Pharmacology 19: 223 -227 (1979)

An Analyst in Biomedical Research Sidney Udenfriend Roche Institute of Molecular Biology, Nutley, N.J.

Key Words. Opioid peptides • /3-Endorphin - Enkephalin • Fluorescamine • Pro-opiocortin • Fluorescence • Interferon • High performance liquid chromatography Abstract. Fluorescamine is a nonfluorescent substance that reacts with primary amines to yield

I chose this title because I consider it most fitting for a volume dedicated to Bernard Brodie. It was in his laboratory that I learned about the importance of quantative analysis in research and, most important, that new dis­ coveries are prompted by newly developed analytical methods. During our first years to­ gether we not only developed many new analyt­ ical procedures, but a philosophy and an ap­ proach to analysis that have guided me through­ out my career. This volume provides me with the opportunity to present the developments which led to the most recent analytical method­ ology in my laboratory and its exciting applica­ tions. My long interest in fluorescence, which was also fostered by Dr. Brodie, led me several years ago to investigate the assay for phenylalanine that is widely used in the diagnosis of phenyl­ ketonuria (11). The reagents used in the assay are ninhydrin and a peptide that react with

phenylalanine to yield fluorescence. Out of these studies on the phenylalanine reaction came fluorescamine (13), a truly amazing reagent. Fluorescamine is a nonfluorescent sub­ stance that reacts with primary amines to yield intensely fluorescent products. At pH 9 and at room temperature the reaction with primary amines occurs in milliseconds and excess re­ agent is hydrolyzed to nonfluorescent products within seconds. Another important advantage is that ammonia, a serious source of contamin­ ation in amine assays with most reagents (i.e., ninhydrin and o-phthaldialdehyde) yields little fluorescence with fluorescamine. Soon after the discovery of fluorescamine my colleagues began developing automated in­ strumentation for its application to the assay of amino acids, peptides and proteins (12). We have developed automated amino acid analyzers that routinely carry out assays below the 100pmol level and give meaningful data with as

Downloaded by: Boston University - 1/14/2019 3:51:20 AM

intensely fluorescent products. We have utilized high performance liquid chromatography along with fluorescamine assay for many studies relating to the biochemistry of the opioid peptides.

little as 10—20 pmol. Fluorescamine has also been applied to the automated on-line monitor­ ing of chromatographic columns used for the purification of proteins and peptides. Again, picomole amounts are sufficient for detection and, when preparative chromatography is car­ ried out with nanomole quantities, the aliquots required for monitoring are negligible. Recently developed methods for sequencing in the picomole range complement methods developed in our laboratories and make possible a complete microchemistry for proteins and peptides. Microchemical procedures should not be considered mere affectation nor limited to un­ usual situations. They make possible research projects that would not otherwise be possible or else too costly to undertake. In the 1950s microfluorometric methods made possible the research that led to our vast knowledge of the biochemistry and pharmacology of catechol­ amines. Amines, steroids, cyclic nucleotides and prostaglandins are among the many types of chemical messengers secreted within multicel­ lular organisms to transmit information from one cell to another. Hormones, neurotrans­ mitters and lymphokines represent but some of the huge number of chemical messengers in­ volved in regulatory processes. Although low molecular weight chemical messengers were the first to be discovered and have been studied most intensively, it is now fairly clear that peptide and protein messengers are far more numerous than any other chemical form of messenger. Over 40 peptide and protein messen­ gers have been completely characterized and there is evidence of many more. Each year adds to the list of established peptide and protein messengers. At first glance it would appear that peptides and proteins are the more complex types of


messengers. Acetylcholine and noradrenaline are indeed simpler molecules than /3-endorphin and corticotropin. However, the overall path­ way of biosynthesis of a molecule such as noradrenaline requires several intermediates, each requiring an enzyme and cofactors. All the above require many genetic determinants. By contrast corticotropin as well as /3-endorphin arise from a common intermediate (see below) which may be the direct product of translation. Protease action liberates the two peptide hor­ mones. Because they are so closely related to the product of translation, protein messengers probably represent the most primitive types of chemical messengers developed by multicellular organisms. There is one thing that all chemical messen­ gers have in common. They are highly active and only small amounts are required to initiate a sequence of cellular reactions. In the case of the catecholamines, micromethodology, starting with spectrofluorometric assay, made possible the many advances in our knowledge concerning their biosynthesis, metabolism, stor­ age and release. We considered the assays for catecholamines quite sensitive. However, there are 3—4 nmol of noradrenaline/g of striatum. By contrast the Leu-enkephalin content of the striatum is only about 200 pmol/g. The stri­ atum is actually one of the richest sources of enkephalin. Peptide messengers are generally found in very low molar concentration. Fur­ thermore, the specific information in peptide messengers is in their unique amino acid se­ quence and there is no chemical reaction to distinguish one peptide from another. Sensitive assays of individual peptide and protein messen­ gers are available. One of the most widely used is radioimmunoassay. While its sensitivity is extremely high (femtomole range) its inherent specificity is always open to question. Antisera, as generally prepared, are a mixture of anti-

Downloaded by: Boston University - 1/14/2019 3:51:20 AM


An Analyst in Biomedical Research



Fig. 1. Separation of known opioid peptides on a single chromatographic column. The enkephalins (10 nmol) and the endorphins (2 nmol) were loaded on the RP-18 column in 1 ml of equilibration buffer. The eluting buffer was 0.5 M formic acid adjusted to pH 4.0 with pyridine. The gradient was 0-20% npropanol (------ ). Fractions were collected at 3-min intervals. Peptides were detected using the flúorescamine system.

bodies directed to different portions of the peptide molecule. Such antisera cannot distin­ guish (absolutely) the antigen from its precur­ sors or metabolites. Furthermore, the presence of even part of an antigenic sequence in a totally unrelated protein can lead to significant error if that protein is present in amounts far greater than the antigen. Recognizing this limi­ tation of radioimmunoassay, some have com­ bined it with column chromatography. Gel filtration, which is most widely used for this purpose, does not, however, possess adequate resolution to insure specificity. High perfor­

mance liquid chromatography (HPLC) is now applicable to peptides and proteins. A typical analytical column provides over 1,000 theoret­ ical plates and can resolve peptides or proteins that are difficult to separate by any other means. An example of the high resolution of HPLC is shown in figure 1 (3). a-Endorphin is completely resolved from 7 -endorphin although the two contain the same sequence of 16 amino acid residues; 7 -endorphin has an additional leucine residue at the carboxy terminus. Of even greater interest is the resolution of Met5endorphin from Leus-cndorphin. The combina­ tion of HPLC with radioimmunoassay should provide the sensitivity and selectivity required for meaningful research. The sensitivity of the fluorescamine pro­ cedure makes it possible to isolate and charac­ terize active peptides from small amounts of tissue. As an example, mammalian /3-lipotropin was originally isolated from thousands of pitu­ itary glands. With our fluorescent procedures one camel pituitary was sufficient. Relatively

Downloaded by: Boston University - 1/14/2019 3:51:20 AM

10 9

few rat pituitary glands (ca. 40) were adequate for characterization of the opioid peptides (6). The ability to work with small amounts of tissue and to use laboratory animals makes it possible to control proteolytic activity which is a serious problem with material collected at autopsy or at the slaughterhouse. We have utilized HPLC along with fluorescamine assay for many studies relating to the biochemistry of the opioid peptides. In our first studies we showed that the (3-endorphin (3,500 MW) in rat pituitaries is identical with the (3-endorphin isolated from sheep, beef and camels (7). This was important since the rat is the species in which most physiological and pharmacological studies are carried out. We also purified rat (3-lipotropin (ca. 10,000 MW) and demonstrated a large precursor (ca. 30,000) in rat pituitaries (6). That precursor was subse­ quently shown to contain the corticotropin sequence as well (4, 8). Because it contains both sequences we have named it pro-opiocortin. Partial sequencing of mouse pro-opiocortin has already been achieved using cloning procedures (5). We have recently purified camel pro-opiocortin to homogeneity (1) and plan to sequence it by microsequencing techniques. It will also permit us to isolate the proteases that cleave the molecule into its two biologically active components. When the same methodology was applied to striatal and intestinal extracts Met-enkephalin and Leu-enkephalin were identified, as was expected. However, none of the larger peptides, that are so plentiful in the pituitary gland, were observed. Instead of the 30,000 MW pro-opio­ cortin we found a much larger protein con­ taining within it the opiate sequence (2). The striatal ‘precursor’ differs not only in size (40,000 -50,000 MW) from pro-opiocortin but also in its amino acid sequence near the active site.


On treatment with trypsin pro-opiocortin, as well as (3-lipotropin ((3-LPH) and the endor­ phins, yield the nonapeptide (3-LPH 61—69. The striatal ‘precursor’ does not yield this nonapep­ tide but does yield other trypsin-resistant active peptides. Recently we have isolated a heptapeptide fragment from beef adrenal medulla which has opioid activity. It contains the metenkephalin sequence plus two additional residues that do not appear in the known pituitary opoid peptides (13). Apparently (3-endorphin is not a precursor of Met-enkephalin even though they apparently share the same active pentapeptide sequence. Obviously both are present in tissues and both trigger isolated opiate receptors. Our interpreta­ tion is that pituitary (3-endorphin is a hormone and like cortiotropin reaches its receptors (still unknown) via the blood. The enkephalins, on the other hand, are found at nerve endings and presumably have neuroregulatory functions there. (3-Endorphin is relatively stable in blood and tissue fluids, a requirement for a hormone. In contrast, the enkephalins are rapidly inactivated in tissue fluids, which is required for the action of a neuroregulatory substance. It should be noted that the stability of (3-endorphin com­ pared to enkephalin is not in keeping with a precursor role. Being active, and more stable, if it were at the same receptor sites in nerve endings it would compete with enkephalin. Obviously, we still have much to learn of the biochemistry as well as the physiology of the recently discovered opiate peptides. The fluorescamine HPLC methodology is being put to other uses where quantities of active peptide are limited. The most notable success by other colleagues in our Institute was the recent purification of human leukocyte interferon to homogeneity and its initial charac­ terization (9, 10).

Downloaded by: Boston University - 1/14/2019 3:51:20 AM


References 1 Kimura, S.; Lewis, R.V.; Gerber, L.D.; Brink, L.; Rubinstein, M.; Stein, S., and Udenfriend, S.: Purification to homogeneity of camel pituitary pro-opiocortin, the common precursor of opioid peptides and corticotropin. Proc. natn. Acad. Sci. USA (in press, 1979). 2 Lewis, R.V.; Stein, S.; Gerber, L.D.: Rubinstein, M., and Udenfriend, S. : High molecular weight opioid-containing proteins in striatum. Proc. natn. Acad. Sci. USA 75: 4021-4023 (1978). 3 Lewis, R.V.; Stein, S., and Udenfriend, S.: Separa­ tion of opioid peptides utilizing high performance liquid chromatography. Int. J. Peptide Protein Res. (in press, 1979). 4 Mains, R.E.; Eipper, B.A., and Ling, N.: Common precursor to corticotropin and endorphins. Proc. natn. Acad. Sci. USA 74: 3014-3018 (1977). 5 Nakanishi, S.; Inoue, A.; Kita, T.; Numa, S.; Chang, A.C.Y.; Cohen, S.N.; Nunberg, J., and Schimke, R.T.: Construction of bacterial plasmids that con­ tain the nucleotide sequence for bovine cortico­ tropin (3-lipotropin precursor. Proc. natn. Acad. Sci. USA 75: 6 021-6025 (19 7 8). 6 Rubinstein, M.; Stein, S.; Gerber, L.D., and Uden­ friend, S.: Isolation and characterization of the opioid peptides from rat pituitary: (3-lipotropin. Proc. natn. Acad. Sci. USA 74: 3052- 3055 (1977). 7 Rubinstein, M.; Stein, S„ and Udenfriend, S.: Isolation and characterization of the opioid pep­ tides from rat pituitary: /3-endorphin. Proc. natn. Acad. Sci. USA 74: 4969 -497 2 (19 7 7).


8 Rubinstein, M.; Stein,' S., and Udenfriend, S.: Characterization of proopiocortin, a precursor to opioid peptides and corticotropin. Proc. natn. Acad. Sci. USA 75: 669 - 671 (197 8). 9 Rubinstein, M.; Rubinstein, S.; Familletti, P.C.; Gross, M.S.; Miller, R.S.; Waldman, A.A., and Pestka, S.: Human leukocyte interferon purified to homogeneity. Science 202: 1289- 1290 (1978). 10 Rubinstein, M.; Rubinstein, S.; Familletti, P.C.; Miller, R.S.; Waldman, A.A., and Pestka, S.: Human leukocyte interferon: production, purifica­ tion to homogeneity, and initial characterization. Proc. natn. Acad. Sci. USA (in press, 1979). 11 Samejima, K.; Dairman, W.; Stone, J., and Uden­ friend, S.: Condensation of ninhydrin with alde­ hydes and primary amines to yield highly fluo­ rescent ternary products. Analyt. Biochcm. 42: 237-247 (1971). 12 Stein, S.; Bohlen, P.; Stone, J.; Dairman, W., and Udenfriend, S.: Amino acid analysis with fluorescamine at the picomole level. Archs Biochem. Biophys. 155: 202-212 (1973). 13 Stern, A.S.; Lewis, R.V.; Kimura, S.; Rossier, J.; Stein, S., and Udenfriend S.: Opioid peptides in striatum and adrenal medullary granules. Proc. natn. Acad. Sci. USA (in press, 1979). 14 Udenfriend, S.; Stein, S.; Bohlen, P.; Dairman, W.; Leimgruber, W., and Weigele, M.: Fluorescamine: a reagent for assay of amino acids, peptides, pro­ teins, and primary amines in the picomole range. Science 178: 871-872 (1972). Sidney Udenfriend, Roche Institute of Molecular Biology, Nutley, NJ 07110 (USA)

Downloaded by: Boston University - 1/14/2019 3:51:20 AM

An Analyst in Biomedical Research

An analyst in biomedical research.

Pharmacology 19: 223 -227 (1979) An Analyst in Biomedical Research Sidney Udenfriend Roche Institute of Molecular Biology, Nutley, N.J. Key Words. O...
625KB Sizes 0 Downloads 0 Views