Protein sequencing method

A protein sequencing method utilizing a composition of matter which comprises a D-C-B-A reagent wherein A is a moiety which can react with and bind to a terminal amino acid of a protein and can result in removal of the terminal amino amino acid, B is a moiety which provides steric separation between C and A, C is a nucleophilic moiety which can be detected, and D is a moiety which protects the C moiety from degradation or other modification, and is labile in acidic media and stable in neutral or basic media. The protein sequencing method and reagent can be used in micro sequencing apparatus.

FIELD OF THE INVENTION 
The invention relates to sequencing a polypeptide or protein molecule in 
order to determine its amino acid sequence. More particularly, the 
invention relates to a novel composition of matter and a method for the 
micro sequencing of very small amounts of protein. 
BACKGROUND OF THE INVENTION 
Proteins (polypeptides) are naturally occurring compounds that are composed 
of long chains of amino acids. Proteins are found throughout living things 
and function as hormones, structural elements, enzymes, immunoglobulins, 
and other constituents of living things. Research regarding the structure 
and functions of proteins often requires that the amino acid sequence 
(primary structure) of the protein be determined. In order for a protein 
or parts of a protein such as somatostatin, insulin, endorphins, etc. to 
be synthesized the sequence of amino acids must be determined before a 
synthesis can be attempted. In the search involving the function of 
proteins such as immunoglobulins, enzymes, viral coat proteins, and 
cell-surface proteins, the primary structure of the polypeptide must be 
determined in an attempt to elucidate the mechanism action of the protein. 
The primary sequence of amino acids in proteins or polypeptides is commonly 
determined by stepwise chemical degradation process in which single amino 
acids are removed one by one from the end of the polypeptide for 
identification. The Edman degradation is the preferred method, while other 
methods have been developed and can be used in certain instances. In the 
Edman degradation amino acid removal from the end of the protein is 
accomplished by reacting the N-terminal amino acid residue with a reagent 
which allows selective removal of that residue from the protein. The 
resulting amino acid derivative is converted into a stable compound which 
can be chemically removed from the reaction mixture and identified. 
Many physiologically active proteins are present in organisms at such 
extremely small concentrations that only very small amounts of the 
proteins can be obtained for sequencing analysis. Most current chemical 
sequencing methods are done with an amount of protein in the 5-100 
nanomole (5.times.10.sup.-9 to 10.sup.-8 mole) range. It has been reported 
that micro sequencing of polypeptides by reverse phase high pressure 
liquid chromatography using ultraviolet light detection means has been 
accomplished with protein samples in the range of 50-500 picomole 
(5.times.10.sup.-11 to 5.times.10.sup.-10 mole) range. Other methods used 
in the micro sequencing of polypeptides involve radio labeling of the 
peptide or reagent, intrinsic radio labeling of the polypeptide, and 
enhanced UV detection of sequence degradation products, and others. While 
we will not discuss the details of these methods, each method has its 
limitations and restrictions. They have not been used with overall 
satisfactory results. The current best art is computer-aided UV detection 
of PTH amino acids from a microsequencer. 
Many techniques have been developed in recent years for the analytical 
separation of polypeptides or proteins having high physiological activity 
in very low quantities of about 10.sup.-12 mole (1 picomole), about 50 
nanograms of a 50,000 molecular weight protein. A sequencing method 
developed specifically for such small amounts of proteins is desirable. 
One detection method which has an inherent sensitivity in the femtomole 
(10.sup.-13 to 10.sup.-15 mole) range is fluorescence. Attempts have been 
made to use fluorescent isothiocyanates in the Edman degradation, however 
overall success in micro sequencing of small amounts of protein has not 
been demonstrated to date. 
Accordingly a substantial need exists for a micro sequencing reagent and 
method adapted for the sequencing of amounts of protein in the low 
picomole to sub-picomole range of protein. 
BRIEF SUMMARY OF THE INVENTION 
We have found a novel protein micro sequencing reagent which can be used in 
sequencing to determine the primary structure of very small amounts of 
protein. The novel micro sequencing reagent comprises a compound having 
the formula A-B-C-D wherein A comprises a chemical functional group which 
can be used to react with a terminal amino acid and which after chemical 
processing can cooperate in the removal of the terminal amino acid from 
the polypeptide chain. B comprises a moiety which connects group A with 
group C and can also function as a ultraviolet light absorbing compound 
and/or as a reactivity-modifying moiety for group A. Group C comprises a 
moiety capable of detection at extremely low concentration or a moiety 
which can be reacted with a group capable of providing the low detection 
limits required in sequencing very small amounts of protein. D is a group 
which protects the C group from degradation or other undesirable reaction 
during the reaction of the sequencing reagent with the terminal amino 
acid. The micro sequencing reagent must be able to react with the 
N-terminal amino acid and cleave the terminal amino acid from the 
polypeptide chain. The D group is removed from the reaction product of the 
amino acid and sequencing reagent exposing the C group which can then be 
used for further reaction with appropriate detection means, or which can 
be itself detected. 
For amino terminal sequencing: 
"A" may be but is not limited to such primary amine-reactive species as 
--N.dbd.C.dbd.S, --N.dbd.C.dbd.O, 
##STR1## 
for carboxyl terminal sequencing: 
"A" may be but is not limited to such carboxyl reactive species as --R--OH 
where R may be either an alkyl or aryl group; 
For any mode of sequencing: 
"B" may be any alkyl chain which may or may not be branched and/or any aryl 
ring and/or rings with akyl substitution and/or substitutions at the meta 
and/or para positions; 
"C" may be any nucleophilic moiety such as --NH--, --S--, --O-- but is not 
limited to these groups; 
"D" may be but is not limited to acid (or base) labile protecting groups 
such as t-butyloxycarbonyl, benzyl, carbobenzyloxy carbonyl, etc. A wide 
variety of protecting groups is known in the art. 
An aspect of the invention is the protein micro sequencing reagent. A 
second aspect of the invention is a method for sequencing small quantities 
of protein using the novel protein micro sequencing reagent of the 
invention. A third aspect of the invention is a method of preparation of 
protein micro sequencing reagents. 
DETAILED DISCUSSION 
A preferred protein micro sequencing reagent comprises a reagent that can 
be used in the Edman sequencing degradation reactions. The micro 
sequencing reagent comprises a reagent having the following formula: 
(L--NH).sub.n --R--N.dbd.C.dbd.S wherein n is an integer, L is a group 
which is labile in acid but is stable in neutral and basic media, and R is 
an aliphatic, aromatic, or mixed aliphatic, aromatic group which 
sterically and electronically separates the nitrogen group from the 
isothiocyanate group, preserving the desired reactivity. The reagent is 
designed such that during micro sequencing of a polypeptide the NH group 
is protected from reaction with the isothiocyanate amine-specific reactive 
moiety during the initial coupling step of the micro sequencing reagent 
with the N-terminal amino acid. After coupling is complete and unreacted 
micro sequencing reagent has been removed the labile L group attached to 
the amino group can be removed simultaneously with the cyclization step in 
the sequencing reaction giving a thiazolinone produced in the cleavage of 
the N-terminal amino acid from the protein. The amino group in acid 
solution appears as an inert protonated cationic moiety during cleavage 
and removal of the L group, and during conversion to the thiohydantoin 
derivative. After chromatographic separation the protonated cationic 
moiety can be deprotonated and converted to a nucleophilic amino group. 
The amino group can then be reacted with a fluorogenic reagent such as 
4-phenylspiro[furan-2(3H),1'-phthalan]-3,3'-dione(fluorescamine) or 
o-phthalaldehyde, (OPA) or other fluorogenic reagents. The resulting 
attached fluorescent moiety can be detected using manual or automated 
fluorimetry taking advantage of the fluorescent properties of the attached 
fluorescent moiety. 
A preferred L group comprises groups having the formula 
##STR2## 
wherein R is a branched chain alkyl, or aryl group having from about 1 to 
about 20 carbon atoms such as tertiary butyl, tertiary amyl, 
1-methylcyclohexyl, biphenylisopropyl, etc. The use of the preferred L 
group results in micro sequencing reagents which are generally freely 
soluble in organic solvents commonly used in Edman degradation methods. 
While the invention is directed primarily to sequencing of proteins 
beginning at the N-terminal amino acid, the general approach and many of 
the variations can also be adapted to sequencing proteins beginning at the 
C terminal amino acid. 
Protein Sequencing 
Any peptide or protein which is to be amino terminally sequenced must have 
a free N-terminal amino acid. Protected or blocked N-terminal amino acids 
must be converted to free N-terminal amino acids before sequencing can 
begin. The protein can be sequenced in free solution or on a variety of 
well known solid support media. Excess sequencing reagent (formula 
L--NH--[CH.sub.2 ].sub.n --R--NCS) is added to the protein in the presence 
of base or after neutralization of the amino group to the protein. After 
sufficient time to insure complete reaction between the sequencing reagent 
and the N-terminal amino acid, the unreacted reagent and other chemicals 
or by-products are removed by washing. The resulting reaction product 
between the sequencing reagent and the protein can have the following 
formula (II) 
##STR3## 
In order to effect cyclization of the reaction product and concommittant 
cleavage of the N-terminal amino acid from the protein, the reaction 
product is treated with anhydrous acid. Simultaneously with the 
cyclization-cleavage, the L group on the amino group is removed, resulting 
in a species (III) and a free terminal amino acid on the peptide chain 
which can then be subjected to another cycle of the degradation. The 
cyclized reaction product is removed from the residual protein by washing 
with suitable solvents. The washes are collected and the recovered 
thiazolinone (III) is converted to the stable thiohydantoin (IV), by 
treatment with aqueous or alcholic acid at elevated temperatures. 
##STR4## 
The resulting substituted reaction product (IV) is subjected to 
chromatographic analysis, most commonly reverse phase high pressure liquid 
chromatography (HPLC). Subsequent to the separation, the reaction product 
can be detected by ultraviolet absorbance if desired or preferably can be 
reacted with a fluorogenic reagent (such as OPA or fluorescamine), under 
appropriate reaction conditions, and detected by fluorimetry. Both UV and 
fluorescent detection can be performed in the same experiment, 
consecutively in the order given. Standard methods, equipment, solvents, 
buffers, reaction sequences of manual and automatic sequencing can be used 
with minor modifications where necessary to accomodate the nature of the 
resulting reaction product. Preferred automated methods include the 
spinning cup, solid phase, and gas liquid phase sequencing methods. 
Further, common pre or post-column chromatography derivatization 
techniques and flow fluorimetry can be used for the fluorescence detection 
of the reaction product. Adjustment of reaction conditions such as 
reagent, buffer, pH, flow rate, delay time, excitation, and detection 
wavelengths may be necessary to optimize sensitivity. The following 
Examples contain descriptions of two micro sequencing reagents, the use in 
sequencing proteins, and a best mode.

EXAMPLE I 
Synthesis of 4-(tertiary-butyloxycarbonylamino)phenylisothiocyanate 
##STR5## 
Into a glass Erlenmeyer flask at ambient temperature was placed 1 gram 
(4.81 millimole) of t-butyloxycarbonyl-1,4-phenylenediamine 
##STR6## 
dissolved in 20 milliliters of chloroform. Into the vessel was added 28 
milliliters of aqueous 1 M sodium bicarbonate. The aqueous and chloroform 
phases were rapidly stirred with a magnetic stirrer until the phases were 
intimately contacted. Into the rapidly stirred biphasic mixture was added 
2.21 grams (19.2 millimole) thiophosgene 
##STR7## 
Copious amounts of solid deposited and rapidly redissolved. The mixture 
was permitted to react at ambient temperature for 2 hours. The reaction 
was monitored by removing microliter aliquots of the reaction mixture for 
thin layer chromatography analysis (silica gel GF, 250 micron thickness; 
developed in CHCl.sub.3 :MeOH: HOAC, 85:10:5 by volume; visualized by 
short wave ultraviolet, and by spraying with 0.1% ninhydrin in n-butanol 
solvent followed by heat. Starting material, having an Rf of 0.45 and 
instantly visualized by ninhydrin and heat, disappeared giving a principal 
compound Rf 0.9 slowly visualized by ninhydrin and heat, and a minor 
compound Rf 0.85. The reaction appeared to be complete within about 15 
minutes. After about 2 hours, mixing was terminated and the phases 
separated. The chloroform layer containing the product was washed with 
three 20 milliliter volumes of water, dried over anhydrous magnesium 
sulfate and subjected to rotary evaporation at reduced pressure. (Caution: 
use of a hood is recommended; thiophosgene vapor is present.) Into the 
rotary evaporator flask was placed three 10 ml portions of dichloromethane 
which were removed in turn using reduced pressure. The crude reaction 
product was a cream color solid, soluble in acetone, methanol, benzene and 
ethyl acetate, but insoluble in petroleum ether. The liquid product was 
recrystallized from hot petroleum ether:ethyl acetate (20:2). The 
resulting white crystalline product was washed with petroleum ether and 
dried under vacuum. The synthetic route yielded 0.251 grams (21% of 
theoretical). A repeat run on a 6.8-fold scale gave a 49% yield of the 
same compound. Infrared analysis showed the strong band at 2100 cm.sup.-1 
characteristic of isothiocyanates, and the band at 1690 cm.sup.-1 
characteristic of urethane carbonyl. 
The neutralization of hydrochloric acid liberated during the synthesis of 
the isothiocyanate is extremely important since the acid can result in the 
removal of the t-butyloxycarbonyl group. Accordingly, the bi phasic 
reaction system in which the bicarbonate neutralizes the acid produced in 
the reaction in the chloroform phase is critical to forming the micro 
sequencing reagent. 
EXAMPLE II 
Synthesis of 4-(tertiary butyloxycarbonylaminomethyl) phenyl isothiocyanate 
##STR8## 
Into a glass Erlenmeyer flask was placed 5 grams (26.5 millimoles) of 
4-nitrobenzylamine hydrochloride suspended in 10 milliliters tertiary 
butanol and 5 milliliters of water. Concentrated aqueous sodium hydroxide 
2.16 milliliters (27 millimoles) was added to the suspension followed by 
10 milliliters of dimethyl formamide. Into the resulting dark colored 
solution having some suspended solids was placed 5.8 grams (26.6 
millimoles) di-tertiarybutyldicarbonate dissolved in 10 milliliters of 
tertiary butanol, drop-wise with stirring over a period of 30 minutes. The 
reaction was allowed to continue overnight. Considerable solid was 
deposited. At the completion of the reaction the reaction mixture was 
diluted with 40 milliliters of water and the crude mixture was extracted 
with three 50-ml volumes of petroleum ether. The aqueous layer, titrated 
to pH 2 in the presence of ethyl acetate, was extracted with three 
60-milliliter portions of ethyl acetate which were combined and dried with 
anhydrous magnesium sulfate combined with the petroleum ether layers. The 
crude product, 4(t-butyloxycarbonylaminomethyl)nitrobenzene, obtained from 
the combined layers were analyzed by TLC chromatography as described above 
which showed the product (Rf 0.9; slowly ninhydrin positive) to be about 
97% pure. The crude solid was dissolved in 50 milliliters of 90% by volume 
acetic acid in water in a glass Erlenmeyer flask. 8.6 grams of zinc dust 
(132.5 millimoles) was added to the vigorously stirred solution. The 
progress of the reaction was monitored chromatography as discussed. The 
zinc reduction was complete after about 1 hour at ambient temperatures. 
The starting material (Rf 0.9, slowly ninhydrin-positive) being replaced 
by a compound of Rf 0.65, instantly ninhydrin-positive. This is the 
direction of Rf change expected for the change chemical nature; 
furthermore, the product showed colored decomposition products on silica 
gel GF, characteristic of an aniline. Two hours after the reaction began 
the mixture was diluted with 300 milliliters of water, titrated to pH 6 
with aqueous sodium hydroxide in the presence of 200 milliliters of ethyl 
acetate. The aqueous phase was extracted with three 100 milliliter volumes 
of ethyl acetate. The combined ethyl acetate layers were dried and solvent 
was removed with a rotary evaporator under reduced pressure yielding a 
thick yellow oil. The crude 4-(tertiarybutyloxycarbonyl aminomethyl) 
aniline was converted to the isothiocyanate following the same procedure 
described in Example I using 40 milliliters chloroform, 60 milliliters 1 
molar aqueous sodium bicarbonate, 2.21 grams (19.2 millimoles) 
thiophosgene. The reaction yielded 1.73 grams of product. The product was 
freely soluble in acetonitrile and dichloromethane. Infrared analysis 
showed the characteristic strong isothiocyanate bands at 2100 cm.sup.-1 
and the urethane carbonyl at 1690 cm.sup.-1. 
EXAMPLE III 
USE IN SEQUENCING 
4(t-butyloxycarbonyl-aminomethyl)phenylisothiocyanate (BAM PITC) was 
dissolved in acetonitrile at a concentration of 5% (w/v) and placed in the 
coupling reagent reservoir and delivery system of an unmodified solid 
phase sequencer (Sequemat Mini 15). The peptide 
Leu-Ala-Gly-Val-Leu-Ala-Gly-Val-Phe coupled to 
aminomethyl-copoly(styrene-1% divinylbenzene) resin (15 mg, about 5 
nanomoles of peptide) was loaded in a standard 3 mm.times.100 mm glass 
reaction column where it was mixed with sufficient glass beads (240-280 
mesh) to prevent clogging of the column. The remainder of the reaction 
column was filled with the same glass beads. A standard solid phase Edman 
degradation was performed according to the following protocol: 
______________________________________ 
Step Functions Channel Time 
______________________________________ 
0 Start 8 0-1 
1 MeOH 1,8 1-5 
2 Buffer 3,8 5-6 
3 Buffer/PITC 3,4,8 6-19 
4 Buffer 3,8 19-23 
5 MeOH 1,8 23-31 
6 DCE 2,8 31-35 
7 MeOH 1,8 35-39 
8 DCE 2,8 39-43 
9 MeOH/F.C. 1,9,8 43-45 
10 TFA/Collect/Reag. Part. 
5 45-54 
11 TFA/Collect/Channel 15/Reag. Part 
5,15 54-56 
12 TFA/Collect/Reag. Part. 
5 56-62 
13 MeOH/Collect 1 62-63 
14 Rest 8 63-64 
15 MeOH/Collect 1 64-65 
16 End E.P. 65-0 
______________________________________ 
At each cycle of the degradation, the cleaved 
amino-methyl-amino-thiozolinone derivative was washed from the reaction 
column in the trifluoroacetic acid used to effect the cleavage, and was 
collected in a conical glass centrifuge tube (3 mm.times.150 mm) in the 
fraction collector. The delivery line to the fraction collector was washed 
with methanol which was also collected in the same tube. After completion 
of the sequencing, the fractions were blown to dryness under a stream of 
nitrogen at 50.degree. C. 1M HCl in methanol (1 mL) was added and the 
tubes heated at 65.degree. C. for 10 minutes to effect conversion to the 
PTH derivative. The samples were again reduced to dryness under a stream 
of nitrogen at 50.degree. C. For analysis, the residue in each tube was 
taken up in 300 uL of methanol. 
EXAMPLE IV 
SEQUENCING 
4-(t-butyloxycarbonyl-aminomethyl(phenylisothiocyanate prepared as in 
Example II was used to sequence the peptide 
Leu-Ala-Gly-Val-Leu-Ala-Gly-Val-Phe covalently attached to 
aminomethyl-poly-(styrene-1% divinylbenzene) resin (15 mg, about 5 
nanomole). The procedures described in Example II were followed, with the 
exception that each cycle the cleaved aminomethyl-anilinothiozolinone in 
trifluoroacetic acid and the subsequent methanol washes were delivered to 
the fraction vessel of a standard P-6 auto convertor where the following 
operations were performed automatically. The TFA and methanol were removed 
under a stream of nitrogen, conversion to the PTH derivative was effected 
with 1M HCl in methanol which was subsequently removed under a stream of 
nitrogen. All operations were performed at 65.degree. C. The resulting PTH 
derivative was rinsed with several lots of methanol into a conical 
centrifuge tube in the fraction collector. The standard P-6 program was 
used. After completion of the sequencing run, the fractions in each tube 
were reduced to dryness under a stream of nitrogen at 50.degree. C. and 
the residue taken up in 300 microliters methanol for analysis. 
EXAMPLE V 
DETECTION 
Aniline, benzylamine, and phenethylamine were used as models for the 
NH.sub.2 (CH.sub.2).sub.n PTH derivatives, where n=0, 1, 2 respectively. 
Aliquots of these compounds were dissolved in methanol and spotted on TLC 
plates (silica gel GF, 250, microns) (100, 200, and 500 picomole of each 
in 1, 2, 5 uL methanol, respectively). The plates were sprayed with buffer 
and with fluorogenic reagent: 
Fluorescamine 
Buffer: 1% triethylamine in acetone (v/v) 
Reagent: 0001% fluorescamine in acetone (w/v) 
O-phthalaldehyde 
Buffer: 1% triethylamine+0.05% beta-mercaptoethanol in acetone (v/v) 
Reagent: 10 mg OPA in 33 mL acetone. 
The plates were examined under a long wave length UV lamp. The three 
compounds each gave a bright green fluorescence on a dark purple 
background after reaction with fluorescamine; this took several minutes to 
reach maximum intensity and was stable for hours at room temperature. One 
hundred picomole of each compound was readily detected. With OPA, 
phenethylamine and benzylamine gave a pale blue fluorescence; this 
developed very rapidly (seconds) and was not long lived (faded noticeably 
after 30 minutes). To the eye, the OPA response was lower, with 100 
picomoles barely detectable. Aniline did not react with OPA under these 
conditions. 
A combined reagent spray was also used for fluorescamine detection: 
Combined Reagent and Buffer for Fluorescamine 
0.001% fluorescamine, plus 
0.01% triethylamine in acetonitrile. 
This spray, used on samples of 500 picomole each of aniline, benzylamine, 
and phenethylamine with fresh reagent gave the same strong green 
fluorescence under the long wave length UV lamp for each compound as 
obtained with the separate reagents in acetone. However, 41/2 hours after 
being made up the single spray gave a noticeably higher background which 
became high enough to significantly interfere when used 71/2 hours after 
being made up. 
The PTH derivative from cycle #2 (Ala) of the sequencing in Example 2 was 
spotted on a TLC plate (as above) (20 uL of 300 uL in methanol; 333 
picomole based on 5 nanomole sequenced). Spraying with either 
fluorescamine or OPA gave a positive response with fluorescence of about 
the expected intensity compared with the response of an equal amount of 
benzylamine. The response was much stronger with fluorescamine than with 
OPA. This indicates that the derivatives obtained from 
sequencing/conversion contain a free amino group in the expected amount. 
HPLC. 
Aniline, benzylamine, and phenethylamine were separated by reverse phase 
HPLC on a C18 micro Bondapak column using an isocratic system consisting 
of 20% methanol in 10 mM potassium phosphate, pH 2.5, pumping at 0.7 mL 
per minute. The compounds were detected by UV absorbence at 214 nm, and 
eluted at 4.93, 5.71, 6.80 minutes, respectively. After passage through 
the UV detector, the effluent stream was mixed with buffered OPA reagent 
(475 mL 0.8 M potassium borate, pH 10.5; helium degassed, plus 400 mg OPA 
in 2mL MeOH+ 1mL beta-mercaptoethanol, kept under helium [fresh daily]) 
pumping at 0.7 mL/min. The combined effluent stream passed through a delay 
coil (50 feet of 1/10,000 inch I.D. tubing) to a flow fluorimeter (Gilson 
Model 121; Corning designation excitation filter 7-60, emission filter 
450-7C). Aniline did not fluoresce under these conditions. Benzylamine and 
phenethylamine gave strong fluorescence signals, with benzylamine giving 
twice the signal of phenethylamine. Benzylamine gave full scale detection 
(greater than 18 cm) from a baseline with 1 mm peak-to-peak noise for 50 
picomoles injected. 
A similar experiment was performed with fluorescamine detection. Aniline 
and benzylamine were separated by reverse phase HPLC on a C18 micro 
Bondapak column using an isocratic system consisting of 10% acetonitrile 
in 10 mM potassium phosphate, pH 5.6, pumping at 1.4 mL per minute. The 
compounds were detected by UV absorbence at 214 mm. After passage through 
the UV detector, the effluent stream was mixed with "buffered" 
fluorescamine reagent (15 mg fluorescamine in 100 mL acetonitrile 
containing 0.15% (v/v) triethylamine)--used within hours of being made 
up--pumping at 0.7 mL/min. The combined effluent stream passed through the 
delay coil described above and was detected in the same flow fluorimeter. 
Both compounds gave strong fluorescence signals under these conditions. 
EXAMPLE VII 
Analysis and Detection of Sequencing Products 
The aminomethyl-PTH derivatives derived from the sequencing described in 
Example 2 were analyzed by reverse phase HPLC using two supelcosil C18 
analytical columns in series with an isocrate eluant consisting of 33.3% 
(v/v) acetonitrile, 0.4% (v/v) dichloroethane, in 20 mM sodium acetate, pH 
4.7, flow rate 1.2 mL/min. Detection was by UV absorbance at 254 nm. 1/20 
of each sample was injected. UV absorbing derivatives were detected in the 
expected amounts (compared with analysis of an identical sequencer run 
using PITC reagent). The elution positions were somewhat earlier for the 
aminoethyl-PTH derivatives compared with the PTH derivatives. This is 
consistent with the expected effect of an additional ammonium-methylene 
moiety. Also as expected, the effect was more pronounced for more 
hydrophobic PTH. See Table. Repetitive stepwise yields for the sequencing 
run were 0.92, 0.91 based on Ala.sup.2,6, Gly.sup.3,7, respectively. The 
control run using PITC gave repetitive stepwise yields of 0.91, 0.94. 
Thus, the Bam-PITC is as efficient as PITC for sequencing under the 
conditions used. 
TABLE 
______________________________________ 
Amino Acid 
PITC Run Bam PITC Run 
(Sequencer 
Elution time/Amount 
Elution time/Amount 
Cycle) (area units) (area units) 
______________________________________ 
Leu (1) 30.13 min/384 16.20 min/432 
Ala (2) 9.65 min/426 6.45 min/479 
Gly (3) 7.52 min/500 5.70 min/326 
Phe (9) 26.36 min/-- 13.70 min/-- 
______________________________________ 
Large amounts of a UV absorbing impurity were present in some cycles, 
obscuring parts of the chromatogram. This is typical of the type of 
impurity occasionally present in sequencing. 
A further aliquot (1/30) of the same set of samples was reanalyzed under 
modified HPLC conditions: C18 microBondapak column, isocratic elution with 
10% acetonitrile in 10 mM potassium phosphate, pH 5.65, flow rate 
0.80mL/min. Detection was by UV at 254 nm, and subsequently the effluent 
stream was mixed with "buffered" fluorescamine reagent (15 mg in 100 mL 
acetonitrile containing 0.15% (v/v) triethylamine kept under an inert 
atmosphere) at 0.40 mL/min. The combined effluent stream passed through 
the delay coil and fluorimeter described in Example VI. Filters used were: 
Corning designation: excitation 7-51; emission 3-71. The UV and 
fluorescence signals were plotted simultaneously on a two pen 
integrator/printer/plotter. Strong fluorescence peaks corresponding to the 
UV absorbance peaks of the aminomethyl-PTH derivatives were observed. Most 
significantly, the strongly UV-absorbing impurity was overwhelmingly 
evident in the UV detection channel, but was not detected in the 
fluorescence signal. This illustrates the particular utility of the 
Bam-PITC reagent in that the UV absorbing impurity from commonly used 
sequenator reagents did not appear in fluorescence detection, UV detection 
can be used for macroscale sequencing and fluorescence detection for 
microscale sequencing with the same reagent. 
While only certain embodiments of our invention have been described in 
specific detail it will be apparent to those skilled in this art that 
other specific embodiments may be practiced, and many changes may all be 
made within the spirit of the invention, and it is intended that all such 
embodiments and changes be considered within the scope of the invention 
which resides wholely within the claims hereinafter appended.