Non-protein Compounds Research Articles

Computational Models for 5αR Inhibitors for Treatment of Prostate Cancer: Review of Previous Works and Screening of Natural Inhibitors of 5αR2

Taking into consideration the high importance of the drug target 5-α-reductase (5αR) in prostate cancer in this work we are going first to review previous works and discuss works related to the computer aided drug design of 5αR inhibitors. We report new results in the in silico screening of natural 5αR inhibitors. Traditionally, drugs were discovered by testing compounds synthesized in time consuming multi-step processes against a battery of in vivo biological screens. Promising compounds were then further studied in development, where their pharmacokinetic properties, metabolism and potential toxicity were investigated. Here we present a study on herbal lead compounds and their potential binding affinity to the effectors molecules of major disease like Prostate Cancer. Clinical studies demonstrate a positive correlation between the extent of 5αR type 2 (5αR2) and malignant progression of precancerous lesions in prostate. Therefore, identification of effective, well-tolerated 5αR inhibitors represents a rational chemo preventive strategy. This study has investigated the effects of naturally occurring non-protein compounds berberine and monocaffeyltartaric acid that inhibits 5αR type2. Our results reveal that these compounds use less energy to bind to 5αR and inhibit its activity. Their high ligand binding affinity to 5αR introduce the prospect for their use in chemopreventive applications; in addition they are freely available natural compounds that can be safely used to prevent prostate cancer.

Computer aided screening of inhibitors to 5-α reductase type 2 for prostate cancer.

Traditionally, drugs are discovered by testing compounds synthesized in time consuming multi-step processes against a battery of invivo biological screens. Promising compounds are then further studied in development, where their pharmacokinetic properties, metabolism and potential toxicity were investigated. Here, we present a study on herbal lead compounds and their potential binding affinity to the effectors molecules of major disease like Prostate Cancer. Clinical studies demonstrate a positive correlation between the extent of 5-α reductase type 2 (isoform 2) and malignant progression of precancerous lesions in prostate. Therefore, identification of effective, well-tolerated 5-α reductase inhibitors represents a rational chemo preventive strategy. This study has investigated the effects of naturally occurring nonprotein compounds berberine and monocaffeyltartaric acid that inhibits 5-α reductase type 2. Our results reveal that these compounds use less energy to bind to 5-α reductase and inhibit its activity. Their high ligand binding affinity to 5-α reductase introduces the prospect for their use in chemopreventive applications. In addition, they are freely available natural compounds that can be safely used to prevent prostate cancer.

Palytoxin found in Palythoa sp. zoanthids (Anthozoa, Hexacorallia) sold in the home aquarium trade.

Zoanthids (Anthozoa, Hexacorallia) are colonial anemones that contain one of the deadliest toxins ever discovered, palytoxin (LD50 in mice 300 ng/kg), but it is generally believed that highly toxic species are not sold in the home aquarium trade. We previously showed that an unintentionally introduced zoanthid in a home aquarium contained high concentrations of palytoxin and was likely responsible for a severe respiratory reaction when an individual attempted to eliminate the contaminant colonies using boiling water. To assess the availability and potential exposure of palytoxin to marine aquarium hobbyists, we analyzed zoanthid samples collected from local aquarium stores for palytoxin using liquid chromatography and high resolution mass spectrometry and attempted to identify the specimens through genetic analysis of 16S and cytochrome c oxidase 1 (COI) markers. We found four specimens of the same apparent species of zoanthid, that we described previously to be responsible for a severe respiratory reaction in a home aquarium, to be available in three aquarium stores in the Washington D.C. area. We found all of these specimens (n = 4) to be highly toxic with palytoxin or palytoxin-like compounds (range 0.5–3.5 mg crude toxin/g zoanthid). One of the most potent non-protein compounds ever discovered is present in dangerous quantities in a select species of zoanthid commonly sold in the home aquarium trade.

Glomalin-related soil protein contains non-mycorrhizal-related heat-stable proteins, lipids and humic materials

Glomalin is reportedly a stable and persistent protein produced in copious quantities by mycorrhizal fungi and may be an important pool of organic N in soil. Glomalin-related soil protein (GRSP), however, is only operationally defined by its extraction method, and has been only poorly characterized at best. The goal of this study was to characterize the molecular structures within GRSP. Synchrotron-based X-ray absorption near-edge structure (XANES) spectroscopy and pyrolysis field-ionization mass spectrometry (Py-FIMS) revealed that GRSP contains a consortium of proteins along with many impurities. Employing proteomic techniques, we found that glomalin itself may be a thioredoxin-containing chaperone; however, no homologies with proteins or DNA of mycorrhizal origin were detected. Proteomics techniques further revealed that this fraction contains large amounts of soil-related heat-stable proteins and proteins of non-mycorrhizal origin. Results of this research show that the current extraction procedure that defines GRSP yields a mixture of compounds and thereby overestimates glomalin stocks when quantified using the Bradford assay. The chemical nature of glomalin has yet to be conclusively determined; it is unlikely that the chemical structure of glomalin can be elucidated from the mixture extracted as GRSP. Instead, an investigation into the specific biochemistry of immunoreactive assays currently used to define GRSP, followed by proteomic characterization of monoxenic mycorrhizal cultures may be required to advance our understanding of the chemical nature and agronomic significance of GRSP in soils. ► Glomalin-related soil protein (GRSP) extracts contained many co-extracted, non-protein compounds. ► Proteomics-based assessment of GRSP showed no homology to any proteins of AMF origin, instead showing homology with thioredoxin and with heat-stable soil proteins. ► Implications are that protein databases do not yet contain glomalin-related sequences, or that glomalin is homologous to non-AMF soil proteins.

The in vitro antibiofilm activity of selected marine bacterial culture supernatants against Vibrio spp.

The aim of the work is to investigate the effect of marine bacterial culture supernatants on biofilm formation of Vibrio spp., a major menace in aquaculture industries. Vibrio spp. biofilm cause life-threatening infections in humans and animals. Forty-three marine bacterial culture supernatants were screened against the hydrophobicity index, initial attachment and biofilm formation in Vibrio spp. Twelve culture supernatants showed antibiofilm activity. The bacterial culture supernatants S8-07 (Bacillus pumilus) and S6-01 (B. indicus) inhibited the initial attachment, biofilm formation and dispersed the mature biofilm at 5% v/v concentration without inhibiting the growth. Analysis by light microscopy and confocal laser scanning microscopy showed that the architecture of the biofilm was destroyed by bacterial supernatants when compared to the control. The bacterial supernatants also reduce the surface hydrophobicity of Vibrio spp. which is one of the important requirements for biofilm formation. Further characterization of antibiofilm activity in S8-07 culture supernatant confirmed that it is an enzymatic activity and the size is more than 10 kDa and in S6-01, it is a heat-stable, non-protein compound. Furthermore, both the supernatants failed to show any biosurfactant activity. The culture supernatants of S8-07 and S6-01 with promising antibiofilm property have potential for application in medicine and marine aquaculture.

Alternative protein sources in diets for Japanese flounder Paralichthys olivaceus (Temminck and Schlegel): II. Effects on nutrient digestibility and digestive enzyme activity

An 8-week feeding trial was conducted to compare the effects of dietary protein sources on nutrient digestibility and digestive enzyme activity. Four experimental diets were formulated to contain one of the following as the sole protein source: fish meal (FM), fish protein concentrate (FPC), soy protein concentrate (SPC) and soy protein isolate (SPI). Each diet was randomly assigned to triplicate aquaria stocked with 25 Japanese flounder (Paralichthys olivaceus) each. The dry matter, crude protein and energy digestibility and individual amino acid availability of the SPC-based diet were significantly lower than those of the other diets. The crude lipid digestibility of soy protein-based diets was significantly lower than that of the FM-based diet. The pepsin/protease activity was significantly higher in fish fed fish protein-based diets compared with fish fed soy protein-based diets. The lipase activity in fish fed the SPI-based diet was the highest among the dietary treatments. These results indicate that P. olivaceus can effectively digest the protein from FPC and SPI (but not SPC) as well as FM. The low protein digestibility and amino acid availability of the SPC-based diet may be related to the non-protein compounds present in SPC, whereas the low-lipid digestibility of soy protein-based diets may contribute to the undigested soy protein fractions and/or the alcohol-soluble components.

Characterization and Fermentability of an Ethanol Soluble High Molecular Weight Coffee Fraction

Brews from differently roasted Arabica coffees were shown to contain 8-12% ethanol soluble substances with molecular masses greater than 2 kDa, possibly contributing to their dietary fiber contents. About 13% of these substances were nondigestible carbohydrates, mainly arabinogalactans. The nondigestible high molecular weight ethanol soluble fraction (HESF) of the medium roasted coffee brew was further characterized and subjected to in vitro fermentation with human fecal bacteria. In addition to carbohydrates, HESF contained proteins/peptides (approximately 20%), but the main fraction was composed of structurally unknown Maillard reaction products. From NMR spectroscopy, we conclude that intact caffeic and ferulic acid derivatives were not incorporated into the melanoidins to a significant extent. Stepwise ultrafiltration and gel filtration indicated a large variation in the molecular weights of HESF constituents. Coffee HESF was shown to be less fermentable by fecal bacteria than soluble coffee fiber isolated by the enzymatic-gravimetric methodology, and because of its lower carbohydrate content, less short-chain fatty acids were produced during the fermentation. Total cell counts, destructive chemical analysis, and NMR spectroscopy indicated that coffee carbohydrates are the preferred substrates for colonic microbiota. However, NMR spectra, absorbances at 405 nm, and nonprotein nitrogen contents showed that noncarbohydrate and nonprotein compounds were also utilized to some extent but the bacterial species involved in this degradation remain to be identified.

Fate of soil bacteria and fungi in the gut of earthworms

The responses of soil microorganisms to the action of gut fluids of three earthworm species, Aporrectodea caliginosa, Lumbricus terrestris and Eisenia fetida were investigated. It was found that the midgut fluid taken form anterior part of the digestive tract could suppress formation of colonies of bacteria, inhibit the germination of spores and reduce the radial growth rate of some fungal colonies. Heating the midgut fluid at 98 °C for 10 min did not eliminate its suppressive activity. This suggests that a non-protein compound ( s) is involved in this action of the midgut fluid. The suppressing effects were shown to be selective towards soil bacteria. However, the responses of microbial cells to the midgut fluid did not correspond to the taxonomic affiliation of the microorganisms tested. The mechanism of the suppressing effect might be a destruction of microbial cell membranes, as demonstrated for the gut of soil millipedes. The selective activity of the gut fluid of earthworms could be a significant factor for the animal's nutrition as well as for regulating the steady state of the intestinal microbial community, and modification of microbial communities in soil.

Urinary Sulfur Excretion and the Nitrogen/Sulfur Balance Ratio Reveal Nonprotein Sulfur Amino Acid Retention in Piglets

We evaluated the use of urinary sulfur (S) excretion as a measure of sulfur amino acid (SAA) catabolism and the nitrogen/sulfur (N/S) molar balance ratio as an indicator of nonprotein SAA storage in growing piglets. After confirming that an intravenous dose of sulfate is fully recovered in urinary sulfate, we measured urinary S recovery after an intravenous dose of methionine in 6 piglets fed an adequate protein (AP) diet and 6 piglets fed a low protein (LP) diet with normal energy provision. As measured over 48 h, recoveries of the methionine load as urinary total S was 106% in the AP group but only 69% in the LP group (P < 0.05). On the baseline diets the N/S balance ratio in the AP group was 36, whereas that in the LP group was 30 (P < 0.05); immediately after the methionine load, this ratio remained constant in the AP group but decreased further, to 26 (P < 0.05) in the LP group. These results indicate that protein-deficient piglets accumulate relatively more S than N from their diet, and under these conditions a significant portion of the S derived from a methionine load is retained in nonprotein compounds. Urinary S excretion, a simple nontracer measurement, can provide an accurate measure of SAA catabolism, and the N/S balance ratio is a potentially useful indicator of changes in nonprotein SAA stores of growing piglets.

Extracellular factors responsible for the adaptation ofPseudomonas fluorescens batch cultures to unfavorable conditions

The culture liquid filtrate of an exponential-phasePseudomonas fluorescens batch culture added to anotherP. fluorescens culture at the moment of inoculation was found (1) to prevent or diminish cell adsorption of the flask walls, (2) to enhance the intensity of cell respiration, (3) to shorten the period of adaptation of LB-grown cells to growth in glucose-containing mineral M9 medium, (4) to stimulate bacterial growth at supraoptimum temperature (36°C) and pH values (4.8 and 9.2), and (5) to decrease the death rate of bacteria at the supraoptimum growth temperature. These results were interpreted as indicating thatP. fluorescens cultures produce two types of regulatory exometabolites similar to those revealed earlier inEscherichia coli andBacillus subtilis cultures: the direct-action adaptogenic factorX1 capable of increasing bacterial resistance to unfavorable growth conditions (temperature and pH) and factor of accelerated adaptation to new media. Both factors are presumably low-molecular-weight hydrophilic nonprotein compounds.

Influence of Dietary Compounds on Intestinal Immunity

This review focuses on the specific effects of nutrients on the intestinal immune system. The intestinal immune system possesses a number of special characteristics different from those of the systemic immune system, which are summarised in this report. Although some of these characteristics are well known, there is a big gap regarding the specific mechanisms that regulate immunity at intestinal level with respect to the systemic immunity. Because of that, during the last years, there is an increasing interest in elucidating the inner mechanisms that regulate the intestinal immune function, as well as how specific nutrients influence that function. We have reviewed this topic with special emphasis on how human milk and its components influence the development of the intestinal immunity in early infancy compared with formula-fed infants. Interactions between nutrients and the intestinal microora have also been reviewed. Some micronutrients, which are present in human milk and in most of foods, are capable of influencing immune functioning at very low concentrations. The specific action of these micronutrients on some parameters of the intestinal immunity as well as the potential mechanisms of action promoted by them have been considered more in detail. However, few data exist on how other specific nutrients, namely protein and non-protein compounds, lipids, carbohydrates, and others, such as mineral and vitamins, influence the intestinal immunity. Further research in these areas is highly recommended.

(3)H]-trimetazidine mitochondrial binding sites: regulation by cations, effect of trimetazidine derivatives and other agents and interaction with an endogenous substance.

Trimetazidine, an antiischaemic drug, has been shown to restore impaired mitochondrial functions. Specific binding sites for [(3)H]-trimetazidine have been previously detected in liver mitochondria. In the present study we confirm this observation and provide additional evidence for the involvement of these sites in the pharmacological effects of the drug. Inhibition experiments using a series of trimetazidine derivatives revealed the presence of three classes of binding sites. An N-benzyl substituted analogue of trimetazidine exhibited a very high affinity (K(i)=7 nM) for one of these classes of sites. Compounds from different pharmacological classes were evaluated for their ability to inhibit [(3)H]-trimetazidine binding. Among the drugs tested pentazocine, ifenprodil, opipramol, perphenazine, haloperidol, and to a lower extent prenylamine, carbetapentane and dextromethorphan competed with high affinity, suggesting a similarity of high affinity [(3)H]-trimetazidine sites with sigma receptors. [(3)H]-Trimetazidine binding was modulated by pH. Neutral trimetazidine had about 10 fold higher affinity than protonated trimetazidine for its mitochondrial binding sites. Various cations also affected [(3)H]-trimetazidine binding. Ca(2+) was the most potent inhibitor and totally suppressed the binding of [(3)H]-trimetazidine to the sites of medium affinity. An endogenous cytosolic ligand was able to displace [(3)H]-trimetazidine from its binding sites. Its activity was not affected by boiling for 15 min, suggesting a non-protein compound. These data suggest that mitochondrial [(3)H]-trimetazidine binding sites could have a physiological relevance and be involved in the antiischaemic effects of the drug.

Seasonal dynamics and effects of nitrogen supply rate on nitrogen and carbohydrate reserves in cutting-derived Salix viminalis plants

Nutrient storage is an important aspect of resprouting potential and production of Salix viminalis L., a pioneer species used for biomass production in Sweden. Seasonal dynamics of nitrogen (N), protein, soluble carbohydrates, starch, and lipids were studied in roots, cuttings, stems, and leaves during a full growth cycle induced by varying photoperiod and temperature in a growth chamber. Nitrogen was supplied at two rates. Both season and N availability significantly affected storage of N and carbohydrates. Reserves peaked in dormancy, and plants grown at a higher N availability were able to build up larger N reserves, whereas carbohydrate reserves were similar in the two N treatments. All perennial plant organs functioned as storage sites for N. Roots were prominent in carbohydrate storage, in good agreement with the notion of a pioneer species adapted to recurring disturbance by a pronounced resprouting capacity. Roots differed from aboveground plant parts in their exceptionally high starch levels, and in that N storage in roots to a greater extent involved nonprotein compounds. Triglycerides contributed to carbon storage in aerial plant parts but not in roots. Our results suggest that an increased N supply enhance both the accumulation of nutrient reserves and early season growth.

Modulation of nonprotein sulphydryl compounds rhythm with buthionine sulphoximine: relationship with oxaliplatin toxicity in mice.

The relationship between the rhythm in tissue nonprotein sulphydryl groups (NPSH) and that in 1,2-diamine (trans-I)-cyclohexane oxalatoplatinum (1-OHP) toxicity was investigated in a total of 266 male B6D2F1 mice, using buthionine sulphoximine (BSO), an inhibitor of gamma-glutamylcysteine synthetase. Mice were synchronized with an alternation of 12 h light (L) and 12 h darkness (D; LD 12:12), and circadian time was expressed in hours after light onset (HALO). NPSH was measured in liver, jejunum and bone marrow at 0, 8 and 16 HALO. Dosing 1-OHP at these times achieved intermediate. high or low toxicity respectively. The physiological circadian rhythm in NPSH content was statistically significant in all tissues studied, with a maximum at the transition from D to L (0 HALO). BSO administration (450 mg/kg i.p., 4 h before sampling) induced a large depletion in liver and jejunum NPSH at their physiological peak (0 HALO), but exerted no significant effect at their trough (8 HALO). As a result, 24 h rhythm was suppressed in liver and jejunum, but remained similar to the physiological one in bone marrow. BSO enhanced 1-OHP-induced mortality and jejunal toxicity, but exerted no significant effect upon bone marrow toxicity. Despite these differences, 1-OHP remained least toxic at 16 HALO, near the middle of the dark span, which corresponds to maximum activity in the circadian rest/activity cycle. Our results show that mean NPSH levels in liver seem to account for the mean level of 1-OHP toxicity, while jejunal NPSH rhythm plays an important role in the intestinal toxicity rhythm of this drug.

Histological study of mechanisms of adaptive cytoprotection on ethanol-induced mucosal damage in rat stomachs.

Adaptive cytoprotection in the gastric mucosa could be induced by exposure to low concentrations of noxious agents. However, experimental results reported so far were based on macroscopic studies. We aimed to investigate the phenomenon of gastric adaptive cytoprotection of mild irritants and its correlation with intramucosal mucus at the histological level. It was found that histological damage induced by ethanol had a negative correlation with the length of the mucus-secreting layer in the gastric mucosa. Mild irritants such as 20% ethanol and 5% NaCl preserved the 100% ethanol-induced intramucosal mucus depletion, but only the former agent demonstrated a cytoprotective effect against the histological damage, indicating that preservation of intramucosal mucus may not necessarily play a permissive role in adaptive cytoprotection. The capsaicin-sensitive sensory afferent neurons, sensory chemoreceptors, muscarinic receptors, alpha2-adrenoceptors and peripheral dopamine D2-receptors were found to be the components of the autonomic nervous system involved in the cytoprotective processes of 20% ethanol. Endogenous mediators including nitric oxide, prostaglandins, and possibly nonprotein sulfhydryl compounds also seemed to participate in such protection. Nevertheless, 0.3 M HCl did not show any effect either on mucosal damage or depletion of intramucosal mucus induced by absolute ethanol. These findings suggest that only 20% ethanol shows histological cytoprotection, which would involve various components of the autonomic nervous system and endogenous mediators. Furthermore, this investigation also implies a new perspective: that in order to study a true adaptive cytoprotection, histological examination of the gastric mucosa should be performed.

Quantification of Interferences in the Direct Measurement of Proteins in Wines From the Champagne Region Using the Bradford Method

At this time, the Bradford method is the most commonly used method in enology for direct protein quantification because of its rapidity and its reproducibility. This paper considers interferences which may falsify the estimation of protein in champagne Pinot noir and Chardonnay wines. Ultrafiltration with 10 kDa MWCO membrane shows that nonprotein compounds largely react with the Coomassie Brilliant Blue (CBB) and can produce an A_{595 nm} equal to 30% to 90% of the initial value. Ethanol and exogenous and endogenous phenolic compounds are principally responsible for these interferences. They act indirectly favoring the neutral form of the dye. However, interferences from ethanol and phenolics are not additive. Furthermore, the interaction is negative. Gelatins used in enology react 50 times less than BSA with the dye reagent. Both bentonite and vegetable charcoal treatments partially eliminate proteins and phenolics. The ratios A_{520 nm}/A_{595 nm} (eq. BSA estimated by the direct Bradford method) increases on increasing bentonite fining and decreases on charcoal treatment. For these reasons, the deproteinization effects of enological treatments are, at present, very difficult to estimate with direct measurement. A modified Bradford method involving of A_{595 nm} before and after ultrafiltration (3 and 10 kDa MWCO) is proposed as a more accurate measure of wine protein content.

Transmural distribution of antioxidant defences and lipid peroxidation in the rabbit left ventricular myocardium

The left ventricular subendocardial and subepicardial layers of six perfused rabbit hearts were tested for enzymatic and non-enzymatic antioxidant defences and for lipid peroxidation. The subendocardium showed significantly lower catalase activity and contents of non-protein thiol compounds and vitamin E associated with a higher degree of lipid peroxidation. The activities of Cu,Zn- and Mn-superoxide dismutases, glutathione reductase, gamma-glutamylcysteine synthetase and gamma-glutamyl transpeptidase showed no significant transmural differences, and Se-independent glutathione peroxidase activity was not detectable in either layer. Comparable results were observed in another group of six unperfused rabbit hearts. In five H2O2-perfused rabbit hearts, lipid peroxidation was higher, and myocardial creatine phosphokinase activity lower, in the subendocardium than in the subepicardium. In this group, only the subendocardium had significantly higher lipid peroxidation levels than the control hearts. Thus, a lower antioxidant capacity and a greater oxidative stress are present in the rabbit subendocardium. These findings could provide insight into the problem of subendocardial vulnerability to free radical-mediated processes, such as occurs in ischaemia-reperfusion injury.

The cuticle of the nematode Caenorhabditis elegans: A complex collagen structure

The cuticle of the nematode Caenorhabditis elegans forms the barrier between the animal and its environment. In addition to being a protective layer, it is an exoskeleton which is important in maintaining and defining the normal shape of the nematode. The cuticle is an extracellular matrix consisting predominantly of small collagen-like proteins that are extensively crosslinked. Although it also contains other protein and non-protein compounds that undoubtedly play a significant part in its function, the specific role of collagen in cuticle structure and morphology is considered here. The C. elegans genome contains between 50 and 150 collagen genes, most of which are believed to encode cuticular collagens. Mutations that result in cuticular defects and grossly altered body form have been identified in more than 40 genes. Six of these genes are now known to encode cuticular collagens, a finding that confirms the importance of this group of structural proteins to the formation of the cuticle and the role of the cuticle as an exoskeleton in shaping the worm. It is likely that many more of the genes identified by mutations giving altered body form, will be collagen genes. Mutations in the cuticular collagen genes provide a powerful tool for investigating the mechanisms by which this group of proteins interact to form the nematode cuticle.

Genetic engineering of commercially useful biosynthetic pathways in transgenic plants

In many economically important plant species, the chemical composition of one or more non-protein compounds determines the value of the plant and may have an important role in protecting the plant from environmental stress, including pests, drought, salt, temperature and light. A number of potential opportunities exist whereby the range or amount of such valuable compounds can be increased by genetic engineering.

A comparison of Lowry, Bradford and Smith protein assays using different protein standards and protein isolated from the marine diatom Thalassiosira pseudonana

Two spectrophotometric assays for protein commonly used in marine research (Coomassie stain, “Bradford”; alkaline copper, “Lowry”) and a more recent assay which has not been applied in this field (bicinchoninic acid, “Smith”) were compared for homogenates of the marine diatom Thalassiosira pseudonona using bovine serum albumin (BSA) as a standard. When homogenates were prepared by precipitating protein with trichloroacetic acid (TCA) and redissolving in 1 N NaOH, the protein content estimated by the Lowry and Smith assays agreed closely, but was consistently 20% higher than that indicated by the Bradford assay. To determine if this difference was due to the choice of a protein standard, protein from T. pseudonana was purified and compared to BSA, bovine gamma-globulin (BGG), and casein. The reactivity of the purified protein (expressed as the slope of the absorbance vs protein concentration curve) did not differ between cultures grown at high or low irradiance. For the Smith and Bradford assays the reactivity of BSA was not significantly different from algal protein, but for the Lowry assay, algal protein was significantly higher in reactivity than BSA. BGG was not significantly different in reactivity from algal protein for the Lowry and Smith assays, but BGG gave significantly lower absorbances than algal protein in the Bradford assay. These results suggest that BSA is a suitable standard for algal protein in the Bradford assays, while BGG is preferable for the Lowry assay. Either protein standard could be used for the Smith assay. Differences in purified algal protein reactivity compared to BSA could not account for the differences among the assays, nor could interference by chlorophyll a. Precipitating protein with TCA prior to analyses gave lower protein than direct analyses of homogenates for the Lowry and Smith assays, but no differences were found for the Bradford assay. As a result, the Lowry and Smith assays indicated up to 60% greater protein than the Bradford if TCA precipitation was not performed. This may be due to removal of free amino acids and small peptides which are less reactive in the Bradford assay. The 20% higher protein found in the Lowry or Smith vs Bradford assays may be due to different assay sensitivity to small peptides or other compounds which are precipitated along with proteins by TCA. Although the Smith assay is substantially simpler to perform than the Lowry, there appear to be no quantitative differences in the results. It remains unclear which spectrophotometric assay is most accurate, but the Bradford assay is faster and simpler, and is less likely to be affected by non-protein compounds found in marine phytoplankton.