Accuracy of the Apple Watch Oxygen Saturation Measurement in Adults and Children with Congenital Heart Disease.

As new customer health devices have been spread throughout the consumer market in recent years, it now needs to be evaluated if they also fulfill the requirements of clinical use. The Apple Watch Series 6 provides a new health feature with its oxygen saturation measurement. The aim of this prospective, investigator-initiated, single-arm study was to compare transcutaneous oxygen saturation measurements using the Apple Watch 6 with the conventional method of pulse oximetry in patients with congenital heart disease. Patients of any age presenting at the Leipzig Heart Center, Department for pediatric cardiology, were included. After obtaining informed consent, the routine oxygen saturation measurement with the pulse oximeter was taken and simultaneously three measurements with the Apple Watch. A total of 508 patients were enrolled. Comparing children and adults in terms of measurement success shows a statistically significant difference with a higher proportion of unsuccessful measurements in children, but no difference concerning correct versus incorrect Apple Watch measurements. Noticeable, strapping on the watch properly around the patient's wrists significantly improved the measurements compared to a watch only laid on. The study demonstrated that oxygen saturation measurement with the Apple Watch 6 is not yet up to the medical standard of pulse oximetry, too large a proportion of the measurements remain either unsuccessful or incorrect. While a high proportion of unsuccessful measurements in children can be attributed to movement, the cause in adults usually remains unclear. Further influencing factors on a correct, or successful measurement could not be found.

Open Field Exercise Testing in Pediatric Congenital Heart Disease Patients: A Subsumption of Cardiovascular Parameters.

Heart failure is a common phenomenon in congenital heart disease patients. Cardiopulmonary exercise testing is used for a reliable assessment of heart failure but is still challenging, especially for young children. Implementing mobile cardiopulmonary exercise testing (CPET) can close that diagnostic gap. While average values for healthy children have already been published, this study aims to describe typical ranges of cardiovascular performance parameters of young children with congenital heart disease performing an 8-min running cardiopulmonary exercise test. Children aged 4-8 years with common congenital heart defects after corrective surgery (Tetralogy of Fallot; transposition of the great arteries and univentricular hearts after palliation) were included. The outdoor running protocol consisted of slow walking, slow jogging, fast jogging, and maximum speed running. Each exercise was performed for 2 min, except the last, in which children were instructed to keep up maximal speed as long as possible. A total of 78 children (45 male/33 female, mean age 6,24) with congenital heart disease participated in the study, of which 97% completed the CPET successfully. A detailed description of participating patients, including data on cardiac function and subjective fitness levels, is given to help physicians use this method to classify their patients. This study presents a typical range for cardiovascular performance parameters in a population of 4-8-year-old children with congenital heart disease tested in a newly developed outdoor running protocol for CPET.

Into the Blue: First in Man Data on Diving Physiology in Fontan Patients.

Swimming and diving are popular recreational activities. As congenital heart disease, especially patients with univentricular hearts after Fontan palliation are thought to have reduced physiologic capacities for compensation of submersion-associated physiologic demands, current guidelines put restraints on this group of patients. Although these restrictions on doctoral advice place a significant burden on affected patients, it is especially interesting that these guideline recommendations are merely based on physiologic assumptions, i.e., expert consensus. A recent study by Paech et al. presented the first in vivo data on the effects of immersion in Fontan patients, stating no major adverse events in their study group as well as comparable physiologic adaption as reported in the literature for healthy people. Yet, submersion was not reflected in this study, and the current study therefore aimed to conduct a first study for the evaluation of the effects of submersion and apnea diving in Fontan patients. A control group of healthy adults as well as patients recruited from the Heart Center Leipzig, Department of pediatric cardiology underwent a standardized diving protocol including a static as well as dynamic apnea phase. Physiologic data were recorded. This study presents the first structured data on diving physiology in Fontan patients compared to healthy probands. There were no adverse events. The physiologic response to diving seems to be comparable between healthy probands and Fontan patients. Although, healthy probands did reach a much better performance, the basic mechanisms of physiologic adaption seem comparable.

Accuracy of the Apple Watch iECG in Children With and Without Congenital Heart Disease.

The development of smart technologies paves the way for new diagnostic modalities. The Apple Watch provides an FDA approved iECG function for users from 22 years of age. Yet, there are currently no data on the accuracy of the Apple Watch iECG in children. While arrhythmias are a frequent phenomenon in children, especially those with congenital heart disease, the increasing spread of smart watches provides the possibility to use a smart watch as mobile event recorder in case of suspected arrhythmia. This may help to provide valuable information to the treating physician, without having the patient to come to the hospital. Necessary treatment adjustments might be provided without timely delay. The aim of this study was therefore to evaluate the agreement of measured values of rate, interval, and amplitude with those obtained by a diagnostic quality ECG recording to an Apple Watch iECG in children with and without congenital heart disease. In this prospective, single-arm study, consecutive patients aged 0-16 years presenting to the Heart Center Leipzig, Department for pediatric cardiology were included. After obtaining informed consent from participants' parents, a 12-lead ECG and an iECG using an Apple Watch were performed. Cardiac rhythm was classified, amplitudes and timing intervals were measured and analyzed in iECG and 12-lead ECG for comparability. These measurements were performed blinded to the patients' history by two experienced pediatric cardiologists. Patient demographic data, medical and cardiac history were assessed. 215 children between 0 and 16 years were enrolled. Comparison of amplitudes and timing intervals between ECG and iECG showed excellent correlation (K > 0.7, p < 0.01) in all parameters except for the p-waves. Automatic rhythm classification was inferior to manual interpretation of ECG / iECG, while iECG interpretation was reliable in 94.86% of cases. The study demonstrates equal quality of the Apple Watch derived iECG compared to a lead I in 12-lead ECG in children of all age groups and independent from cardiac anatomy.

The Fontan and the Sea: First-in-Man Data on Swimming and Diving Physiology in Fontan Patients.

While swimming represents a popular recreational activity, the immersion of the human body into the water requires a complex physiologic adaption of the whole cardiopulmonary and circulatory system. While this sport is regarded as beneficial, especially in cardiovascular patients, current guidelines hypothesized a possible hazardous effect of swimming and especially diving in patients with univentricular hearts after Fontan palliation. Yet, actual data to underline or contradict these assumptions are lacking. Therefore, this study aimed to conduct a first feasibility study for the evaluation of these effects on Fontan physiology and elucidate the gap of evidence currently preventing patients after Fontan palliation from being restricted from swimming or diving on doctoral advice. Patients recruited from the Heart Center Leipzig, Department of pediatric cardiology, underwent spiroergometry treadmill testing followed by a spiroergometry swimming stress test in a counter current pool. Physiologic data were recorded. A short apnea diving test was performed. The current study found similar physiologic reactions comparing treadmill and swimming exercise stress testing. Heart rate response and oxygen uptake were comparable on land and in the water. This study presents the first-in-man data on swimming and diving in Fontan patients. In this small study cohort of three Fontan patients, there were no adverse events triggered by swimming and breath-hold diving seen. Basically, the physiologic response to exercise was comparable on land and in the water.

Drug-Induced Loss of Preexcitation in Pediatric Patients with WPW Pattern During Electrophysiologic Study.

Ablation of accessory pathways (AP) is one of the most often performed procedures in pediatric electrophysiology. In pediatric patients these procedures are mostly performed in anaesthesia or sedation. In some of these patients who are referred for electrophysiologic (EP) study, we could observe disappearance of the preexcitation, i.e. antegrade conduction of an AP during introduction of sedation. As a suppression of AP conduction capacities has been reported as negative side effect of propofol and other anaesthetics, the aim of this study was to evaluate risk factors for drug-induced suppression of AP conduction properties. Consecutive, pediatric patients with Wolff-Parkinson-White (WPW) pattern referred for EP study in the period of 2016-2017 were reviewed in retrospect. Patients with complex congenital heart disease were excluded. An entire chart review including ECG, bicycle stress testing, and periprocedural data was performed. In 4 of 37 patients included into the study, loss of preexcitation could be observed during sedation. Data analysis showed weaker conduction capacities of the AP as a risk factor (p = 0.009). Interestingly, absolute (p = 0.11) or adjusted to body weight (p = 0.92) drug doses were not a relevant risk factor. Patients with WPW and weaker conduction capacities of the AP, as implied by an early disappearance of preexcitation during exercise stress testing, seem to be more prone to drug-induced suppression of an AP.

Rate Control by Transoesophageal Atrial Overdrive Pacing for Refractory Supraventricular Tachycardia with Severe Ventricular Dysfunction: A Bridge to Recovery.

Tachyarrhythmia-induced cardiomyopathy (TIC) is a rare, yet life-threatening phenomenon in children. TIC has been defined as myocardial dysfunction that is entirely or partially reversible after control of the responsible tachyarrhythmia and is typically caused by an incessant supraventricular tachycardia. In cases of unsuccessful termination of the tachycardia, cardiogenic shock may occur. Several authors favour the use of mechanical circulatory support in such cases. Particularly with regard to these partly severe cases, the authors would like to present the option of transoesophageal overdrive pacing (TOP) as a tool for initial heart rate control and hemodynamic stabilization of patients with TIC refractory to conventional strategies of tachycardia termination. Chart review for patients receiving TOP during the last 5 years in two centres was performed. A case series of successful TOP including technique and outcomes is depicted. Rate control through TOP is a safe and effective option to restore acceptable hemodynamics in infants with refractory supraventricular tachycardia and severe impairment of ventricular function. It presents the possibility of immediate heart rate control and offers time for myocardial recovery and safe implementation of antiarrhythmic drug therapy.

QRS Width as a Predictor of Right Ventricular Remodeling After Percutaneous Pulmonary Valve Implantation.

Recent data showed a right ventricular dyssynchrony in patients with tetralogy of Fallot (TOF). Percutaneous pulmonary valve implantation (PPVI) has become an important procedure to treat a pulmonary stenosis and/or regurgitation of the right ventricular outflow tract in these patients. Despite providing good results, there is still a considerable number of nonresponders to PPVI. The authors speculated that electrical dysfunction of the right ventricle plays an underestimated role in the outcome of patients after PPVI. This study aimed to investigate the influence of right ventricular electrical dysfunction, i.e., right bundle branch block (RBBB) on the RV remodeling after PPVI. The study included consecutive patients after correction of TOF with or without RBBB, who had received a PPVI previously at the Heart Center of the University of Leipzig, Germany during the period from 2012 to 2015. 24 patients were included. Patients without RBBB, i.e., with narrow QRS complexes pre-intervention, had significantly better RV function and had smaller right ventricular volumes. Patients with pre-interventionally QRS width below 150 ms showed a post-interventional remodeling of the right ventricle with the decreasing RV volumes (p = 0.001). The parameters of LV function and volume as well as RV ejection fraction remained unaffected by RBBB. The presented data indicate that the QRS width seems to be a valuable parameter in the prediction of right ventricular remodeling after PPVI, as it represents both electrical and mechanical functions of the right ventricle and may serve as an additional parameter for optimal timing of a PPVI.

Development of an AI based automated analysis of pediatric Apple Watch iECGs.

Introduction: The Apple Watch valuably records event-based electrocardiograms (iECG) in children, as shown in recent studies by Paech et al. In contrast to adults, though, the automatic heart rhythm classification of the Apple Watch did not provide satisfactory results in children. Therefore, ECG analysis is limited to interpretation by a pediatric cardiologist. To surmount this difficulty, an artificial intelligence (AI) based algorithm for the automatic interpretation of pediatric Apple Watch iECGs was developed in this study. Methods: A first AI-based algorithm was designed and trained based on prerecorded and manually classified i.e., labeled iECGs. Afterward the algorithm was evaluated in a prospectively recruited cohort of children at the Leipzig Heart Center. iECG evaluation by the algorithm was compared to the 12-lead-ECG evaluation by a pediatric cardiologist (gold standard). The outcomes were then used to calculate the sensitivity and specificity of the Apple Software and the self-developed AI. Results: The main features of the newly developed AI algorithm and the rapid development cycle are presented. Forty-eight pediatric patients were enrolled in this study. The AI reached a specificity of 96.7% and a sensitivity of 66.7% for classifying a normal sinus rhythm. Conclusion: The current study presents a first AI-based algorithm for the automatic heart rhythm classification of pediatric iECGs, and therefore provides the basis for further development of the AI-based iECG analysis in children as soon as more training data are available. More training in the AI algorithm is inevitable to enable the AI-based iECG analysis to work as a medical tool in complex patients.

Blood pressure response to dynamic exercise testing in adolescent elite athletes, what is normal?

Background: In general, only few studies are dedicated to blood pressure behavior under physical stress in children and adolescents. Even less is published about the blood pressure behavior of young high-performance athletes on the ergometer. For this reason, we evaluated the blood pressure behavior under stress compared to non-athletes in a large collective (n = 739) of young high-performance athletes (age 10-20 years, mean 15.8 years, male 442, female 297) of different sports. A complete echocardiographic examination was available in all athletes. Result: Regardless of gender, the young competitive athletes achieved significantly higher maximum blood pressure values than investastigated populations from previous studies. Based on the data obtained, blood pressure percentiles are now defined explicitly for junior athletes across sports as well as age- and gender-dependent, which did not exist in this form of normal values for the special clientele of young competitive athletes. The echocardiographic examinations demonstrated stress-induced cardiac adaptation adaptations in the majority of athletes, which thus correlate with the comparatively higher stress blood pressures compared to non-athletes. Conclusion: For the first time, blood pressure percentiles for exercise tests on the ergometer for age groups and gender in high performance athletes are defined based on a comparatively large collective of young competitive athletes. Upper limits were determined, in particular for systolic blood pressure under stress, and categorized according to gender and age. Performance diagnosticians and physicians are now enabled to make a more accurate assessment of the corresponding blood pressure regulation of young athletes under exercise conditions.

[Fixed bearing unicondylar arthroplasty in medial osteoarthritis of the knee]. / Schlittenprothese mit fixiertem Tibiainlay bei medialer Gonarthrose.

OBJECTIVE: The unicondylar prosthesis replaces the medial femerotibial compartment only, the part presenting with osteoarthritic changes. The remaining compartments of the knee present less osteoarthritic changes and thus can be preserved. INDICATIONS: Osteoarthritis of the medial femorotibial compartment is the ideal indication for unicondylar arthroplasty. The knee should show an intraarticular deformity, which means the malalignment is caused by the osteoarthritic changes of the medial compartment. CONTRAINDICATIONS: Malalignment of >5°, flexion contracture of >10°, mediolateral instability and symptomatic osteoarthritis of a second compartment should be considered as contraindications for unicondylar arthroplasty. SURGICAL TECHNIQUE: In the current article, implantation of the BalanSys® system is presented. Femoral bony resection is solely ligament balanced. The technique allows creation of an optimal extension and flexion gap. Bone cuts were performed using a soft tissue tension device for measuring the extension and flexion gap. POSTOPERATIVE MANAGEMENT: Full weight bearing on crutches is allowed immediately after surgery without restriction in flexion. Crutches are recommended for 4 weeks in order to compensate for neuromuscular deficits. Anticoagulation is recommended for 11-14 days according to the AWMF guidelines (S3 guidelines, Release:15 October 2015). RESULTS: The clinical follow-up after 2 years showed 87 ± 13 points in the knee score and 80 ± 10 points in the function score. The mean range of motion increased from 113°±24° prior to surgery to 122°±23° after surgery. A preoperative extension deficit of 10° was observed in 9 patients and reduced postoperatively in 3 patients.

The crystal structure of the Bacillus lentus alkaline protease, subtilisin BL, at 1.4 A resolution.

The crystal structure of subtilisin BL, an alkaline protease from Bacillus lentus with activity at pH 11, has been determined to 1.4 A resolution. The structure was solved by molecular replacement starting with the 2.1 A structure of subtilisin BPN' followed by molecular dynamics refinement using X-PLOR. A final crystallographic R-factor of 19% overall was obtained. The enzyme possesses stability at high pH, which is a result of the high pI of the protein. Almost all of the acidic side-chains are involved in some type of electrostatic interaction (ion pairs, calcium binding, etc.). Furthermore, three of seven tyrosine residues have potential partners for forming salt bridges. All of the potential partners are arginine with a pK around 12. Lysine would not function well in a salt bridge with tyrosine as it deprotonates at around the same pH as tyrosine ionizes. Stability at high pH is acquired in part from the pI of the protein, but also from the formation of salt bridges (which would affect the pI). The overall structure of the enzyme is very similar to other subtilisins and shows that the subtilisin fold is more highly conserved than would be expected from the differences in amino acid sequence. The amino acid side-chains in the hydrophobic core are not conserved, though the inter-residue interactions are. Finally, one third of the serine side-chains in the protein have multiple conformations. This presents an opportunity to correlate computer simulations with observed occupancies in the crystal structure.

Strategy and implementation of a system for protein engineering.

This paper describes an overall view of an industrial protein engineering project from conception to successful completion. The choice of rational design was determined by the availability of an excellent three-dimensional crystal structure and the availability of information in the literature to define a strategy. The design strategy was refined extensively during the course of the project. The development of methods for mutagenesis, expression, verification, purification, and characterization of mutant enzymes is dictated in part by the enzyme property one chooses to modify and must be rapid yet accurate. Such an approach would be applicable to improve the stability of any other protein or enzyme. Using this approach, we successfully increased the stability of subtilisin BL over 10-fold at 50 degrees C with an overall success rate greater than 60%.

Unusual ligand binding at the active site domain of an engineered mutant of subtilisin BL.

As an attempt to recruit the third calcium binding site of thermitase into subtilisin BL, a Bacillus lentus alkaline protease (BLAP), the amino acid sequence from position 50 to 60 and position 92 was modified to the equivalent amino acids in thermitase. The resulting protein, designated BLAPm109, exhibited unusual biochemical features. Peptide mapping and gel electrophoresis revealed that two protein species co-purify in a ratio of about 1:1. Form 1 consisted of a single polypeptide of 269 amino acid residues. Form 2 was the same protein but with an internal peptide bond cleavage at the C-terminus of position 54. On electropherograms a dimer of Form 1 and Form 2 was also detectable. A zymogram showed that all three molecular species were catalytically active. From this protein mixture, crystals suitable for X-ray analysis were nevertheless obtained. SDS-PAGE of protein recovered from a crystal revealed that only Form 2 appears. in the crystal. The space group for this crystal was P21 with unit cell dimensions of a=42 angstroms, b=58 angstroms, c=47 angstroms and beta = 106.3 degrees. Examination of the preliminary electron density map revealed that the "thermitase loop" from 50 to 60 departs from the surface of the protein and winds through the active site of a symmetry-related copy of the asymmetric unit.

Hypertrophic intraventricular flow obstruction after very-low-dose dexamethasone (Minidex) in preterm infants: case presentation and review of the literature.

The use of dexamethasone in preterm infants developing bronchopulmonary dysplasia has been proven to be effective. Hypertrophic cardiomyopathy is a frequently reported, although transient, side effect of high-dose dexamethasone administration. The recent introduction of very low dexamethasone dose, called 'Minidex', promised equal effectiveness compared to high-dose dexamethasone without relevant side effects. Our study presents two patients developing hypertrophic cardiomyopathy with intraventricular cardiac obstruction after administration of 'Minidex'. Marked cardiac side effects may occur even during very-low-dose dexamethasone treatment in preterm neonates. Betablocker and discontinuation of dexamethasone seem to allow spontaneous reversal of myocardial hypertrophy and obstruction. After all, systematic surveys of the incidence of cardiac complications in a larger population of preterm infants treated with very low doses of dexamethasone are needed.

Kinetics of ribulosebisphosphate carboxylase at high protein concentration.

Kinetic parameters of ribulos-1,5-bisphosphate carboxylase/oxygenase (RuBP carboxylase) are usually evaluated in dilute solutions (less than 0.1 mg ml-1). Yet, this enzyme occurs in vivo at 100-200 mg ml-1 and a total protein concentration 300-400 mg ml-1. Enzymes can change their catalytic properties upon 'crowding'. Hence it became of interest to determine whether RuBP carboxylase elicits any properties not observable in dilute solution. Pre-steady state progress curves of fully activated enzyme showed an initial burst followed by a slower rate of product formation. The extent of the burst increased as concentration ratios of RuBP and RuBP carboxylase decreased. The burst corresponds to 1/8 turnover per holoenzyme or 1 turnover per active site. No discontinuity in progress curves was observed with partially activated enzyme.

Peptide mapping of subtilisins as a practical tool for locating protein sequence errors during extensive protein engineering projects.

Protein engineering based on rational design is an iterative process of sequential amino acid residue replacements. This requires a rapid and sensitive method for checking the protein sequence after each round of mutagenesis. As shown with subtilisin BL, acid treatment followed by urea denaturation renders the enzyme degradable by trypsin within 10 min. Separation of the peptides by reversed-phase HPLC produces a map that differentiates even the most conservative alteration on peptides as large as 48 amino acid residues. The method was used to uncover erroneous mutations; to determine the concentration of active protease relative to an internal standard of known specific activity; to measure the rate of oxidation of methionine-216 in the oxyanion hole of subtilisin BL; and to document that under these conditions no other methionine in the molecule is oxidized by hydrogen peroxide.

Active site studies of ribulose-1,5-bisphosphate carboxylase/oxygenase with pyridoxal 5'-phosphate.

There are 16 epsilon-amino groups of lysyl residues which are essential for the activity of ribulose-1,5-bisphosphate carboxylase/oxygenase. These lysyl residues formed a Schiff base with pyridoxal 5'-phosphate which was stabilized by NaBH4 reduction. The stoichiometry of covalently bound pyridoxal 5'-phosphate after NaBH4 reduction was determined spectrophotometrically with a derived molar extinction coefficient of 4800 M-1 cm-1. The incorporation of pyridoxal 5'-phosphate into the protein was accompanied by loss of the carboxylase and oxygenase activities, but the ratio of their activities remained constant. Vmax, but not Km, values were changed by this modification of the amino acid groups. Half of the epsilon-amino groups of lysine appeared to be at the 8 catalytic sites and half at the 8 activator sites for CO2, as indicated by kinetics of reactivation of the enzyme activity during dissociation of the Schiff base between pyridoxal 5'-phosphate and the protein. Reduction with NaB3H4 revealed that all 16 of the lysyl residues were on the large subunit. Ribulose-1,5-bis-phosphate alone protected 16 amino groups from Schiff base formation with pyridoxal 5'-phosphate, and the enzyme activity was fully conserved. NaHCO3 increased and MgCl2 lowered slightly the protective effect of ribulose-1,5-bisphosphate. Modification of sulfhydryl groups by p-chloromercuribenzoic acid inhibited the enzyme and excluded binding of 8 equivalents of pyridoxal 5'-phosphate. Upon removal of the mercuribenzoate groups with excess dithiothreitol, the loss of enzyme activity was exponentially correlated with the binding of 8 mol of pyridoxal 5'-phosphate/mol of enzyme. In contrast to p-chloromercuribenzoic acid, iodoacetamide, which inhibited the catalysis, had no influence on the binding of the 16 pyridoxal 5'-phosphate/mol of enzyme. It is suggested that the CO2 activator site with one epsilon-amino group for binding of CO2 and the catalytic site with one epsilon-amino group of a yet unknown function are located closely together on the large subunits of the enzyme. The results are consistent with a sulfhydryl group as the proton acceptor opposite carbon 3 of ribulose-1,5-bisphosphate, and when this sulfhydryl group is blocked by the bulky mercuribenzoate group, but not by the small carboxyamidomethyl group, pyridoxal 5'-phosphate binding is modified.

Purification and degradation of ribulose bisphosphate carboxylase from soybean leaves.

A rapid method is described for the preparation of up to 500 milligrams of pure ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBP carboxylase) from 250 grams of field-grown soybean leaves. Leaves were extracted in 20 millimolar phosphate (pH 6.9) at 4 degrees C, containing 4% (w/v) polyvinylpolypyrrolidone, 10 micromolar leupeptin, 1 millimolar phenylmethyl sulfonylfluoride, 1 millimolar diethyldithiocarbamate, 5 millimolar MgCl(2), 1 millimolar dithiothreitol, 0.2 millimolar ethylene-diaminetetraacetic acid, 50 millimolar 2-mercaptoethanol. The extract was incubated in the presence of 5 millimolar ATP at 58 degrees C for 9 minutes, then centrifuged and concentrated. Sucrose gradient centrifugation into 8 to 28% (w/v) sucrose on a vertical rotor for 2.5 hours yielded pure enzyme with a specific activity of 1.1 to 1.3 micromoles per minute per milligram protein at pH 8.0, 25 degrees C. Soybean plants of the same line grown (at 400 microeinsteins per square meter per second) in growth chambers yielded enzyme with a specific activity of 0.6 to 0.7 micromoles per minute per milligram protein. During prolonged purification procedures a proteolytic degradation of RuBP carboxylase caused complete loss of catalytic activity. Without destroying the quaternary structure of the enzyme, a 3 kilodalton peptide was removed from all large subunits before further breakdown (removal of a 5 kilodalton peptide) occurred. Catalytic competence of the enzyme was abolished with the loss of the first (3 kilodalton) peptide.

Zymogram of proteases made with developed film from nondenaturing polyacrylamide gels after electrophoresis.

A simple, extremely versatile method for preparing zymograms from proteases after nondenaturing Phast-System gel electrophoresis is described. After completion of the run and before staining, an electropherogram and a piece of developed, single-side coated X-ray film are brought into contact for 5 min at room temperature. The film overlay is then separated and the gel is stained for protein. The zymogram on the X-ray film is generated by simply pouring about 10 ml of a suitable buffer solution at 30 to 50 degrees C over the film strip. Clearing zones appeared within a few seconds to a few minutes depending on protease amount. For the alkaline protease subtilisin BL the lower limit of detectability was 10 ng applied to the gel prior to electrophoresis. For preservation and archiving the zymogram film is simply rinsed with distilled water and air-dried. The film can be cut to size and mounted for a slide projector, or the film can serve as a negative for photographic enlargements. The clearing zone area is proportional to the amount of protease from 10 to 100 ng of protein.