UV Absorption Peak Research Articles

Application of Laser Induced Photoacoustic Spectroscopy to the Determination of 99Tc.

Abstract Laser induced photoacoustic spectroscopy was applied to the determination of 99Tc in a solution coupled with quantitative complexation of Tc with tu (thiourea) to form Tc(tu)6 3+The complexation procedure was adopted to shift the uv absorption peak of pertechnetate to the visible region, Photoacoustic signals due to the 488 n m absorption peak of Tc(tu)6 3+ was analyzed by using a Past Fourrier Transform (FFT) system. The quantitative detection of Tc could be easily performed down to the 10-8 M concentration in this system. This is about 1,000 times more sensitive than the ordinary colorimetrie measurement.

3-Hydroxy-3-methylglutaryl coenzyme A lyase: affinity labeling of the Pseudomonas mevalonii enzyme and assignment of cysteine-237 to the active site.

Pseudomonas mevalonii 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) lyase is irreversibly inactivated by the reactive substrate analog 2-butynoyl-CoA. Enzyme inactivation, which follows pseudo-first-order kinetics, is saturable with a KI = 65 microM and a limiting k(inact) of 0.073 min-1 at 23 degrees C, pH 7.2. Protection against inactivation is afforded by the competitive inhibitor 3-hydroxyglutaryl-CoA. Labeling of the bacterial enzyme with [1-14C]-2-butynoyl-CoA demonstrates that inactivation coincides with covalent incorporation of inhibitor, with an observed stoichiometry of modification of 0.65 per site. Avian HMG-CoA lyase is also irreversibly inactivated by 2-butynoyl-CoA with a stoichiometry of modification of 0.9 per site. Incubation of 2-butynoyl-CoA with mercaptans such as dithiothreitol results in the formation of a UV absorbance peak at 310 nm. Enzyme inactivation is also accompanied by the development of a UV absorbance peak at 310 nm indicating that 2-butynoyl-CoA modifies a cysteine residue in HMG-CoA lyase. Tryptic digestion and reverse-phase HPLC of the affinity-labeled protein reveal a single radiolabeled peptide. Isolation and sequence analysis of this peptide and a smaller chymotryptic peptide indicate that the radiolabeled residue is contained within the sequence GGXPY. Mapping of this peptide within the cDNA-deduced sequence of P. mevalonii HMG-CoA lyase [Anderson, D. H., & Rodwell, V. W. (1989) J. Bacteriol. 171, 6468-6472] confirms that a cysteine at position 237 is the site of modification. These data represent the first identification of an active-site residue in HMG-CoA lyase.

Levels of direct-acting mutagens, total N-Nitroso compounds in nitrosated fermented fish products, consumed in a high-risk area for gastric cancer in southern China

A high gastric cancer mortality in Fujian province (Peoples Republic of China) has been associated with the consumption of certain salted fermented fish products such as fish sauce (FS). We have investigated the levels and nature of N-nitroso compounds (NOC) and genotoxins present, before and after nitrosation, in 49 FS samples collected from villages in this high-risk area, pooled into six samples. The concentrations of total NOC before nitrosation ranged from 0.2 to 16 μmoles/1, and after nitrosation at pH 2 and pH 7, they rose by up to 4800- and 100-fold, respectively. In nitrosated samples, 40–50% of total NOC was not extractable into organic solvents; volatile N nitrosamines accounted for 1–2% and N-nitrosamino acids for 8–16% of total NOC. None of the FS samples exhibited genotoxic activity, but after nitrosation all were weakly active in the SOS chromotest. The highest SOS-inducing potency was observed with nitrosated ethyl acetate extracts of most samples. The formation of methylating agents was measured by incubation of nitrosated FS with DNA and subsequent analysis of 7-methylguanine adduct. 2 of the 6 nitrosated FS samples caused a slight increase in DNA methylation. 1 pooled home-made FS sample (the only one tested) contained tumour promoter-like substances, as measured by expression of certain EBV genes in Raji cells. HPLC fractionation of ethyl acetate extracts of FS samples allowed identification of three UV-absorbing peaks that, upon nitrosation, produced direct-acting genotoxins. This genotoxicity was partly ascribed to the formation of nitrite-derived arene diazonium cations that were characterized by a coupling reaction with N-ethyl-1-naphthylamine and thin-layer chromatography. The presence of nitrite-dependent genotoxins and tumour promoter-like substances supports the hypothesis that FS consumption is a risk factor for gastric cancer in certain areas of China.

Sporeamicin A, a new macrolide antibiotic. I. Taxonomy, fermentation, isolation and characterization.

Sporeamicin A, a novel antibiotic, was isolated from the culture filtrate of an actinomycete. The producing organism, strain L53-18, was taxonomically assigned as a species of the genus Saccaropolyspora. The antibiotic was extracted with chloroform and was then purified by crystallization. It was obtained as colorless prisms from ethanolic solutions. Sporeamicin A exhibited a strong UV absorption peak at 276 nm. The molecular formula of sporeamicin A was determined to be C37H63NO12.

Determination of dietary cadmium-induced metallothioneins in rabbit kidneys and cadmium in metallothioneins by anion-exchange high-performance liquid chromatography coupled with graphite furnace atomic absorption spectrometry

A rapid method is described for the determination of dietary cadmium-induced metallothioneins (MTs) in rabbit kidneys by anion-exchange high-performance liquid chromatography. Rabbit kidney MT-I and MT-II were eluted at ca. 15.0 and 18.8 min, respectively, from a DEAE-5PW anion-exchange column with a Tris—HCl buffer (0.01–0.25 M, pH 8.6) and detected by ultraviolet absorbance at 254 nm. A standard calibration curve was constructed using purified standard MT isoforms, which demonstrated an excellent linear correlation between UV absorbance peak heights and the amounts of MT isoforms. Feeding a dose of cadmium for some days resulted in an increase in MT concentrations in rabbit kidneys, but not in the livers. The cadmium concentrations in MT-I and MT-II elutions were determined by graphite furnace atomic absorption spectrometry. MT-I and MT-II showed some differences associated with the oral intake of cadmium. Dietary cadmium also caused zinc to accumulate in kidneys to some extent. The effects of dietary olcic acid on the synthesis of MTs were also studied. Based on the method of standard additions, the recovery of MTs exceeded 93% and replicated injection of samples yielded a relative standard deviation of 2.4% at an MT level of 280 μg/g.

Isolation of bovine ovarian inhibin, its immunoneutralization in vitro and immunolocalization in bovine ovary

A purification scheme involving gel permeation chromatography, anion exchange chromatography and reversed-phase high performance liquid chromatography (RP-HPLC) was used to isolate from bovine follicular fluid (FF) biologically-active inhibin of molecular weight 32 kDa. Chromatographic fractions were monitored for inhibin-like biological activity (ILA) using a simplified bioassay procedure in which a suppression of total basal FSH production by rat pituitary cells in monolayer culture indicates the presence of ILA. Approximately 3 mg protein having an ILA potency (ED 50 value in in vitro bioassay) of 1.7 ng/ml was obtained from 4 l crude bovine FF (260 g protein; ILA potency 3750 ng/ml) reflecting an approximate 2200-fold purification factor with an overall recovery of about 3%. The isolated material appeared as a single major UV absorbance peak on RP-HPLC and as a single band (32 kDa) when subjected to SDS-PAGE (15% gel) under non-reducing conditions. Under reducing conditions the molecule dissociated into 2 subunits of apparent molecular weight 22 and 14 kDa confirming that it is probably identical to the 31 32 kDa form of bovine ovarian inhibin previously reported by two other independent research groups. An antiserum raised in a chicken against the isolated material completely neutralized the suppressive effects of both 32 kDa inhibin and bovine FF on basal production of FSH by rat pituitary cells in vitro but only partially reversed the suppressive effects of both porcine and human FF. Immunohistochemical staining of sections of bovine ovary and of isolated preparations of bovine granulosa cells using this antiserum confirmed that granulosa cells are a major source of inhibin. The observation that specific immunostaining was not confined to these cells, however, suggests that they may not be the exclusive source of immunoreactive inhibin in the bovine ovary.

Digoxinlike Materials in Human Breast Cyst Fluids

Digoxinlike materials (DLMs) are compounds found in animals that (1) cross-react with digoxin antibodies in specific radioimmunoassay procedures and (2) bind to Na+, K(+)-ATPase and inhibit its activity. DLMs may play a part in the regulation of electrolyte composition. We have determined DLM concentrations in breast cyst fluids and found that these fluids were a rich source of DLMs. Total DLMs were elevated in fluids with type 1 electrolyte composition (high K+ and low Na+ content) when compared to fluids with type 2 composition (low K+ and high Na+ content). Chromatographic studies showed that the basis for the increase was the presence of an additional DLM in the type 1 cyst fluids rather than an increased concentration of the same DLM present in both types of fluids. We propose that this additional DLM may have a role in the accumulation of K+ ions in type 1 fluids. In addition, 7 of 80 cyst fluid samples subjected to high-performance liquid chromatography had a large UV-absorbing peak at 6.4 minutes. Four of these samples were taken from women who later developed breast cancer; information on the subsequent medical history of the other 3 women was unavailable. We suggest that the presence of this material in cyst fluid may be a marker of risk for development of breast cancer.

Detection and characterization of carcinogen—DNA adducts in exfoliated urothelial cells from 4-aminobiphenyl-treated dogs by 32P-postlabelling and subsequent thin-layer and high-pressure liquid chromatography

The recent development of sensitive methods to detect carcinogen-DNA adducts offers a useful biochemical approach to human risk assessment. However, a major obstacle to developing a human biomonitoring method for carcinogen-DNA adducts has been the problem of obtaining target tissue DNA samples by non-invasive means. This work describes a method for the isolation of nanogram quantities of DNA from urothelial cells exfoliated into urine and the detection of carcinogen-DNA adducts from that DNA by 32P-postlabelling methods. Urine samples were collected on ice from dogs treated with 4-aminobiphenyl (ABP) over a 2-week period and pooled according to an experimental plan that involved analysis of cumulative 48- or 72-h samples. The pooled samples were sieved and then washed repeatedly with a sucrose buffer to dissolve contaminating triple phosphate (MgNH4PO4), calcium oxalate and uric acid crystals. DNA was isolated using an enzyme-solvent extraction method with the DNA being co-precipitated from ethanol with glycogen. The DNA was hydrolysed and postlabelled with 32P under conditions of excess ATP so that nucleotides were labelled quantitatively. Adducts observed on the resulting thin-layer chromatograms were identical to those obtained from DNA modified in vitro with N-hydroxy-4-aminobiphenyl and from dog bladder urothelial DNA isolated from the ABP-dosed animals at termination of the experiment. Furthermore, a dose-related increase in ABP-DNA adduct formation was demonstrated. Thus, it appears that the carcinogen-DNA adduct levels in the exfoliated bladder cells are reflective of the levels in the intact urothelium once steady-state levels have been achieved. To establish the identity of the major ABP-urothelial DNA adduct in chronically-treated dogs, the predominant 32P-postlabelled adduct was eluted from the thin-layer chromatograms and co-injected on an HPLC system with a synthetic [3H]N-(deoxyguanosin-8-yl)-4-aminobiphenyl-3',5'-bisphosphate standard. Dual-label analysis of 3H and 32P indicated that both eluted from the column in the same fraction, which coincided with the UV absorbance peak of the synthetic marker. Preliminary experiments with exfoliated urothelial cells from human urines indicate that these methods should have general utility for monitoring humans exposed to urinary bladder carcinogens and for investigations of the biochemical mechanisms by which such adducts are formed in the urothelium.

Orientation evaluation of polyion complex Langmuir-Blodgett films by Fourier transform IR transmission and reflection-absorption spectroscopy

Quantitative evaluation of molecular orientation in Langmuir-Blodgett (LB) films of polyion complexes between two azobenzene-containing fatty acids with different chain lengths (the C 12 and C 8 tail with the common C 5 spacer) and two polycations were performed by comparing the absorption intensities of the Fourier transform IR transmission and reflection-absorption spectra. The results obtained were discussed in connection with the UV absorption peak maxima of the azobenzene chromophores and the limiting areas of the surface pressure-area isotherms of the spread monolayers on the water surface. For LB films of polyion complexes of the fatty acid with the C 12 tail, the self-association forces are so strong that the degree of perpendicular orientation of the molecular chain and the stacking aggregation of the azobenzene chromophores remain high and unchanged from those for its barium salt. The polymer chain is considered to be twisted or folded to fit with the compact cross-section of the fatty acid. For LB films of polyion complexes of the fatty acid with the C 8 tail, however, the degree of orientation and aggregation are degraded from those of its barium salt, the polymer chain being highly extended.

Purification and Properties of Methanobacterium thermoautotrophicum DNA Photolyase

We have purified DNA photolyase from the autotrophic anaerobic archaebacterium Methanobacterium thermoautotrophicum to near homogeneity by a two-column affinity chromatography. The purified enzyme has an Mr = 60,000 and shows near UV absorption peak at 440 nm and a fluorescence emission maximum at 462 nm indicating that it contains 8-hydroxy-5-deazaflavin (coenzyme F420) as an intrinsic chromophore. The photolyase binds with high specificity to thymine dimer in DNA with an equilibrium binding constant, KA = 1.4 x 10(9) M-1, and a dissociation rate constant, koff = 1.4 x 10(-4) s-1 (t1/2 = 43 min). Despite 6-fold higher affinity compared to the folate-containing Escherichia coli photolyase the two enzymes apparently contact the same phosphates around the thymine dimer: the phosphate immediately 5' and the three phosphates immediately 3' to the dimer on the damaged strand and the phosphate across from the dimer in the minor groove on the complementary strand. The absolute action spectrum of the Methanobacterium photolyase in the 400-500-nm region closely matches the absorption of the enzyme-bound F420. The quantum yield (phi) over this region is constant and is approximately 0.2. The value is measurably smaller than the quantum yields reported for other DNA photolyases.

Biosynthesis of Porphyrinogens in Etiolated Euglena gracilis Z. I. Isolation and Purification of an Endogenous Factor Stimulating the Formation of Porphyrinogens

A low molecular weight, heat-stable factor has been purified from Euglena gracilis supernatant fraction by employing gel filtration, cation and anion exchange and paper chromatography. This endogenous compound stimulates porphobilinogenase (PBG-ase) (EC 4.3.1.8) activity, an enzyme of the porphyrin biosynthetic pathway. 10-7 ᴍ folic acid and 10-4 ᴍ 6-biopterin produced a significant activation, equivalent to 2-4 units of the purified factor. Elution patterns from the columns and fluorescence and UV absorption peaks suggest that this compound is a pteridine. This conclusion is further supported by the fact that both, folic acid and 6-biopterin can replace the action of the isolated factor on PBG-ase. The mechanism of stimulation is discussed.

Mechanisms of inhibition of Gaeumannomyces graminis var. tritici by fluorescent pseudomonads

The take‐all fungus Gaeumannomyces graminis var. tritici reduced the weight of wheat plants grown in tubes containing sterilized sand and plant nutrient solution. Fluorescent pseudomonads, when added to the tubes, inhibited the take‐all fungus and increased plant weight. Iron (as FeNaEDTA) had no effect on the inhibition of the fungus by the pseudomonads. Some of the pseudomonads produced a compound in the wheat rhizosphere with a UV absorption peak at 365 nm, but the inhibition of G. graminis by pseudomonads was not proportional to the UV absorbance of rhizosphere extracts. A yellow crystalline compound, absorbing at 365 nm, was extracted from broth cultures and shown to be toxic to G. graminis under acid conditions. This compound is considered to be phenazine‐I‐carboxylic acid.

Purification and partial sequence of rabbit polymorphonuclear leukocyte-derived lymphocyte proliferation potentiating factor resembling IL-1 beta.

A rabbit polymorphonuclear leukocytes (PMN)-derived lymphocyte proliferation-potentiating factor (PMN factor) was finally purified to homogeneity. PMN factor was released from early inflammatory peritoneal exudate cells (98% of PMN) stimulated with kaolin under roller bottle culture conditions. PMN factor was purified by large sequential scale steps, using membrane-type ion exchangers and gel filtration, followed in this order by HPLC steps with cationic ion exchangers and a hydroxylapatite column. Homogeneity was manifested based on the criteria of a single m.w. 18, 500 band on silver-stained polyacrylamide gel, a superimposable activity on a UV absorbance peak in analytic HPLC gel filtration, and detection of a single amino-terminal sequence. The homogeneous PMN factor had an isoelectric value of 7.2 and an activity of 1.9 x 10(7) U/mg in the thymocyte comitogenic assay. PMN factor stimulated one-half of the maximal response of thymocyte proliferation at 2.8 x 10(-12) M. Because of similarities in the physicochemical properties, specific activity, and amino-terminal sequence between rabbit PMN factor and human IL-1 beta, this PMN factor is therefore considered to be a rabbit IL-1 beta.

Affinity chromatography of naturally occurring folate derivatives

In a previous report (1980, in Methods in Enzymology, Vol. 66, p. 686, Academic Press, New York) we described the preparation and use of purified milk folate-binding protein covalently linked to a Sepharose matrix. The present study was undertaken to test the capacity of this preparation to purify quantitatively folates from tissue extracts. In experiments that employed tissue extracts containing biologically synthesized [ 3H]folates, recovery of radioactivity from columns of the immobilized folate-binding protein (affinity columns) was consistently 90–95%. Folates eluted from the affinity column were practically pure. Anion-exchange chromatography of the purified fraction from rat liver extract yielded a number of uv-absorbing peaks which corresponded to the elution profile based on 3H counts, with respect to both the position and the area of the peaks. Treatment of the original extracts with folylpolyglutamate hydrolases resulted in the shift of these peaks to lower retention times, which corresponded to mono- or diglutamyl folate derivatives. Similar results were obtained with extracts from Lactobacillus casei. The purification of tissue folates by affinity chromatography allows determination of folate activity by direct physicochemical methods, which is particularly useful for analysis of folate composition in tissues.

A neurosecretory hyperglycemic hormone from the sinus gland of the mexican crayfish Procambarus bouvieri (ortmann)—II. Structural comparison of two isoforms of the hormone

1. 1. The hyperglycemic activity of a crude extract of Procambarus bouvieri (Ortmann) sinus glands was resolved into two UV-absorbing peaks by means of a single step of reverse-phase high performance liquid chromatography (RP-HPLC) on a μ-Bondapak-Phenyl column. These peaks have been designated Crustacean Hyperglycemic Hormones CHH-B and CHH-C in the order of elution. 2. 2. The ratio CHH-B/CHH-C was approximately 3:1, both in area under the curve and in protein content. 3. 3. A structural comparison of the two isoforms of the CHH showed a substantial homology manifested in molecular weight (6000–6200), pI (4.79), number of residues (52–53), number of cysteines (4), number of acid residues, including their amides (12), number of basic residues (8), missing amino acids (methionine, histidine and tryptophane), amino end (blocked) and carboxyl end (isoleucine). 4. 4. The only clear difference between the two isoforms of the CHH is their degree of hydrophobicity which might be due to minor differences in the number of neutral hydrophobic residues and/or postranslational modifications of the type amidation/deamidation of acid residues which cannot be detected in acid hydrolysates.

タンパク質の高性能等速電気泳動

For the rapid and sensitive analysis of proteins, and to obtain reproducible results, an apparatus for isotachophoresis, Shimadzu IP-2A, was modified and the electrophoretic conditions were standardized. The apparatus was modified as follows; 1) Two peristaltic pumps were used instead of N2 gas pressure system, for rinsing and refilling the capillary tube and the electrode chambers. 2) The flow lines were simplified into 3 lines, instead of 10 lines of the commercial apparatus. The electrophoretic conditions were changed as follows; 1) Hexane was overlayed on the electrolyte solutions to minimize the solubilization of CO2, instead of the use of Ba(OH)2. 2) The inner surface of the capillary tube was coated with hydroxypropyl methyl cellulose (HPMC), instead of using HPMC-containing leading electrolyte. The amount of the ampholyte mixture (Ampholine pH 3.5-10) to be applied was standardized to be 20μg. From these improvements and standardization, the interruption of electrophoresis by N2 gas bubbles or by the precipitation of BaCO3 were avoided and the procedure of isotachophoretic analysis was simplified. Serum proteins as low as 3μg could be separated into 20 UV-absorbing peaks within 16min of running time in good reproducibility.

Strain-dependent inhibition of nitrous oxide reduction in denitrifiers by yeast extract

Pseudomonas sp. type strain H8 (ATCC 35402) was shown to be capable of denitrification but unable to reduce N2O in a complex medium containing 0.15% yeast extract. When added to a defined medium, autoclaved or filter-sterilized yeast extract from various suppliers was confirmed to inhibit N2O reduction by H8. The inhibition increased to 90% when the yeast extract concentration was increased to 0.5% in the medium. Only one of seven other denitrifying pseudomonads tested was similarly inhibited. Fractionation of yeast extract on a Sephadex G-10 column showed that the component responsible was a small molecule (molecular weight <700) that eluted with the first uv absorption peak. The inhibition was reversible by the addition of 10 μM Cu2+. Reduced glutathione was assayed in the first fraction of the Sephadex eluate and was found to mimic the effect of yeast extract. It is proposed that yeast extract inhibits N2O reduction by certain denitrifiers through glutathione complexing of Cu2+ which is a cofactor of N2O reductase.

Purification of the yeast PHR1 photolyase from an Escherichia coli overproducing strain and characterization of the intrinsic chromophores of the enzyme.

We have placed the PHR1 gene of Saccharomyces cerevisiae under the transcriptional and translational control of the tac expression cartridge. Under inducing conditions Escherichia coli cells harboring plasmids carrying this construct accumulate approximately 8% of total cellular protein as the Phr1 photolyase. Using a strain devoid of E. coli photolyase activity, we have obtained milligram quantities of the yeast enzyme at greater than 95% purity and have characterized the enzyme. Phr1 photolyase is a monomer in solution with an Mr of 60,000, has a turnover number of 0.7 dimers min-1 molecule-1 in vitro, exhibits absorbance maxima at lambda = 277 and 377 nm, and has a fluorescence excitation maximum at 390 nm and an emission maximum at 475 nm. The near UV absorbance peak is shown to reflect the contributions of two intrinsic chromophores which are noncovalently bound to the enzyme. Spectroscopic, fluorescence, and thin layer chromatographic studies indicate that one of these chromophores is 1,5-reduced FAD rather than 4a,5-reduced FAD as previously proposed (Iwatsuki, N., Joe, C. O., and Werbin, H. (1980) Biochemistry 19, 1172-1176), while the other chromophore has properties similar to the second chromophore of E. coli photolyase. The fact that yeast and E. coli photolyases are similar both with respect to amino acid sequence and chromophore composition provides strong evidence that the enzymes share a common action mechanism which may also be utilized by photolyases from other organisms throughout the phylogenetic tree.

The use of TSK size exclusion columns in determination of the quality and quantity of humus in raw waters and drinking waters

The usefulness of TSK G2000SW and TSK G3000SW columns, used alone or in tandem, were tested for separating humus in different types of raw waters and drinking waters by size exclusion chromatography. The raw water chromatograms showed characteristically from 5 to 7 UV-absorption peaks for surface water samples and from 2 to 4 peaks for ground water samples. The chromatograms of drinking water samples varied greatly depending on the amounts of humus present in the raw waters and on the effectiveness of the treatment processes. If ground water was used as raw water, the drinking water sample sometimes contained more organic matter than the raw water sample. Only 20 pl of untreated water sample was needed for one chromatogram, and the analysis time was 20 minutes for single columns and 40 minutes for the tandem column. The linear regressions of the sum of five peak heights of TSK G3000SW chromatograms to TOC-,KMnO4-, and colour values gave correlation coefficients of 0.954, 0.959, and 0.915, respectively. The correlation of peak heights to colour values was not linear. In the fractionation experiments, the main part of the observed mutagenic activity eluted from the tandem column in one peak, and the rest of the mutagenicity eluted in the far end of the chromatogram.

Identification of Pea Gibberellins by Studying [14C]GA12-Aldehyde Metabolism

Experiments were designed to test the hypothesis that the labeled products recovered from plant tissue incubated with [(14)C]GA(12)-7-aldehyde ([(14)C]GA(12)ald) would serve as appropriate [(14)C]markers for the recovery of naturally-occurring gibberellins (GAs). The [(14)C]GA(12)ald (about 200 millicuries per millimole) was synthesized from pumpkin endosperm using [4,5-(14)C]mevalonic acid. It was added to the adaxial surface of isolated pea cotyledons at 22 days after flowering. Products recovered after 0.5 and 4.0 hour incubations yielded four major peaks which were separated by high performance liquid chromatography (HPLC). These products were purified by multiple-column HPLC using on-line radioactivity detection. They were then added as [(14)C]markers to two unlabeled pea extracts. In general, preparative HPLC followed by further HPLC purification resulted in a single UV-absorbing peak co-eluting with each [(14)C]marker. These [(14)C] and UV-absorbing peaks were shown to contain GA(53), GA(44), GA(20), GA(19), and GA(17) by GC-MS. The finding of GA(53) is novel; all others have previously been found in pea. Endogenous GAs of pea were thus readily detected using [(14)C]GA(12)ald metabolites as [(14)C]markers to recover naturally occurring GAs suggesting that the method may be applicable in detecting naturally occurring GAs in other species.