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A high mild traumatic brain injury (mTBI) incidence rate exists in military and sport. Hypopituitarism is an mTBI sequela; however, few studies have examined this phenomenon in those with an mTBI history. This cross-sectional study of Special Operations Forces combat soldiers aimed 1) to relate anterior pituitary gland volumes (actual and normalized) to insulin-like growth factor 1 (IGF-1) concentrations, 2) to examine the effect of mTBI history on anterior pituitary gland volumes (actual and normalized) and IGF-1 concentrations, and 3) to measure the odds of demonstrating lower anterior pituitary gland volumes (actual and normalized) or IGF-1 concentrations if self-reporting mTBI history.Anterior pituitary gland volumes were manually segmented from T1-weighted 3D brain MRI sequences; IGF-1 serum concentrations were quantified using commercial enzyme-linked immunosorbent assays. Correlations and linear regression were used to determine the association between IGF-1 serum concentration and anterior pituitary gland volume (n = 74). Independent samples t-tests were used to compare outcomes between mTBI groups and logistic regression models were fit to test the odds of demonstrating IGF-1 concentration or anterior pituitary volume less than sample median based on mTBI group (n = 54).A significant linear relationship between the subjects' anterior pituitary gland volumes and IGF-1 concentrations (r72 = 0.35, P = 0.002) was observed. Soldiers with mTBI history had lower IGF-1 concentrations (P0.001) and lower anterior pituitary gland volumes (P = 0.037) and were at greater odds for IGF-1 serum concentrations less than the sample median (odds ratio = 5.73; 95% confidence interval = 1.77-18.55).Anterior pituitary gland volume was associated with IGF-1 serum concentrations. Mild TBI history may be adversely associated with anterior pituitary gland volumes and IGF-1 concentrations. Longitudinal IGF-1 and anterior pituitary gland monitoring may be indicated in those who report one or more mTBI.

Our aim in this study was to psychometrically test resilience assessments (Ego Resiliency Scale [ER89], Connor-Davidson Resilience Scale [CD-RISC 25], Responses to Stressful Experiences Scale [RSES short-form]) and describe resilience levels in a Special Operations Forces (SOF) combat sample.Fifty-eight SOF combat Servicemembers either entering SOF (career start; n = 38) or having served multiple years with their SOF organization (mid-career; n = 20) self-reported resilience, mild traumatic brain injury (mTBI) history, and total military service.All resilience metrics demonstrated acceptable internal consistency, but ceiling effects were found for CD-RISC and RSES scores. ER89 scores were moderate on average. ER89 scores were higher in SOF career start than mid-career Servicemembers (ηρ2 = 0.07) when accounting for the interaction between SOF career stage and total military service (ηρ2 = 0.07).SOF mid-career Servicemembers had similar ER89 resilience scores with more total military service. The SOF career start combat Servicemembers had higher ER89 measured resilience with less total military service only, potentially showing a protective effect of greater service before entering SOF.The ER89 may be a more optimal military resilience metric than the other metrics studied; longitudinal research on SOF combat Servicemember resilience is warranted.

Military resilience research is increasing due to the growing literature associating resilience with stress adaptation. This study aimed to investigate which physiological stress adaptation components were associated with resilience in Special Operations Forces combat service members. Special Operations Forces combat service members (n = 117) self-reported resilience (ER89) and lifetime clinician-confirmed mild traumatic brain injury history. Participants also underwent transcranial Doppler ultrasonography to measure middle cerebral artery velocity during rest and a breath-holding task. Neither resilience nor mild traumatic brain injury history was significantly associated with middle cerebral artery velocity percent increase following breath-holding; younger Special Operations Forces combat service members had a higher percent increase in middle cerebral artery velocity following a breath-holding task. Resilience was negatively associated with time to return to baseline middle cerebral artery velocity following peak velocity; whereas, mild traumatic brain injury history did not have a significant association. The Special Operations Forces combat service members that scored higher in resilience tended to return to baseline middle cerebral artery velocity following peak velocity faster than their less resilient counterparts. More resilient Special Operations Forces combat service members recovered faster from physiological stress (breath-holding) than less resilient counterparts. This is the first study to investigate resilience and cerebrovascular stress response and recovery in this population. Our initial findings indicated that the Ego Resiliency Scale may be an optimal resilience psychometric and should be used to evaluate effective military resilience trainings, which aim to improve performance and mental health.

The purpose of this study was to investigate how concussion history affects neurovascular coupling in Special Operations Forces (SOF) combat Soldiers. We studied 100 SOF combat Soldiers [age = 33.5 ± 4.3 years; height = 180.4 ± 6.0 cm; 55 (55.0%) with self-reported concussion history]. We employed transcranial Doppler (TCD) ultrasound to assess neurovascular coupling (NVC) via changes in posterior cerebral artery (PCA) velocity in response to a reading and a visual search task. Baseline TCD data were collected for 2 min. NVC was quantified by the percent change in overall PCA response curves. We employed linear mixed effect models using a linear spline with one knot to assess group differences in percent change observed in the PCA velocity response curves between SOF combat Soldiers with and without a concussion history. Baseline PCA velocity did not significantly differ (t98 = 1.28, p = 0.20) between those with and without concussion history. Relative PCA velocity response curves did not differ between those with and without a concussion history during the reading task (F1,98 = 0.80, p = 0.37) or the visual search task (F1,98 = 0.52, p = 0.47). When assessing only SOF combat Soldiers with a concussion history, differential response to task was significantly greater in those with 3 or more concussions (F1,4341 = 27.24, p < 0.0001) relative to those with 1-2 concussions. Despite no main effect of concussion history on neurovascular coupling response in SOF combat Soldiers, we observed a dose-response based on lifetime concussion incidence. While long-term neurophysiological effects associated with head impact and blast-related injury are currently unknown, assessing NVC response may provide further insight into cerebrovascular function and overall physiological health.

The stellate ganglion block (SGB) procedure has been used successfully for over 10 years to treat post-traumatic stress symptoms in thousands of US military service members, civilians, and veterans in select hospitals in Europe and North America. Primarily through targeting the autonomic nervous system, the SGB procedure serves as an invaluable adjunct to trauma-focused psychotherapy. Without published best practices for emerging therapies, clinicians are left on their own to determine how best to apply new treatments to their patient populations. The aim of this qualitative research was to compile attitudes and recommendations from therapists with expertise in using SGB for treating symptoms of post-traumatic stress disorder, so that their experiences could be disseminated widely to clinicians without SGB expertise. An 18-item survey was developed and distributed electronically to a group of behavioral health professionals of various specialties between May and June 2020. Of surveyed behavioral health clinicians with personal experience incorporating SGB into their trauma-focused psychotherapy, 95% of respondents would recommend SGB to a colleague as a useful tool for the treatment of trauma-related disorders. SGB was rated at least as useful as the most valuable interventions listed in the American Psychological Association Clinical Practice Guideline for the Treatment of Post-traumatic Stress Disorder with 100% of respondents characterizing SGB as ‘Very Beneficial’ or ‘Somewhat Beneficial’, and 0 respondents characterizing SGB as ‘Not Helpful’ or ‘Harmful’. Given the feedback from this study, behavioral health providers should consider using SGB in conjunction with standard trauma-focused care.

Background Special Operations Forces (SOF) combat soldiers are frequently exposed to blast and blunt neurotrauma, most often classified as mild traumatic brain injury (mTBI). Repetitive mTBI may increase the risk of developing long-term neurological sequelae. Identifying changes in neuroinflammatory biomarkers before chronic conditions emerge could serve as preliminary evidence of developing neuropathology. Objective To determine the effects of mTBI history, lifetime mTBI incidence, and recency on blood biomarker concentrations of axonal protein neurofilament light (NfL), glycolytic enzyme neuron-specific enolase (NSE), astrocyte-expressed S100 calcium-binding protein B (S100B), and neurotrophic cytokine interleukin-6 (IL-6) in healthy, active duty SOF combat soldiers. Methods Self-reported mTBI history/recency and fasted blood samples were collected in this cross-sectional study of 104 asymptomatic SOF combat soldiers. Biomarker concentrations were quantified using commercial enzyme-linked immunosorbent assays. Mann-Whitney U and Kruskal-Wallis tests were used to compare groups. Post hoc tests with appropriate corrections were conducted as warranted. Results Soldiers with mTBI history had higher NSE concentrations than those without (z = -2.60, P = .01). We also observed significant main effects of lifetime mTBI incidence on NSE (χ(3) = 9.52, P = .02) and S100B (χ(3) = 8.21, P = .04) concentrations and a significant main effect of mTBI recency on NfL concentration (χ(2) = 6.02, P = .049). Conclusion The SOF combat soldiers with mTBI history had increased NSE. Longitudinal studies in this population are needed due to between-subject heterogeneity in biomarker concentrations. The NfL concentrations in our SOF combat soldiers-regardless of mTBI history or recency-were similar to values previously reported in civilian acute TBI patients.

Introduction Special Operations Forces (SOF) combat arms and combat support Soldiers are at risk for impaired mental health, such as mood- and stress-related disorders, due to operational and training demands. Additionally, these individuals experience high risk for sustaining mild traumatic brain injury (mTBI). These mTBIs have also been linked to negative psychological outcomes, such as anxiety and depressive symptoms. Studying mental illnesses and their related symptoms alone does not fully address mental health, which may be better understood by 2 separate but overlapping continua measuring both mental illness and subjective well-being (ie, emotional, psychological, and social well-being). Due to the lack of research in this area, current mental health symptoms in active SOF combat Soldiers in relation to mTBI warrants investigation. Materials and Methods In this study, 113 SOF combat and combat support Soldiers completed self-report psychological and mTBI history measures during an in-person laboratory setting. These psychometric measures included (1) psychological distress (Brief Symptom Inventory 18), (2) anxiety (Generalized Anxiety Disorder 7-item), (3) posttraumatic stress (PTSD Checklist for DSM-5), (4) somatization (Patient Health Questionnaire-15), and (5) subjective well-being (Mental Health Continuum Short Form). Results On average, SOF combat Soldiers endorsed moderate well-being and low psychological distress, somatization, posttraumatic stress, and anxiety. Most SOF combat Soldiers had sustained 1 or more mTBI. We observed mTBI history had significant effects on each dependent variable in the expected directions. History of more mTBIs, controlling for age, was associated with lower subjective well-being as well as higher psychological distress, somatization, posttraumatic stress, and anxiety symptoms. Conclusion Although SOF combat Soldiers reported relatively adaptive mental health symptoms across participants, there was considerable variance in the measures reported. Some of the variance in mental health symptoms was accounted for by mTBI history while controlling for age, with reporting higher numbers of lifetime mTBIs and older age being associated with worse mental health symptoms. Longitudinal investigations into these associations and their impact on Soldier performance is warranted.

The purpose of this study was to investigate how concussion history affects neurovascular coupling in Special Operations Forces (SOF) combat Soldiers. We studied 100 SOF combat Soldiers [age = 33.5 ± 4.3 years; height = 180.4 ± 6.0 cm; 55 (55.0%) with self-reported concussion history]. We employed transcranial Doppler (TCD) ultrasound to assess neurovascular coupling (NVC) via changes in posterior cerebral artery (PCA) velocity in response to a reading and a visual search task. Baseline TCD data were collected for 2 min. NVC was quantified by the percent change in overall PCA response curves. We employed linear mixed effect models using a linear spline with one knot to assess group differences in percent change observed in the PCA velocity response curves between SOF combat Soldiers with and without a concussion history. Baseline PCA velocity did not significantly differ (t

The present study investigated Special Operations Forces (SOF) combat Servicemember mental health at different SOF career stages in association with resilience.Fifty-eight SOF combat Service Members either entering SOF (career start; n=38) or multiple years with their SOF organization (mid-career; n=20) self-reported mild traumatic brain injury (TBI) history, resilience, subjective well-being, depression, anxiety, and posttraumatic stress. Poisson regression analyses were employed to test SOF career stage differences in each mental health symptom using resilience, while accounting for other pertinent military factors.There were significant interaction effects of SOF career stage and resilience on mental health symptoms. SOF career start combat Servicemembers endorsed lower depression and posttraumatic stress and higher subjective well-being with higher resilience, but these associations between resilience and mental health symptoms were not seen in SOF mid-career Servicemembers.Although preliminary, the adaptive association between resilience and mental health seemed to be blunted in combat Servicemembers having served multiple years in SOF. This information informs research to provide evaluation tools to support prophylactic performance and long-term health preservation in military populations.

The purpose of this study was to investigate how concussion history affects cerebrovascular reactivity (CVR) in Special Operations Forces (SOF) combat soldiers. We studied 104 SOF soldiers [age = 33.5 ± 4.3 years; height = 179.7 ± 6.3 cm; 59 (56.7%) with self-reported concussion history]. We employed transcranial Doppler (TCD) ultrasound to measure middle cerebral artery (MCA) velocity. Baseline TCD data were collected for 2 min. Changes in MCA velocity were measured in response to five breath-holding trials and five hyperventilation trials. Cerebrovascular reactivity was quantified by the breath-holding index (BHI), vasomotor reactivity reserve (VMRr), and percent change in overall response curves. Independent t tests were employed to assess group differences in BHI, and VMRr values. We employed mixed effects models with quadratic mean structures to assess group differences in percent change MCA velocity response curves. There were no significant group differences in BHI (t102 = 0.04, p = 0.97) or VMRr (t102 = -0.33, p = 0.75). There were no group differences in relative MCA velocity response curves during the breath-holding task (F1,5092 = 0.19, p = 0.66) or during the hyperventilation task (F1,5092 = 0.41, p = 0.52) between SOF soldiers with and without a self-reported concussion history. If CVR deficits exist immediately post-concussion, our study suggests that these deficits recover over time in this population. While long-term neurophysiological effects of blast-related injury are currently unknown, assessing CVR response may provide further insight into cerebrovascular function and overall physiological health following blast exposure.

U.S. Army Special Operations Forces (SOF) soldiers deploy frequently and conduct military operations through special warfare and surgical strike capabilities. Tasks required to execute these capabilities may induce physical and mental stress and have the potential to degrade soldier physiological status. No investigations have longitudinally characterized whether combat deployment alters anthropometrics or biochemical markers of physiological status in a SOF population of frequent deployers.Effects of modern combat deployment on longitudinal changes in anthropometrics and physiological status of elite U.S. Army SOF soldiers (n = 50) were assessed. Changes in measures of body composition, grip strength, physiological status, and health behaviors from baseline to postdeployment were determined with paired t test and McNemar's statistic. Baseline measures were obtained between 4 and 8 weeks before deployment. Deployment length was a uniform duration of time between 3 and 6 months (all soldiers completed the same length of deployment). Post hoc analyses determined change in body mass within quartiles of baseline body mass with paired t test and associations between change in sex hormone-binding globulin (SHBG) and change in body mass with correlation coefficient. The study was approved by the Human Use Review Committee at the U.S. Army Research Institute of Environmental Medicine, Natick, Massachusetts.In response to deployment, increases in lean mass (77.1 ± 7.6 to 77.8 ± 7.5 kg), maximum grip strength (57.9 ± 7.2 to 61.6 ± 8.8 kg), and conduct of aerobic (156 ± 106 to 250 ± 182 minutes/week) and strength training (190 ± 101 to 336 ± 251 minutes/week) exercise were observed (p0.05). Increases in serum SHBG (35.42 ± 10.68 to 38.77 ± 12.26 nmol/L) and decreases in serum cortisol (443.2 ± 79.3 to 381.9 ± 111.6 nmol/L) were also observed (p0.05). Body mass changes were dependent on baseline body mass. Soldiers in the lowest quartile of baseline body mass increased body mass (75.6 ± 2.6 vs. 76.6 ± 2.8 kg, p = 0.03), as did those in the second quartile (81.6 ± 2.0 vs. 83.7 ± 3.5 kg, p = 0.02). Those in the third quartile also tended to increase body mass (89.2 ± 2.6 vs. 90.9 ± 3.3 kg, p = 0.05), while those in the upper quartile tended to decrease body mass (98.5 ± 3.6 vs. 96.7 kg, p = 0.06). Change in SHBG was inversely correlated with change in body mass (r = -0.33, p = 0.02). There were no changes in fat mass, body fat percentage, waist circumference, neck circumference, total testosterone, calculated bioavailable or free testosterone, high-sensitivity C-reactive protein, tumor necrosis factor-α, interleukin-1β, or interleukin-6. Inflammatory markers were skewed toward lower values.Overall, physiological status of elite SOF soldiers characterized by multiple prior deployments was minimally impacted by combat deployment, in the absence of major unit casualties. The majority experienced some adaptive changes, including increased lean mass, grip strength, time spent engaged in exercise, and decreased levels of the stress hormone cortisol. Mechanisms contributing to inverse correlations between change in SHBG and change in body mass may be further clarified. Future investigations may also more fully characterize the degradation and optimization of health and physiological status of SOF training and deployment cycles with in-theater data collection and repeated measures.

Long chain fatty acids (LCFAs), a major source of cellular energy, are solubilized and transported in the blood by binding to serum albumin. Changes in human serum albumin's (HSA's) UV absorption and characteristic reactivity with pyridoxal-5'-phosphate appear to reflect a concerted change in its structure upon binding five equivalents of myristate. Isothermal titrations with myristate and other LCFA anions are also consistent with the presence of five strong, interacting, binding sites. Although HSA is usually thought to have many independent LCFA anion binding sites, just five interacting sites appear to account for the changes in structure that accompany its binding of myristate.

Rate constants of 0.0054 and 0.021 M(-1)s(-1) for the reactions of acrylamide with human serum albumin (HSA) and glutathione (GSH), respectively, were determined under physiological conditions by following the loss of their thiol groups in the presence of excess acrylamide. Based on these in vitro values, reactions with these thiols appear to account for most of acrylamide's elimination from the body. Combined with data from other studies, these results should be useful for assessing the health risk of dietary acrylamide.

In this study, we synthesized a zwitterionic DTNB derivative, 5-(2-aminoethyl)-dithio-2-nitrobenzoate (ADNB), and characterized its reactions with several cationic, anionic, and neutral thiols. Reactions with ADNB, unlike those with DTNB, are relatively insensitive to electrostatic environments and ionic strengths. At relatively low ionic strength, rate ratios, k ADNB / k DTNB , varied from 0.22 for reactions with low-molecular-weight cationic thiols to 3.0 for those with low-molecular-weight anionic thiols. A k ADNB / k DTNB ratio of ∼200 for Cys-34 of BSA appears to reflect a very anionic environment. k ADNB / k DTNB ratios of ∼6 and ∼1, respectively, for canine and equine serum albumins, which have Glu-82 → Asp and Glu-82 → Ala substitutions suggest Glu-82 is the most important anionic residues affecting the reactivity of Cys-34 in BSA. k ADNB / k DTNB ratios appear to be useful for characterizing electrostatic environments of thiol groups in proteins.

The chemical modifications of proteins are reviewed. Subjects include: (1) modifications done to study protein function; (2) two naturally occurring modifications: carbonylamine reactions and reactions with nitric oxide; (3) applications for bioconjugation and mass spectral analysis; and (4) modifications of food proteins.

Model dithiol compounds were S-nitrosated and the kinetics of their decompositions were studied. Rate constants for intramolecular homolysis of dinitrosated dithiols varied from 1.6 to 10 × 10−3 s−1. Rate constants for intramolecular heterolysis of mononitrosated dithiols varied from 4 to 32 × 10−4 s−1. Similar reactions may account for the effects of NO and nitrovasodilators on some enzymes.

4,4'-Dipyridyl disulfide (4-PDS) and sodium arsenite have been used to develop a rapid and sensitive assay for monothiols in the presence of dithiothreitol (DTT). The procedure is similar to that of Zahler and Cleland (J. Biol. Chem. 243, 716-719, 1968) but involves the use of a lower pH and 4-PDS, which is more effective than 5,5'-dithiobis-(2-nitrobenzoic acid) at low pH. Background reactions that interfere with the determination of slow reacting thiol groups are much reduced in the new assay procedure. The reduction of oxidized glutathione by DTT at three different pH values and a partial and the complete reduction of bovine serum albumin by DTT have been characterized in order to demonstrate the use of this procedure. In the first two cases monothiol groups were readily detected without interference from DTT. Lower than expected numbers of thiol groups in the case of three reduced and denatured model proteins appeared to reflect their partial complexation by arsenite. Equilibrium and kinetic constants for the formation and dissociation of the DTT-arsenite complex from pH 5 to 8 are presented.

Background In early 2007, U.S. Army helicopter aviators deployed to Afghanistan complained of increased pain frequency during flight. The aviators attributed this pain to body armor, which had not been worn prior to deployment. This study was conducted to investigate these complaints. Methods A retrospective study of pain frequency among 68 deployed helicopter aviators was performed using self-reported categorical pain ratings and flight times abstracted from a safety questionnaire. To assess the association of substantial increases in flight time on reported pain frequency, study subjects were divided into a group reporting a decrease or increase of 1 h or less in average daily flight hours during deployment as compared to predeployment (level flight hours, LFH) and a group reporting an increase of greater than 1 h in average daily flight hours (increased flight hours, IFH). Results A significantly higher proportion of aviators in the IFH group reported an increase in pain frequency as compared to LFH (81.5% vs. 61.1%, respectively). The relative risk (RR) of increased pain frequency associated with IFH was greatest in the lower back (RR = 1.80), legs (RR = 2.60), arms (RR = 9.11), and the groin (RR = 12.1). Discussion This study provides preliminary evidence that complaints of increased pain frequency during deployment were associated with substantial increases in flight times. Further study is warranted to investigate and confirm the underlying causes of this pain.

Potentially dangerous nitrosamines have been shown to result from the reaction of sodium nitroprusside with several drugs under physiological conditions (pH 7.3 and 37°C). In each case the products were identical to those produced upon reaction with nitrous acid at much lower pH values. Reaction rates were shown to reflect a first order dependence on both amine and nitroprusside concentrations and to increase at higher pH values, approximately in proportion to concentrations of unprotonated amine. Fast reactions of sodium nitroprusside with reduced glutathione, cysteine, and ascorbate suppress but do not prevent the conversion of amines into N-nitrosamines. These results show sodium nitroprusside to be very potent nitrosating agent under physiological conditions and suggests nitrosamines may be formed during its normal pharmacological administration.

The S-nitroso derivative of 5-thio-2-nitrobenzoate was synthesized from 5,5'-dithiobis(2-nitrobenzoic acid) and partially characterized. Although relatively unstable, it is easy to prepare and reacts very rapidly with thiols and thiol groups of proteins to give corresponding S-nitrosothiols and 5-thio-2-nitrobenzoate dianion. The latter's easy spectrophotometric detection makes such reactions easy to follow and to quantitate.

The transfer of nitroso groups from S-nitroso-L-cysteine (1) and six other simple S-nitrosothiols to Cys 34 of bovine serum albumin (2) has been followed using Ellman's reagent, 5,5'-dithio-bis (2-nitrobenzoate) (3), to detect the resulting thiols. The described method utilizes the low reactivity of (3) with (2) and the high extinction coefficient of 2-nitro-5-thiobenzoate produced upon its reaction with thiols to follow the transfer of nitroso moieties at low concentrations where other procedures are not feasible. A second-order rate constant of 6400 M-1 s-1 obtained for the reaction of (2) with S-nitrosomercaptoethylamine is approximately 10 times faster than that for its reaction with (1), approximately 40 times faster than that for its reaction with S-nitrosoglutathione, and consistent with Cys 34 being located in a narrow crevice in close proximity to an anionic charge.

Gary Means1, Cristóbal S Berry-Cabán2, Kurt Hammermeuller11Department of Family Medicine, 2Department of Research, Womack Army Medical Center, Fort Bragg, NC, USABackground: A review of the literature and an extensive Medline search revealed that this is the first case report of the use of guaifenesin to increase sperm motility.Case: A 32-year-old male presented for an infertility evaluation. He reported an inability to conceive with his wife after 18 months of unprotected intercourse. A semen analysis was performed that included spermatozoa count, liquefaction, morphology, motility, viscosity and volume. Initial results of the semen analysis demonstrated low sperm count and motility. The provider offered treatment with guaifenesin 600 mg extended release tablets twice daily. Two months after guaifenesin therapy the semen analysis was repeated that demonstrated marked improvement in both total sperm count and motility.Conclusion: Evidence for the effectiveness of guaifenesin is almost entirely anecdotal. Given the mechanism of action of guaifenesin, it is not clear from this case why the patient demonstrated such a large improvement in both sperm count and motility. Additional studies of the effects of guaifenesin on male fertility could yield information of the medication's effect on men with normal or decreased total sperm counts.Keywords: sperm motility, guaifenesin, infertility, male pregnancy

The rapid reaction of diisopropylfluorophosphate with a tyrosine residue of human serum albumin at 0.02 m ionic strength involves prior rapid reversible binding characterized by a dissociation constant of 3.6 × 10 −3 m and an apparent p K a of 8.3. The rapid reaction of p -nitrophenyl acetate with human serum albumin (G. E. Means and M. L. Bender, 1975, Biochemistry 14 , 4989–4994) appears to involve the same tyrosine residue and is thus stoichiometrically inhibited by prior reaction with diisopropylfluorophosphate. Both reactions are strongly inhibited by decanoate anion, strongly retarded at higher ionic strength, and reflect strong rapidly reversible binding and abnormally low tyrosine p K a values. This reactive tyrosine residue thus appears to be located in a primary binding site for small apolar anions and to be closely associated with several cationic groups.

Monomer and dimer fractions of human serum albumin (HSA) obtained from charcoal-treated Fraction V HSA have very similar fluorescence and circular dichroism spectra, but the dimer neither binds l -tryptophan nor reacts rapidly with p -nitrophenyl acetate. The latter reaction presumably occurs in a major binding site of the monomer as many strongly bound ligands including l -tryptophan, small fatty acid anions ( e.g., S.-W. M. Koh and G. E. Means, 1979 , Arch. Biochem. Biophys. 192 , 73–79), and several drugs ( e.g, N. P. Sollenne and G. E. Means, 1979 , Mol. Pharmacol. 15 , 754–757) all decrease rates of reaction in direct proportion to their concentration. Binding data for those ligands indicate the presence of less than one binding site per molecule of charcoal-treated Fraction V HSA, and thus appear to reflect only its content of albumin monomer. The absence of that binding site in the dimer may reflect its inclusion within the dimer interface.

The importance of carbohydrate and protein structure on the nonenzymatic glycosylation of proteins is discussed on the basis of limited model compound studies. Rates of giycosyiation presumably depend on the proportion of sugar present in the carbonyl form, on the amine basicity, and on its steric accessibility. The thermodynamic stability of the resulting glycosylamino moieties, which presumably depend in part on the protein structure may, however, be the most important factor in determining the extent of their reaction.

Human serum albumin reacts very rapidly with p-nitrophenyl acetate (NphOAc). Rapid acetylation of the protein accompanies and largely accounts for the easily observed rapid formation of of p-nitrophenolate ion. One group is acetylated much faster than all others. It appears to be located in a high affinity binding site for small fatty acid anions, to have a pKa of 8.7, and a limiting bimolecular rate of reaction with NphOAc of approximately 3 X 10(4) M-1 sec-1 at alkaline pH values. Rapid reversible binding appears to be a major contributor to the high reaction velocity.

A new method is described for the immobilization of biologically active proteins onto several types of organic polymer beads. First, the soluble protein is modified by reaction with an excess of a hydrophobic imidoester, for example methyl 4-phenyl-butyrimidate, at ca. pH 9 and 0 degrees . Excess imidoester and side products resulting from imidoester hydrolysis are separated from the hydrophobic protein derivative by exclusion chromatography or dialysis. A suspension of polymer beads (e.g. Amberlite XAD-7) is then added to a solution of the modified protein at room temperature or below and stirred gently for 1-2 h. The polymer beads are allowed to settle, separated from the solution by decantation or filtration, washed, and resuspended in an appropriate buffer. Quantitative adsorption of protein to the polymer beads is observed under such conditions. The synthesis of seven hydrophobic imidoesters and their use for the immobilization of trypsin onto several types of porous polymer beads is described. The immobilizations of trypsin, yeast alcohol dehydrogenase, and E. coli asparaginase by this procedure with high recoveries of catalytic activity, suggests that it will be applicable to a large number of biologically active proteins.

Reductive methylation has little or no detectable effect on the catalytic or physicochemical properties of bovine trypsin but reduces its susceptibility to autolysis. Increased stability after methylation appears to result from the conversion of trypsin-susceptible lysine residues to trypsin-resistant epsilon-N,N-dimethyllysine residues. Reductively methylated trypsin is easily prepared and may be useful in place of trypsin where autolysis is otherwise difficult to control.

Amino groups of proteins can be alkylated by reaction with a variety of aldehydes or ketones in the presence of several different mild reducing agents. Because reductive alkylation occurs under mild conditions and has relatively minor effects on most proteins, it is becoming one of the more important methods for protein modification. This report discusses some of the recent applications of this reaction.

Small unbranehed fatty acid anions inhibit the fast reaction between p-nitrophenylacetate and human serum albumin. Plots of reactivity versus fatty acid anion-albumin ratios resemble simple binding isotherms from which corresponding dissociation constants have been calculated. For the homologous fatty acid anions, butyrate through decanoate, dissociation constants decrease from 3.2 × 10−4 to 1 × 10−7 m , respectively, by uniform increments per methylene group according to the relationship −ΔG °(kcal) = 0.804n + 2.30, where n is the number of constituent methylene groups. Small fatty acid anions thus appear to interact primarily with a single, relatively uniform apolar binding site with a capacity sufficient for nine methylene groups. Fatty acid anions larger than decanoate interact significantly with other sites and do not obey the same relationship. The reactivity of diluted human serum with p-nitrophenylacetate was found to be one-third to one-half of that expected for its content of serum albumin, but as in vitro, it could be completely inhibited by small amounts of decanoate.

Summary New assays for carboxypeptidase A and carboxypeptidase B were developedusing polypeptides prepared from proteins as substrates. The substrates were prepared by treating casein with either α-chymotrypsin or trypsin and then reductively methylating the amino groups of the resulting mixtures of peptides with NaBH 4 and HCHO. Reductively methylated chymotrypsin-treated casein and reductively methylated trypsin-treated casein were relatively specific substrates for carboxypeptidase A and carboxypeptidase B, respectively. Hydrolysis of COOH-terminal amino acids was quantitatively measured by reaction of newly formed amino groups with trinitrobenzenesulfonic acid. Controls containing the substrates without additions of the carboxypeptidases gave very low blank values with trinitrobenzenesulfonic acid because of the unreactivity of this reagent with dimethylamino groups. As little as 0.1 μg of either enzymes was adequate for assay. The pH optima for both enzymes were similar to the optima of these enzymes on dipeptides, but the pH profiles were broader. Carboxypeptidase B did not hydrolyze COOH-terminal e- N,N -dimethyllysines, but it rapidly hydrolyzed COOH-terminal homoarginines.