 T-14 T-9 Va1-A1a-Trp-Lys-Ala-Asp-Ser-Ser-Pro-Val-Lys-A1a-G1y-Va1-G1u- 150 160 T-7 T-4 Thr-Thr-Thr-Pro-Ser-Lys-G1n-Ser-Asn-Asn-Lys-Tyr-Ala-A1a-Ser- 170 T-16 T-1 Ser-Tyr-Leu-Ser-Leu-Thr-Pro-G1u-G1n-Trp-Lys-Ser-His-Arg-Ser- 180 190 T-8 T-13 Tyr-Ser-Cys-Gln-Val-Thr-His-Glu-Gly-Ser-Thr-Val-Glu-Lys-Thr- 200 T-10 Val-A1a-Pro-Thr-G1u-Cys-Ser. 210 214
Figure )24 53 that was sequenced, protein SH (106)c The variable region of protein
WHITE is similar to other proteins of subgroup II in its deletion at residue 96 and insertion of the Asp-Val-Gly triplet at positions 27a,
27b, and 27c. In addition, protein WHITE pos-sesses certain residues common only to proteins of subgroup II. These linked amino acid residues are definite at positions 3(Ala); 17(Ser), 23(Thr), 25(Thr),
57(Val), and 90(Tyr), and possible at positions 8(Ala), 18(Ile),
29(Tyr), 38(His), 50(Ser), and 88(Ser)~ These latter seven residues are modified by individual specific exchanges and might be included in the type of antibody-combining site variation that Oudin termed
"idiotype" (107). Prot·ein WHITE exhibits 84% amino acid sequence homology with protein NEI, 84% with protein VIL, and 78% with protein
BO. The percentage of homology among these four subgroup II proteins ranges from 73% to 84%. When these are compared to light chains of other ,subgroups, the range is from 53% to 65% with the most extensive variability occurring in three specific hypervariable (CDR) areas including positions 24 to 34 (L-1), positions 50 t6 56 (L-2), and positions 89 to 97 (L-3). It is particularly significant that proteins
WHITE and NEI have identical tetrapeptides in the L-1 (positions 30 to 34) and L-3 (positions 93 to 96) regions. The A-L-1 sequence
Ser-Tyr-Asn-Phe is unique for NEI and WHITE and does not occur in eighteen other A~L-1 sequences (112). Likewise, the A-L-3 sequence
Asn-Ser-Thr-Arg found in both NEI and WHITE does not occur in sixteen other A-L-3 sequences (113). It appears that these L-1 and L-3 sequences are linked. Thi~ high percentage of homology and the strict regularity of variability between these lambda chains from different individuals would be contrr3ry to both the somatic-mutation 54 theory of random hypermutation of a small number of genes, and Kabat's
theory of random insertion from "minigenes"~ These data lend more
support to the multigene germ line theory of consecutive ~ene duplica
tion and subsequent point mutation of a large number of inherited
genes.
An unusual structural feature of protein WHITE is the presence
of covalently bound carbohydrate. Although all classes of human
immunoglobulins consistently have carbohydrate conjugated to specific
sites in the constant regions of their heavy chains, carbohydrate residues havi been detected on the variable region of only 6% to
15% of kappa and lambda light chains (12,94,108,109,110). The occurrence of carbohydrate on light chains is not associated with any particular subgroup, chain type, or position within the variable region. Sox and Hood have proposed that the tripeptide Asx-X-Ser/
Thr, where X represents any amino acid, and the third residue is either Ser or Thr, must be present before glycosylation can occur
(12). The carbohydrate of protein WHITE is covalently attached to the asparagly residue in the tripeptid~ Asx-Ser-Thr (~ositions 93 td 95) an~ therefore, supports the hypothe~is of Sox and Ho6d. It remains to be determined whether the occurrence of carbohydrate on light chains is by chance and simply occurs because of the presence of the characteristic glycosylation tripeptide, or whether it serves some functional purpose ·in secretion or antibody s·pecificity. SUMMARY
1. Bence Jones protein WHITE is a lambda type light chain consist ing of 216 amino acidsG In the native state protein WHITE is a disulfide bridged dimer with a molecular weight of approximately 46,000 daltons.
2. Sequence homology with other light chains places protein WHITE into subgroup II of lambda light chains.
3G Isotypically protein WHITE may be classified as OZ , Kern-, MCG-, MZ-, EV-, WAY-, and WEIR-.
4. The primary structure of Bence Jones protein WHITE has been determinedo It is significant that proteins WHITE and NEI have identical tetrapeptides in the L-1 and L-3 regions unique to these two proteins. The occurrence of these linked areas supports the multigene germ line theory of antibody diversity.
5. Bence Jones protein WHITE is one of the 6% to 15% of light chains that have carbohydrate attached.
6. The WHITE carbohydrate is covalently attached to an asparagyl residue that is part of an Asx-Ser-Thr tripeptide (positions 93 to 95), and therefore supports the theory that Asx-X-Ser/ Thr is a recognition triplet for the transglycosylas·e enzyme.
55 REFERENCES
1. Lederberg, J. 1959. Genes and antibodies. Science 129: 1649.
2. Smithies, 0. 1967. Antibody variability. Science 157: 267.
3. Edelman, G.M. and Gally, J. 1967. Somatic recombination of ~uplicated genes: A hypothesis of the origin of antibody diversity. P.N.AeS. 57: 353.
4~ Edelman, G.M. and Gall, W.E. 1969. The antibodj problem. ·Ann. Rev. Biochem. 38: 415.
5. Milstein, C. and Munro, A.J. 1970. , The genetic basis of antibody specificity. Ann. Rev. Micro 24: 335.
6. Jerne, N.K. 1971. The somatic generation of immune recognition. Eur. · J. Imm. 1: 1.
7. Dreyer, W.J., Gray, W. and Hood, L. 1967. The genetic, molecular and cellular basis pf,antibody formation: Some facts and a unifyin~ hypothesis. Cold Spring Harbor Sym. Quant. Bio1. 32: 353.
8. Hood, L. and Prahl, J.W. 1971. The immune system: A model for differentiation in higher organisms. Adv_. Imm. 14: 291.
9. Hood, L. and Talmage, D.W. 1970. Mechanism of antibody diversity: Germ line basis for variability. Science 168: 325.
10. Langer, B., Steinmetz-Kayne, M. and Hilschmann, N. 1968. Die vollstandige aminosaurquenz des Bence-Jones-Proteinn NEW (lambda-Typ.), Subgruppen im. variablen teil bei immuno globulin-1-Ketten vom lambda-Typ~ Hoppe Seylers z. Physiol. Chern. 349: 945.
11. Bull. World Health Org. 1969. 41: 975.
12. Sox, H.C. and Hood, L. 1970. Attachment of carbohydrate to the variable region of myeloma immunoglobulin light chain. P.N.A.S. 66: 975.
13. Porter, R.R. 1967. The structure of antibodies. Sci. Amer. 217: 81.
14. Delaney, R., Fellows, R.E., Hill, R.L. and Lebevitz, H.E. 1966. The evolutionary orgins of the immunoglobulins. P.N.A.S. 56: 1762.
15. Tiselius, A. and Kabat, E.A. 1939. An electrophoretic study of immune sera and purified antibody preparations. J. Exp. Med. 69: 119.
56 57
16. Grabar, Po·and Williams, C.A. 1953. Methode permittant l'etude conjuguee des proprietes electrophoretiques et immunochimiques d'un melange de proteines: Application au serum sanguin. Biochem. Biophys. Acta 10: 193.
17. Slater, R.J., Ward, S.M. and Kunkel, H.G. 1955 .. Immuno logical relationships among the myeloma proteins. J. Exp. Med• 101: 85.
18. Rowe, D.S. and Fahey, J.L. 1965e A new class of human immunoglobulins. J. Exp. Med. 121: 171.
19. Ishizaka, X., Ishizaka, T .. and Hornbrook, M.Ms 1966e Physiochemical properties of reaginic antibody. J. Imrnunol. 97: 840.
20. Porter, R.R~ 1959. The hydrolysis of rabbit gamma globulin and antibodies with crystalline papain. Biochem .. J. 21.= 119.
21. Ed e lma n , . G • M• and Po u l i k , M• D • l 9 61 • Studies on the struc tural units of the gamma globulins. J. Exp. Med. 113: 861.
22. Press, E.M. 1967. The amino acid sequence of the N-terminal . 84 resiques of a human heavy chain of immunoglobulin G (DAW). Cold Springs Harbor Symp. Quant. Biol. 32: 45.
23. Hill, R.L., Lebovitzi H.E., Fellows, R.E., Jr. and Delaney, R., in Nobel Symposium 3, Gamma Globulins (edited by J. Killander), Al~qvis~ and Wiksell, Stockholm 1967, p. 109.
24. Press, E.M.·and Piggot, P.J. 1967. The chemical structure of the heavy chains of human immunoglobulin G. Biochem. J. 104: 30C.
25. Putnam, F.W. 1969. Immunoglobulin structure: Variability and homology. Science 163: 633.
26. Grey, H.M. and Kunkel, H.G. 1964. H chain subgroups of myeloma proteins and normal 7S gamma globulin. J. Exp. Med. 120: 253.
27. Vaerman, J.P. and Heremans, J.F. 1966. Subclasses of human immunoglobulin A based on differences in the alpha polypeptide chains. Science 153: 647.
28. Feinstein, D. and Franklin, E.G. 1966. Two antigenically distinguishable subclasses of human A myeloma protein differing in their heavy chains. Nature 212: 1496.
29. ·Kunkel, H.G. and Prendergast, R.A. 1966. Subgroups of gamma-A immune globulins. Proc. Soc. Exp. Bio1. Med-. 122: 910. 58 30. Milstein, C. and Pink, J.R.L. '1970. Structure and evolu tion of immunoglobulins. Prog. Biophys.. Mol. Biol. 21: 209.
3le Fahey, J.L. and Terry, W.D. 1964. Subclasses of human gamma globulin based on differences in the heavy polypeptide chains. Science 146: 400.
32. Fudenberg, H.H., Pink, J.R.L., Stites, D.P. and Wang, A.C. 1972. Basic Immunogenetics. Oxford University Press. New York p. 15.
33. Wells, J.V., Fudenberg, H.H. and Givol, D. 1973o Localization of idiotypic antigenic determinants in the Fv region of murine myeloma protein MOPC-315. P.N.A.S. 70: 1585.
34. Frangione, B., Milstein, C. and Franklin, E.C. 1968. Intrachain disulphide bridges in immunoglobulin G heavy chains. Biochem. J. 106: l5o
35. Frangione, Bo and Milstein, C. 1967. Disulphide bridges of immunoglobulin G-1 heavy chains. Nature 216: 939.
36. Pink, J.R.L. and Milstein, C. 1967~ Disulphide bridges of a human IgG protein. Nature 216: 941.
37. Rutishauser, u., Cunningham, B.A., Bennett, C., Konigsberg, W. and Edelman, G. 1968. Amino acid ··sequence of the Fe region of a human IgG-1 protein. P.N.AoS. 61: 1414.
38. Edelman, G.M., Cunningham, B~A., Gall, W.E., Gottlieb, P.D., Rutishauser, U. and Waxdal, M.J. 1969. The covalent structure of an entire IgG-1 immunoglobulin ·molecule. P.N.A.S. 63: 78.
39. Jones, H.B. 1848. On a new substance occurring in the urine of a patient with mollities ossium. Philos. Trans. Royal Soc. Land. 55: 62.
40. Edelman, G.M. and Gally, J.A. 1964. A model for the 7S antibody molecule. P.N.A.S. 51: 846.
41. Hilschmann, N. and Craig, L.C. 1965. Amino acid sequence studies with Bence-Janes proteins. P.N.A.S. 53: 1403.
42 •. Korngold, L. and Lipari, R. 1956. Multiple-myeloma protein. III. The antigenic relationship of Bence Jones proteins to normaL gamma globulin and multiple myeloma serum proteins. Cancer 9: 262.
43. Fahey, J.L. and Solomon, A. 1963. Two types of gamma myeloma proteins, gamma-1 Macroglobulins, beta-2a myeloma proteins, and B·ence ·Jones proteins identified by two groups of common antigenic determinants. J •. Clin. Invest. 42: 811. 59 44. Mannik, M. and Kunkel, H.G. 1963. Two major types of normal 7S gamma globulin. J. Ex·p. Med. 117: 213.
45. Kabat, E., Wu, T. and Bilofsky, H. 1978. Variable region genes for the immunoglobulin framework are assembled from small segments of DNA-A hypothesis. P.N.A.S. 75: 2429.
46. Wang, A.C., Fudenberg, H.H. Wells, J.V. and Roelcke, D. 1973. A new subgroup of the kappa chain variable region associated with anti-Pr cold agglutinins. Nature 243: 126.
47. Schneider, M. and Hilschmann, NG 1974. Die primarstruktur einer monoklonalen Immun.ogtobulin-L-Kette der subgruppe IV vom K-typ (Bence Jones protein LEN): eine neue subgruppe der L-Ketten vom K-typ. Hoppe-Seylers z. Physiol. Chern. 355: 1164.
48c Solomofi, A. 1976. Bence-Jones proteins and light chains of irnrnunoglobulins. New Eng. J. Med. 294: 17.
49. Solomon, A. and McLaughlin, C.Lo 1969. Bence-Jones proteins and light chains of immunoglobulins. II. Immunochemical differentiation and classification of kappa chains. J. Exp. Med. 130: 1295. . .
50. Sletten, K., Hannestad, K. and Harboe, M. 1974. Immuno chemical detection of the VkiV subgroup. Scancl. J. Immunol. 3: 219.
51. McLaughlin, C.L. and Solomon, A. 1974. Immunochemical classification of nonisolated light chains of immunoglobulins. J. Immunol. 113: 1369.
52. Solomon, A. and McLaughlin, C.L. 1971. Bence Jones proteins and light chains of immunoglobulins. IV. Immunochemical differentiation among proteins within each of the three established K-chain classes. J. Immunol. 106: 120 • . -- 53. McLaughlin, C.L. and Solomon, A. 1971. Bence Jones· proteins and light chains of immunoglobulins. VI. Immunochemical detection of interclass similarities among K-chains. J. Immunol. 101: 1699.
54. McLaughlin, C.L. and Solomon, A. 1972. Bence Jones proteins and light chains of immunoglobul-ins. VII. Localization of antigenic sites responsible for immunochemical heterogeneity of K-chains. J. Biol. Chern. 247: 5017.
55. Oudin, J. 1960. Allotype of rabbit serum proteins. J. Exp. Med. 112: 107.
56. Steinberg, A.G. 1969. Globulin polyrnorphisrns in man. Ann. Rev. Genetics 3: 25. 60 57. Grubb, R .. 1970. The genetic markers of human immunoglobulins .. Mol. Biol. Biochem. Biophys. 9: 1.
58. Natvig, J.B. and Kunkel, H.G. 1973. Human immunoglobulins: classes, subclasses, genetic variants and idiotypes. Adv. Immunol. 16: 1. ·
59o Milstein, C. 1966. Variations in amino acid sequence near the disulphide bridges of Bence Jones proteins. Nature· 209: 3 70.
60.- Baglioni, Ce, Zonta, L.A. and Cioli, D. 1966. Allelic antigenic factor Inv(a) of the light chains of human immunoglobulins: chemical basis. Science 152: 1517.
61. Hood, L. and Ein, D. 1968. Immunoglobulin lambda chain_ structure: two genes, one polypeptide chain. Nature 220: 764.
62. Eulitz, M. 1974. A new subgroup of human L-chains of t.he lambda type: primary structure of Bence Jone& protein DEL. Eur. J. Biochem. 50: 49.
63o Tischendorf, F.W. and Osserman, E.F. 1969. Two antigenic subtypes of human lambda immunoglobulin chains. J. Immunol. 102: 172.
64. Tischendorf, F.W. and Tischendorf, M.M. 1970. Markers of the variable and constant region of human lambda immuno globulin chains. Eur. J. Biochem. 13: 398.
65. Tischendorf, F.W., Tischendorf, M.M. and Osserman, E.F. 1970. Subgroup-specific antigenic marker on immunoglobulin lambda chains:. identification of three subtypes of the variable region. J. Immunol. 105: 1033.
66. Tischendorf, F.W. 1971. Antigenische klassifzierung der - paraproteine und normalen itnmunoglobuline vom Lambda-typ. z. Naturforsch 26: 1292.
67. Appel-la, E. and Ein, D. 1967. Two types of Lambda poly peptide chains in human immunoglobulins based on an amino acid substitution at position 190. P.N.A.S. 57: 1449.
68. Milstein, C., Frangione, B. and Pink, J.R.L. 1967. Studies on the va-riability of immunoglobulin sequence. Cold Spring Harbor Symp. Quant. Biol. 32:. 31.
69. Hess, M. and Hilschmann, N. 1971. Genetischer polymorphismus im konstanten teil vom humanen Immunoglobulin-L-Ketten vom lambda typ, II. Hoppe--Seylers z. Physiol. Chern. 352: 657.
70. Okada, Y., Nozu, Y. and Titani, K. 1972. Amino acid seq uence of a lambda chain of human IgA. Immunochem. 9.: 207. 61
71. Fett, J.W. and Duetsch, H.F. 1974. Primary str~cture of the MCG lambda chain. Biochem. 13: 4102.
72. Ein, D. and Fahey, J.L. 1967. Two types of lambda poly peptide chains in human immunoglobulins. Science 156: 947.
73. Hess, M., Hilschmann, N. and Rivat, L. 1971. Isotypes in human immunoglobulin lambda chains., Nature (New Biol.) 234: 58.
74. Ein, D. 1968. Nonallelic behavior of the OZ groups in human lambda immunoglobulin chains. P.N .• A.S. 60: 982.
75. Gibson, D., Levanson, M. and Smithies, 0. 1971. Hetero geneity of normal human immunoglobulin light chains: nonallelic variation in the constant region of lambda chains. Biochem. 10: 3114.
76. Fett, J.W.·and Deutsch, H.F. 1976. The variability of human lambda chairi c6nstant regions and some relationships to V-region sequences. Immunochem. ll= 149.
77. Putnam, F.W., Titani, Ko and Wikler, M. 1967. Structure and evolution of kappa and lambda light chains. Cold Spring Harbor Symp. Quant. Biol. 32: 9.
78. Burnet, F.M. 1959 .. The Clonal Selection Theory of Immunity. Nashville, Tenn.: Vanderbilt.
79. Brenner, S. and Milstein, C. 1966. Origin of antibody variation. Nature 211: 242.
80. Hilschmann, N. ·1967. Zum mechanismus der antikorperbildung. Hoppe-Seyler z. Physiol. Chern. 348: 1291.
81. Gally, J.A. and Edelman, G.M. 1972. The genetic control of immunoglobulin synthesis. Ann. Rev. Genetics 6: 1.
82. Whitehouse, H.L.K. 1967. Crossover model of antibody variability. Nature 215: 371.
83. Dreyer, W.J. and Bennett, J.C. 1965. The molecular basis of antibody formation: A paradox. P.N.A.S. 54: 864.
84. Cohn, M. in Rutgers Symposium on Nucleic Acids in Immunology. (edited by O.J. Plescia and W. Braun), Springer-Verlag, New York 1968, p. 671.
85. Lennox, E.S. and Cohn, M. 1967. Immunoglobulins. Ann. Rev. Biochem. 36: 365 .
.. 86. Cohn, M. In Nobel Symposium 3, Gamma Globulins (edited by J. Killander), Almquist and Wiksell, Stockholm 1967, p. 615. 62 87. Kohler, G. and Milstein, C. 1975. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256: 354.
88. Milstein, C., Brownlee, G.G., Cartwright, E.M., Jarvis, J.M. and Proudfoot, N.J. 1974_._ Sequence analysis of immuno globulin light chain ~essenger RNA. Nature 252: 354.
89. Gally, J .A. and Edelman, G·.M. 1970.· Somatic translocation of antibody genes. Nature 227: .341.
90. Kabat, D. 1972. Gene selection in hemoglobin and in antibody-synthesizing cells. Science 175: 134.
91. Hozumi, N. and Tone~una, S. 1976. Evidence for somatic rearrangement of immunoglobulin gene~ coding for variable and constant regions. P~N.A.S. 73: 3628.
92. Weber, K. and Osborn, M. 1969. The reliability of mole cular weight determinations by dodecyl sulfate polyacryla mide gel electrophoresis. J. Biol. Chern. 224: 4406.
93.· Ponstingl, H., Hess, ·M. and Hilschmann, N. 1971. Die primarstrl,lktur einer monoklonalen immunoglobulin L-Kette vom lambda typ, subgruppe IV (Bence Jones protein KERN). Hoppe_Seyler z. Physiol.. Chern. 352: 247.
94. Garver, F.A. and Hilschmann, N. 1972. The primary structure of a monoclonal human lambda type immunoglobulin L-chain of subgroup II (Bence Jones- protein NEI). Eur. J. -Biochem. 26: 10.
95. Raftery, M~A. arid Cole, D. 1966. On the aminoethylation of proteins. J. Biol. Chern. 241: 3457.
96. Moore, S. and Stein, W.H. 1954. A modified ninhydrin reagent for the photometric determination of amino acids and related compounds. J. Biol.· Chern. 211: 907.
97. Jepson, J. and Smith, I. 1953. 'Multiple dipping' proc- edures in paper chromatography: a specific test for ~ydroxy- proline. Nature 172: 1100.
98. Smith, I. 1953. Colour reactions on paper chromatograms by a dipping technique. Nature 171: 43.
99. Archer, R. and Crocker, C. 1952. Reactions coloreis specifiques de l'arginine et de la tyrosine realisies apres chromatographie sur papier. Biochem. Biophys. Acta 9: 704.
100. Sanger, F. and Tuppy, H. 1951. The amino acid sequence in the phenylalanyl chain of insulin. Biochem. J. 49: 463. 63 101. Toonnies, G. and Kolb, J.J. 1951. Techniques and reagents for paper chromatography. Anal. Chern. 23: 823.
102. Gray, WeR. 1967. Dansyl chloride procedure. Methods Enz. 11: 139o
103. Woods, K.R. and Wang, K.To 1967. Separation of dansylamino acids by polyamide layer chromatography. Biochern. Biophys. Acta 133.: 369.
104. Wikler, M. and Putnam, F.W. 1970c Amino acid sequence of human lambda chains: III. Tryptic peptides, chyrnotryptic peptides and sequence of protein BO. J. Biol. Chern. 245: 4488.
105. Ponstingl 3 H. and Hilschmann, N. 1969. Vollstandige amino sauresequenz einer lambda kette der subgruppe II (Bence Jones protein VIL). Hoppe Seyler Zc Physiol. Chern. 350: 1148. -
106. Wikler, M., Titani, K., Shinoda, T. and Putnam, F. 1967~ The complete amino acid sequence of a lambda type Bence Jones protein; J. Biol. Chern. 242: 1668.
107. Oudin, J. 1966. The genetic control of immunoglobulin synthesis. Proc. R. Soc. (Biol.) 166: 207.
108. Garver, F.A., Chang, L., Mendicino, J., tsobe, T. and Osserrnan, E.F. 1975. Primary structure of a deleted human lambda type immunoglobulin light chain containing carbohy- drate: Protein Srn X P.N.A.S. 72: 4559. ·
109. Edmundson, A.B.; Shober, F.A. and Ely, K~R. 1968. Char acterization of human L~type Bence Jones proteins containing carbohydrate. Arch. Biochern. Biophys. 127: 725.
110. Spiegelberg, H.L., Abel, C.A. and Fishkin, B.G. 1970. Localization of the carbohydrate within the variable region of light and heavy chains of human garnrna-G myeloma proteins. Biochern. 9: 4217.
111. Seidman, J.G., Leder, A., Nau, M., Norman, B. and Leder P. 1978. Antibody diversity. Science 202: 11.
112. Kiefer, C.R., Patton, H.M., McGuire, B.S. and Garver, F.A. 1980. The V region sequence of A Bence Jones protein WH: Evidence for separate germ-line sets within A subgroups. J. Irnrnunol. 124: 301.
113 • Kabat , E • , Wu , T • and Bi 1 of sky , H • 19 7 6 • Varia b 1e regions of immunoglobulin chains. Bolt, Beranek and Ne\vrnan, Cambridge, Mass.