Enzymatic Agents Research Articles

Standardized reprocessing of reusable colonoscopy biopsy forceps is effective: results of a German multicenter study.

National and international guidelines recommend that a standardized protocol consisting of cleaning, ultrasound cleaning, and sterilization should be used for the reprocessing of endoscopic accessories in order to reduce the risk of transmission of microorganisms. This German multicenter study investigated the efficacy of standardized reprocessing of reusable biopsy forceps used during colonoscopy. Ten endoscopy centers (eight hospitals and two private practices) used 330 biopsy forceps during routine colonoscopy. The forceps were used once, five times, or 20 times for colonoscopy, based on a randomization plan. The reprocessing protocol consisted of manual cleaning with an enzymatic agent, ultrasound cleaning with an enzymatic agent (30 min, 40 degrees C, 47 Hz), neutralization, drying, and sterilization (5 min, 134 degrees C). Aldehydes were not used, and the protocol did not include a disinfection step. The biopsy forceps were sent to three microbiological institutes, based on a randomization plan, to have them tested for the presence of organisms, including identification of bacteria. A total of 318 of the 330 forceps were evaluable; 314 forceps (98.74 %) were sterile after use once, five times, or 20 times. Four forceps were contaminated with Staphylococcus epidermidis (n = 2), Bacillus licheniformis (n = 1) and Corynebacterium aquaticum (n = 2). All of 25 forceps were sterile after being used 20 times. Colonoscopy biopsy forceps can be reliably reprocessed following this standardized protocol, even without aldehydes.

Enzymatic cleaning of inorganic ultrafiltration membranes fouled by whey proteins.

The aim of this work was to study the cleaning of inorganic membranes fouled with whey protein solutions using the enzymatic formulation Alcalase (Novo Nordisk A/S). Hydraulic and chemical methods were considered to characterize the cleanliness of the membranes. Cleaning efficiency was observed to be a function of the operating conditions. The operating conditions tested were the following: recycling versus non-recycling of permeate, pH of the cleaning solution, addition of alkali to regulate the pH, enzymatic agent concentration, and cleaning time. The best conditions to perform the cleaning were related to the best conditions to hydrolyze whey proteins in a discontinuous reactor using the same enzyme preparations. Very high cleaning efficiencies (>90%) were achieved in short operating times (20 min). However, residual matter was observed on the membrane surface.

Zinc-catalyzed oxidation of 5-S-cysteinyldopa to 2,2'-bi(2H-1,4-benzothiazine): tracking the biosynthetic pathway of trichochromes, the characteristic pigments of red hair.

Trichochromes, the peculiar pigments of red human hair, featuring the Delta(2,2)(')-bi(2H-1,4-benzothiazine) skeleton, are known to arise from cysteinyldopas, mainly the 5-S-isomer (5). However, the mode of formation and the direct precursors have remained largely undefined. To fill this gap, we investigated the oxidation of 5 in air or with chemical and enzymatic agents under biomimetic conditions. In the presence of zinc ions, which occur in epidermal tissues at significant concentrations, the reaction course is diverted toward the formation of a labile 3-carboxy-2H-1,4-benzothiazine intermediate (11), which was identified by direct NMR analysis. Structural formulation was supported by characterization of the analogous compound 13 isolated from oxidation of the model 5-methyl-3-S-cysteinylcatechol (12) after methylation. In the further stages of the oxidation, diastereomeric 2,2'-bi(2H-1,4-benzothiazine) 15 and 14 were obtained from 5 and 12, respectively, the reaction proceeding at a higher rate and to a greater extent in the presence of acids. The dimers were shown to readily convert to each other in the presence of acids. In the case of the methylated dimers 14, a 2,2'-bi(4H-1,4-benzothiazine) intermediate (16) was isolated and characterized. In acidic media, trichochrome C (1a), the most abundant in red human hair, was smoothly formed from aerial oxidation of 15, and under similar conditions, trichochrome-related products (17 and 18) were obtained from 14 prior to or after methylation. The presence of 1a and precursors 5 and 15 was investigated by HPLC analysis of red hair samples following mild proteolytic digestion. On the basis of these data, a likely biosynthetic route to trichochrome pigments of red human hair is depicted.

Cryopreservation Increases Availability and Usability of Ovine Adipose-tissue Derived Stem Cells for Regenerative Medicine Search

Aim: Our long-term aim is to develop a living valvular substitute using Regenerative Medicine principles, by seeding decellularized porcine heart valve scaffolds with adult stem cells and conditioning them in bioreactors before implantation. In this study, adult stem cells were isolated from sheep adipose tissue (ADSCs). However, we found it impractical to use cells immediately after propagation and thus, in order to extend their availability in time, a preservation method was needed. Methods: Adipose tissue was harvested from 6 sheep. ADSCs were isolated using enzymatic agents and cultured. The cells were tested for plasticity using chondrogenic, adipogenic and osteogenic differentiation kits and then cryopreserved in DMSO at -1400C. Viability was tested after a 3 week storage using Trypan Blue Staining. Results: Ovine ADSCs exhibited excellent plasticity and differentiation potential. An average of 18 million ADSCs were obtained from each ovine, exhibiting more than 88% viability after a 3-week cryopreservation period followed by thawing. Conclusions: DSMO cryopreservation represents a suitable method for ovine ADSCs for regenerative medicine. This method expands the usage of stem cells in vitro before they are differentiated into more specialized cells, offering large numbers of usable ADSCs with minimal cell loss at any desired time point.

Involvement of protein phosphatases in differential regulation of renal proximal tubular PAH and sodium-dependent dicarboxylate transport.

It has been demonstrated that the basolateral organic allion (PAH) transporter and the sodium-dependent dicarboxylate transporter of rabbit renal proximal tubules are regulated differentially. A variety of protein kinases has been shown to be involved in the regulation of organic anion transport while dicarboxylate uptake, to which the first is coupled functionally, is not influenced by these kinases. This study was undertaken to elucidate whether respective transporter activities are modulated differentially by protein phosphatases as well. The experiments were performed on isolated S, segments of proximal tubules microdissected from rabbit kidneys without the use of enzymatic agents. 3H-PAH was used as marker substance of the PAH transporter, 14C-glutarate as a marker of the sodium dicarboxylate cotransporter. 30 s tubular uptake measurements were performed. Vanadate (10(-3) M), a selective inhibitor of tyrosine phosphatase, did not reduce PAH uptake significantly, while inhibitors of the serine threonine phosphatases 1 and 2A, okadaic acid and calyculin A (10(-6) M, each) induced a significant decrease of 30 s PAH uptake (by 32.3% +/- 7.9% and 25.6% +/- 6.4%) but not a change in dicarboxylatc transport. These findings indicate that, in addition to a variety of protein kinases, serine threonine phosphatases have a role in the regulation of renal basolateral PAH transport. There is no effect of these phosphatases on basolateral 30s gutaltarate transport. Thus, additional evidence for differential regulation of short-time activiity of the transporters for PAH and dicarboxylates is provided.

Role of the calcium/calmodulin‐dependent protein kinase II in the regulation of the renal basolateral PAH and dicarboxylate transporters1

The aim of the present study was to investigate whether the activities of the renal basolateral organic anion transporter (PAH transporter) and the sodium-dependent dicarboxylate transporter are modulated by the calcium/calmodulin-dependent multifunctional protein kinase II (CaM kinase II). The studies were performed on isolated S2 segments of proximal tubules microdissected from rabbit kidneys without the use of enzymatic agents. 3H-PAH was used as marker substance of the anion transporter, and 14C-glutarate as a marker of the sodium/dicarboxylate cotransporter. Because the tubules were not perfused, and hence were collapsed, the tubular uptake of the marker substances reflects transport across the basolateral cell membrane. To obtain uptake rates most closely related to initial transport rates, 30 s tubular uptake measurements were performed. The results show that a selective inhibitor of CaM kinase II, KN93, inhibited tubular PAH uptake. The smallest effective dose was 10(-7) M. An inactive analogue of KN93, KN92, was without effect, even at the high concentration of 10(-5) M. In contrast to PAH transport, tubular 14C-glutarate uptake was not affected by KN93 (10(-5) M). PAH transport was also inhibited after elevation of intracellular Ca2+ by the Ca(2+)-ionophore A 23187 and by the polycationic antibiotic neomycin, but not by the intracellular Ca2+ modulators thapsigargin and ryanodine. The effect of the Ca(2+)-ionophore could be abolished by KN93, but not by Rp-cAMPs, an inhibitor of protein kinase A, indicating that this event was mediated by CaM kinase II, but not by PKA. The results provide the first evidence that, in addition to the protein kinases A and C (previous studies from this lab), CaM kinase II has a role in the regulation of the renal basolateral PAH transporter, whereas the renal basolateral dicarboxylate transporter does not depend on CaM kinase II activity.

The debridement of chronic wounds: a systematic review.

The debridement of chronic wounds: a systematic review.

Basolateral transport of glutarate in proximal S2 segments of rabbit kidney: kinetics of the uptake process and effect of activators of protein kinase A and C.

The kinetics of tubular glutarate uptake, the coupling of glutarate to p-aminohippurate (PAH) transport and the effect of activators of protein kinase A and C on glutarate uptake were studied using isolated S2 segments of proximal tubules microdissected from rabbit kidneys without the use of enzymatic agents. Because the tubules were not perfused, and hence were collapsed, the tubular uptake of [14C]glutarate reflects transport across the basolateral cell membrane. To obtain uptake rates most closely related to initial transport rates, 30 s glutarate uptake measurements were performed. In a first set of experiments it could be shown that preloading proximal S2 segments with glutarate (10(-3 )M) stimulated [3H]PAH uptake indicating that glutarate may be a substrate of the PAH /dicarboxylate exchanger. The kinetic data revealed a Km value of 0. 62 mM and a Vmax value of 84.1 pmol nl-1min-1 for tubular [14C]glutarate uptake across the basolateral cell membrane. In contrast to basolateral PAH transport (previous studies from this laboratory), tubular 30 s [14C]glutarate uptake was not affected by either the phorbol ester phorbol 12-myristate 13-acetate (PMA, 10(-7 )M), an activator of protein kinase C, or by the membrane-permeant analogues of cAMP, dibutyryl cyclic AMP (db-cAMP, 10(-4 )M) and 8-bromoadenosine 3',5'-cyclic monophosphate (Br-cAMP, 10(-4 )M). The results indicate that the protein kinases A and C have no function in the regulation of the renal basolateral dicarboxylate transporter. This finding agrees well with the structural feature of the recently cloned rabbit renal dicarboxylate transporter which does not contain any putative phosphorylation sites for protein kinase C or cAMP-dependent kinase.

Effect of activation of protein kinase A and of protein kinase C on the kinetics of the renal basolateral PAH transporter.

The aim of the present study was to examine the effect of activation of the protein kinase A (PKA) and protein kinase C (PKC) pathways on 3H-p-aminohippurate (PAH) uptake of isolated S2 segments of proximal tubules, microdissected from rabbit kidneys without the use of enzymatic agents. Because the tubules were not perfused, and hence were collapsed, the tubular uptake of 3H-PAH reflects transport across the basolateral membrane. The phorbol ester phorbol 12-myristate 13-acetate (PMA) (10(-7) M), an activator of PKC, significantly increased tubular 3H-PAH uptake with steady state conditions (by 115%), whereas dibutyryl cyclic adenosine monophosphate (db-cAMP) (10(-4) M) and forskolin (10(-4) M) significantly inhibited it (by 42% and 52%, respectively). Kinetic data, which were based on 15 sec PAH uptake measurements, revealed that PMA, after a 10 min incubation period, significantly enhanced Km and Vmax of the PAH transporter (Km from 174 +/- 22 to 447 +/- 91 microM, Vmax from 2.76 +/- 0.24 to 16.67 +/- 1.85 pmol nL-1 min-1), whereas db-cAMP significantly decreased Vmax (from 2.76 +/- 0.24 to 1.82 +/- 0.19 pmol nL-1 min-1). The Km value was also numerically lowered by dibutyryl-cAMP (from 174 +/- 22 to 139 +/- 21 microM), but this change did not reach statistical significance. The data provide evidence that short time activation of the PKC pathway 1) enhances the effectiveness of PAH transport into proximal S2 segments across the basolateral cell membrane, 2) increases the maximum transport rate of the PAH transporter and 3) decreases its affinity for PAH. Activation of the cAMP/PKA pathway induces the opposite effects.

Sterility testing of antimicrobial-containing injectable solutions prepared in the pharmacy

The need for sterility testing of antimicrobial-containing injectable solutions is discussed and specific testing methods are described. Despite their antimicrobial activity, antimicrobial-containing injectable drug products are not necessarily self-sterilizing and can become contaminated. In addition to practicing aseptic technique, pharmacists must perform end-product sterility testing on intravenous solutions to ensure their sterility. The United States Pharmacopeia provides guidelines for the performance and validation of two sterility test methods: membrane filtration and direct transfer to culture media. Membrane filtration is the method of choice for sterility testing of many antimicrobial-containing injectable solutions. After the test article is filtered, the membrane is rinsed with sterile fluid to remove residual antimicrobial agent, cut into two portions, and immersed in two types of culture medium. Visible turbidity of a sample within the appropriate incubation period indicates the presence of a contaminating microorganism. Closed filtration systems minimize false-positive results. In the direct transfer method, samples of the test article are directly inoculated into vessels of culture media, and antimicrobial activity is eliminated by dilution or by deactivation with chemical or enzymatic agents. Sterility testing as well as aseptic technique is needed to ensure the sterility of antimicrobial-containing injectable solutions.

Failure of Dithiothreitol and Pronase to Reveal a False-Positive Cryptococcal Antigen Determination in Cerebrospinal Fluid

A patient with squamous cell carcinoma of the lung and a serum rheumatoid factor (RF) of 1:1,280 had a positive cerebrospinal fluid (CSF) latex agglutination test (LAT) for cryptococcal antigen, in culture-negative, India-ink-negative CSF. Pretreatment of the sample of CSF with 2-mercaptoethanol (2-ME) ablated the antigen titer and established the presence of a false-positive LAT, whereas CSF pretreated with dithiothreitol (DTT) and pronase continued to yield a false-positive result. The differing ability of pronase, DTT, and 2-ME to eliminate interfering substances from CSF has not been previously described. Moreover, because RF is unlikely to cross the blood-brain barrier, the authors postulated that malignant disease was responsible for the patient's false-positive LAT in CSF. Hence, the authors report the case to emphasize that false-positive LAT results in CSF are unlikely to be produced by RF and to underscore the benefit of using enzymatic and sulfhydryl-reducing agents when the validity of the initial test results are in doubt. Such a procedure will optimize the chances of accurately identifying false-positive LAT results in CSF.

Genetic organization of the cSOD microregion of Drosophila melanogaster

Cu/Zn superoxide dismutase (cSOD) is an important enzymatic agent of physiological defense against active oxygen species. Previously, we defined the essential biological role of cSOD in Drosophila through analysis of a cSOD null mutant. In the process of isolating this mutant, we also identified several vital genes in the chromosomal region surrounding the cSOD gene (the cSOD microregion). To further our genetic analysis of cSOD function, we have undertaken a detailed description of the cSOD microregion as defined by the breakpoints of the deficiency Df(3L)lxd9. Examination and correlation of mutations previously recovered with new X-ray induced mutations described in this paper identify a total of 12 vital genes, including cSOD, and one nonvital gene, lxd, in this region. We propose the adoption of a single genetic nomenclature for the genes of this region. Two newly generated hypomorphic alleles of cSOD are described that confer phenotypes similar to cSODn108, confirming the important physiological role of the enzyme in Drosophila.

The Use of Proteolysis with Ficin, for Immunostaining of Paraffin Sections: A Study of Lymphoid, Mesenchymal, and Epithelial Determinants in Human Tissues

In order to maximize staining, modifications of immunostaining methods have included proteolytic enzyme digestion of tissue. The authors performed a study of the effect of ficin in 110 paraffinized specimens, including tonsil, lymph nodes, benign vascular and nerve sheath tumors, and various carcinomas and sarcomas. This agent was compared with pepsin and bromelain, as alternative proteases. A panel of monoclonal and polyclonal antibodies was used, with and without previous digestion by ficin, pepsin, and bromelain. A score was assigned to each stain, based on the number and intensity of reactive cells. Ficin enhanced staining markedly in immunostains with antibodies to keratin and Factor VIII-related antigen (F8RAG). Conversely, it abolished staining for LN-2 (a lymphoid marker) and weakened reactivity for S-100 in nerve sheath tumors. Bromelain produced similar results, except that it enhanced S-100. Pepsin was comparatively less active than ficin and bromelain overall but did produce the greatest amplification of vimentin staining in sarcomas. Digestion with any of the three enzymes failed to influence reactivities of leukocyte common antigen, UCHL-1 (a lymphoid marker), alpha-1-antichymotrypsin, carcinoembryonic antigen, epithelial membrane antigen, and blood group isoantigens. These results may reflect a dissimilar recognition of peptide targets in some antigenic proteins, by ficin, bromelain, and pepsin. Hence, one enzymatic agent is unlikely to produce optimal staining for all determinants. With this proviso, however, ficin appeared to be the best general enhancer for antigens known to require vigorous digestion (e.g., keratin; F8RAG) for optimal reactivity in paraffin sections.

Ultrastructural studies of the effect of human leukocyte extracts on periodontopathic bacteria.

Human gingival crevicular exudate (GE) and leukocyte extracts (LE) were previously shown to be able to release the bulk of radioactivity from a variety of 14C-labeled oral bacteria. The present study demonstrates that the release of radiolabel by these enzymatic agents from two periodontopathic bacteria (Actinobacillus actinomycetemcomitans strain Y4 and Capnocytophaga sputigena strain 4) is accompanied by both cell wall and cytoplasmatic damage. The possible role of bacterial products released by lysosomal enzymes, present in the gingival crevice, is discussed.

Electron transfer loci on blue copper proteins

The chemical properties of the Cr(II)/(III) couple has been successfully employed to final affinity label electron transfer loci on redox proteins. Being strong reductants and able to exchange their ligands sphere fast, the Cr(II) ions can coordinate one or more of the surface residues on the protein while reducing it. Since the Cr(III) produced is effectively substitution inert, any protein residues in the coordination sphere of the Cr(II) during the electron transfer will remain bound to the Cr(III) product. Identification of the Cr(III) binding loci has been achieved primarily through proteolytic cleavage of the different labeled proteins. Spectroscopic methods have been useful in corroborating these assignments. Several single blue copper proteins have been examined by the above approach. These include the bacterial electron carriers azurin from Pseudomonas aeruginosa and Alcaligenes faecalis. Plastocyanin (from French bean and poplar tree) which serves as an electron mediator in the photosynthetic apparatus and stellacyanin obtained from the lacquer sap of Rhus vernicifera have all been studied by this method. More recently it has been shown that even the multicentered blue copper oxide-laccase can be reductively labeled by this procedure. In this latter case, while 3.3 equivalents of Cr(II)aq were required for full reduction of the protein, only 0.7 Cr(III) ions remained bound to laccase after extensive dialysis. This may indicate a single reduction locus for Cr(II) in this protein. The cuprous ions in the Cr(III) labeled plastocyanin, azurin and stellacyanin could be fully reoxidized by inorganic or enzymatic agents. While the original, single Cr(III) ion coordinated to azurin and stellacyanin remains bound through several Cr(II) reduction and reoxidation cycles, one can label plastocyanin with at least two Cr(III) ions in two cycles. In the structure of both plastocyanin and Ps azurin the ‘northern’ end imidazol of His-87 or 117 respectively was considered to be the potential electron transfer site. The analysis of the Cr(III) labeled sites on these two proteins clearly showed that electrons can also be introduced via different loci. These were proposed to proceed via the His 35 region in azurin (Az) and the negative patch on plastocyanin (Pc). To examine whether the latter electron transfer sites are also involved in the biochemical function of these proteins, their reactivities, in the native and Cr(III) labeled forms were compared. For Pc, photoreduction and oxidation by chloroplasts and by photosystem I reaction centers respectively were studied. For azurin the reactions with cytochrome c 551 and Ps. cytochrome oxidase were investigated. It became apparent that the Cr(III) label attenuated the reactivity of both azurin and plastocyanin with only one of their respective partners. This led to the conclusions that on both proteins: (a) There are probably two distinct and physiologically operative electron transfer sites. (b) One of these sites is centered around the respective Cr(III) labeled region. (c) By elimination, the second is at the exposed, homologous imidazol of His-87 or 117 in Pc and Az respectively.

Nonspecific proteases: a new approach to the isolation of adult cardiocytes.

The potent collagenolytic activity of nonspecific proteinases suggested their use as enzymatic agents for the dissociation of single adult heart cells. This was assessed in guinea pig hearts perfused for 1 min with solutions containing hyaluronidase (100-10 000 U/mL), trypsin (100-10 000 U/mL), crude collagenase (100-500 U/mL), or nonspecific protease (0.1-100 U/mL). No rod-shaped cells were observed among the cells isolated with these concentrations of hyaluronidase, trypsin, or crude collagenase. By contrast, 45-80% of the cells released with nonspecific protease (5-10 U/mL) were rod shaped and Ca2+ tolerant. Resting and action potentials recorded from cells dispersed with nonspecific protease were similar to those recorded from cells isolated after prolonged collagenase exposure.

Chemical Debridement of Burns

Experiments were conducted using non-enzymatic chemical agents (with emphasis on certain mercaptans), alone, in conjunction with enzymatic agents and/or other nonenzymatic chemicals for debridement of burns. Both in vitro (rats, pigs, humans) and in vivo (rats, pigs) tests were carried out. N-acetylcysteine, penicillamine and cysteine ethyl ester in low to moderate concentrations accelerate the debriding action of bromelain (an enzymatic preparation from pineapple stems) and in higher concentrations, N-acetylcysteine and penicillamine (cysteine ethyl ester was not tested) cause ready separation of the burn eschar from the underlying tissue before solubilization of the eschar is complete (rat) or has occurred (pig). Debridement of 3 degree burns of rats is complete within 4-6 hours; the take of immediately applied syngeneic skin grafts is complete and permanent. This is first time rapid debridement of 3 degree burns permitting immediate successful skin grafting has been accomplished with known defined chemicals. In pigs there is softening of the 3 degree burn eschar by N-acetylcysteine but little, if any, dissolution of the eschar. However, mechanical separation of the eschar from the underlying tissue is accomplished readily with a wooden throat stick with no bleeding. There is a change in color of the superficial layer of the underlying subcutaneous tissue from yellow-light brown to dark brown-black. The debrided areas begin to granulate promptly. The healing of deep dermal burns of pigs is hastened by the application of N-acetylcysteine for a day (beginning 24 hours after burning) while the healing of moderately deep dermal burns is not modified. Unburned skin is not damaged. There is no apparent systemic toxicity associated with the use of N-acetylcysteine for debridement of 10-15% b.s.a. 3 degree burns of rats or 15-20% b.s.a. 3 degree burns of pigs. Major emphasis has been on N-acetylcysteine because of the potential adverse secondary effect of penicillamine and cysteine ethyl ester; N-acetylcysteine is readily metabolized. The use of a keratolytic agent prior to the application of N-acetylcysteine hastens the latter's action. Sulfamylon and sulfadiazine can be used with N-acetylcysteine without interfering with its debriding action. The effects of the mercaptans are likely due largely to their ability to depolymerize connective tissue proteoglycans and proteins, especially at the interface between living and dead tissue.

Some properties of Ascaris suum allergen A.

The stability of <i>Ascaris suum </i>allergen A to chemical and enzymatic agents has been studied. The allergenic activity measured in the rat passive cutaneous anaphylaxis test was found to be stable to heat at 100 °C, hot acid and periodate at 4 °C but to be destroyed completely by hot alkali. The allergen showed various degrees of resistance to treatment by the proteolytic enzymes trypsin, papain, chymotrypsin, elastase, subtilisin and pronase but was hydrolysed readily by pepsin. The effect of alkali on the allergen has been further studied by thin-layer gel filtration, ultracentrifugation, electrophoresis and amino acid analysis. Although the amino acid composition and sedimentation coefficients remained the same as the native allergen after this treatment, the isoelectric point and electrophoretic mobility were changed.

Tyrosinase activity in subcellular fractions of black and albino mice.

TYROSINASE (EC 1.10.3.1.), a key enzyme in melanogenesis, seems to occur in an inactivated form in various tissues1–3. I have examined the possibility that inhibition, rather than a lack of enzymatic activity, is involved in the absence of melanin synthesis in the albino mouse; the c (albino) locus has up to now been considered the structural locus for tyrosinase4,5. I report here the effects on albino and black murine pigment tissue of physical methods and chemical and enzymatic agents which affect tyrosinase activity6–8.

Interaction of lipids with acid-soluble bovine skin collagen

Acid-soluble bovine skin collagen was found to contain lipids even after exhaustive extractions with chloroform-methanol. To determine if this lipid fraction was bound covalently to collagen, the protein was partially hydrolyzed by chemical or enzymatic agents to yield a series of polypeptide fragments. Each of these polypeptides was isolated and found to be free of any accompanying lipid. It is concluded that the associated lipids of soluble bovine skin collagen are bound by weak intermolecular forces.